an evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals: a report...
TRANSCRIPT
Mutation Research, 115 (1983) 1-72 1 Elsevier Biomedical Press
An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals
A report of the U.S. Environmental Protection Agency Gene-Tox Program *
Andrew J. Wyrobek a, Laurie A. Gordon a, James G. Burkhart b Mary W. Francis c, Robert W. Kapp Jr. d, Gideon Letz e, Heinrich V.
Malling b, John C. Topham f and M. Donald Whorton g Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550 (U.S.A.), b Laboratory of Biochemical Genetics, National Institute of Environmental Health Sciences, Research
Triangle Park, NC 27709 (U.S.A.), c Information Division, Oak Ridge National Laboratory **, Oak Ridge, TN 37830 (U.S.A.), a Biodynamics, Inc., East Millstone, NJ 08873 (U.S.A.), e California State Department
of Health, Berkeley, CA 94704 (U.S.A.), ! Imperial Chemical Industries, Macclesfield (Great Britain), g School of Public Health, University of California, Berkeley, and g Environmental Health Associates, Inc.,
Berkeley, CA 94704 (U.S.A.)
(Received 24 September 1982) (Accepted 13 October 1982)
* Work Group Report prepared for the Gene-Tox Program (Office of Toxic Substances, Office of Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC). The authors are members of the Gene-Tox Work Group on Sperm Tests in Animals and Men. This paper was prepared as an interim report to evaluate the utility of a particular assay system. A.J. Wyrobek was the chairman of the reviewing committee.
** Operated by Union Carbide Corporation under contract W-7405-eng-26 with the U.S. Department of Energy.
By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.
Although the review described in this article has been funded wholly or in part by the U.S. Environmental Protection Agency through interagency agreement DOE 40-1123-80, EPA No. 80-D- X0953 to the Oak Ridge National Laboratory, it has not been subjected to the agency's required peer and policy review, and therefore, does not necessarily reflect the views of the agency and no official endorsement should be inferred. The protocols stated, suggested use of the assay in a screening program, and research recommended should not be taken to represent agency policy on these matters.
This document was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor the University of California nor any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility
0165-1110/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
Contents
S u m m a r y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. D e s c r i p t i o n of s p e r m tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. D e s c r i p t i o n of s p e r m test m e t h o d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. S tudies in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. S tudies in o t h e r m a m m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. The r e p r o d u c t i v e a n d gene t ic imp l i ca t i ons o f chemica l ly i n d u c e d s p e r m a n o m a l i e s
C. Past uses of s p e r m tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. P ro toco l guide l ines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. M o u s e s p e r m m o r p h o l o g y test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a. A n i m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b. E x p o s u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
c. C o n t r o l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
d. S p e r m s a m p l i n g a n d eva lua t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
e. Sta t is t ica l c r i te r ia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. O t h e r s p e r m tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I lL I n t e r p r e t a t i o n o f the reviewed d a t a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. P r e s e n t a t i o n o f d a t a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. S p e r m tests in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. S p e r m tests in o the r m a m m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Cr i t e r i a for a ccep t ab i l i t y o f pub l i shed d a t a . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Stat is t ical e v a l u a t i o n s p r o v i d e d b y or ig ina l a u t h o r s . . . . . . . . . . . . . . . . . . . . . .
D. Cr i t e r i a used by reviewers to c lass i fy agen t s as posit ive, negat ive , or inconc lus ive
induce r s of s p e r m a n o m a l i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. S p e r m tests in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. S p e r m tests in o t h e r m a m m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E. Usefu lness o f s p e r m tests in h a z a r d eva lua t ion . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Test p e r f o r m a n c e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Overa l l p e r f o r m a n c e ; n u m b e r of chemica l s a n d chemica l c lasses tes ted in the
va r ious s p e r m tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. M o u s e s p e r m m o r p h o l o g y test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. A c r o s o m e a b n o r m a l i t y test in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. S p e r m c o u n t s in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . .
4. S p e r m mot i l i ty in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. S p e r m tests in o the r m a m m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. C o r r e l a t i o n wi th m a m m a l i a n m u t a g e n i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Specif ic locus m u t a t i o n ( S L M ) test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. He r i t ab l e t r a n s l o c a t i o n (HT) test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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for the a c c u r a c y , comple teness , o r usefu lness o f a n y i n f o r m a t i o n , a p p a r a t u s , p r o d u c t , o r p rocess
disc losed, o r r ep resen t s tha t its use w o u l d no t i n f r inge pr iva te ly o w n e d r ights . Refe rence here in to a n y
specif ic c o m m e r c i a l p r o d u c t s , process , o r service b y t r ade n a m e , t r a d e m a r k , m a n u f a c t u r e r , or
o therwise , does no t necessar i ly cons t i t u t e or i m p l y its e n d o r s e m e n t , r e c o m m e n d a t i o n , or f a v o r i n g b y
the U n i t e d Sta tes G o v e r n m e n t or the Un ive r s i ty o f Ca l i fo rn ia . The views a n d o p i n i o n s o f a u t h o r s
expressed here in d o no t necessar i ly s ta te o r reflect those of the U n i t e d Sta tes G o v e r n m e n t t he reo f a n d
shal l no t be used for adver t i s ing o r p r o d u c t e n d o r s e m e n t pu rposes .
W o r k p e r f o r m e d u n d e r the ausp ices o f the U.S. D e p a r t m e n t of Ene rgy b y the L a w r e n c e L ive rmore
N a t i o n a l L a b o r a t o r y u n d e r c o n t r a c t n u m b e r W-7405-eng-48 ; a lso by s u p p o r t f r o m the U.S. Env i ron-
m e n t a l P ro t ec t ion A g e n c y , the U.S. N a t i o n a l Ins t i tu te of E n v i r o n m e n t a l H e a l t h Sciences. Imper ia l C h e m i c a l s Indus t r i e s ( G r e a t Br i ta in) a n d o the r sources .
3. Dominant lethal (DL) test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 4. The sensitivity and specificity of the mouse sperm morphology test for germ
cell mutagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 C. Correlation with mammalian carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1. Carcinogenicity list provided by the EPA . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2. Carcinogenicity list developed by the reviewing committee . . . . . . . . . . . . . . 16 3. The sensitivity and specificity of the mouse sperm morphology test for car-
cinogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 4. Importance of testing to lethality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 5. Summary of correlations with carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . 17
D. Comparison of sperm data from man, mouse, and other mammals . . . . . . . . . . . 18
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 A. Strengths and weaknesses of sperm tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 B. The role of animal sperm tests in assessing mutagenic potential . . . . . . . . . . . . . 19 C. The role of animal sperm tests in assessing carcinogenic potential . . . . . . . . . . . . . 20 D. Recommendations for further research, development, and validation of sperm
tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 VI. Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 VII. Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
1. Mouse sperm morphology test: positive compounds . . . . . . . . . . . . . . . . . . . . . 22 2. Mouse sperm morphology test: negative compounds . . . . . . . . . . . . . . . . . . . . . 30 3. Mouse sperm morphology test: inconclusive compounds . . . . . . . . . . . . . . . . . . 46 4. Agents tested for induction of acrosomal abnormalities in mice . . . . . . . . . . . . . 50 5. Agents tested for changes in sperm counts and motility in mice . . . . . . . . . . . . . 50 6. Sperm tests in other mammalian species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 7. Germ cell mutational activity of agents tested in the mouse sperm morphology
test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 8. Carcinogenic activity of agents evaluated in the mouse sperm morphology t e s t . . . 63 9. Comparison of sperm test results for agents tested in more than one mammalian
species including man . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 VIII. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 IX. Appendix I. Transmission of chemically induced sperm defects to the F 1 progeny of
treated mice: the F t sperm morphology test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 B. Test description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
1. Genetic basis of the method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 2. Protocol used in studies reviewed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
C. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Table Al. F I sperm morphology test: Results with chemicals . . . . . . . . . . . . 70
D. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 E. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
S u m m a r y
T h e l i t e r a t u r e o n t h e m o u s e s p e r m m o r p h o l o g y t e s t a n d o n o t h e r s p e r m t e s t s in
n o n h u m a n m a m m a l s w a s r e v i e w e d (a ) to e v a l u a t e t h e r e l a t i o n s h i p o f t h e s e t e s t s to
c h e m i c a l l y i n d u c e d s p e r m a t o g e n i c d y s f u n c t i o n , g e r m - c e l l m u t a g e n i c i t y , a n d c a r c i n o -
g e n i c i t y , a n d ( b ) to m a k e a n i n t e r s p e c i e s c o m p a r i s o n o f r e s p o n s e s to c h e m i c a l s . A
t o t a l Of 71 p a p e r s w e r e r e v i e w e d .
T h e m o u s e s p e r m m o r p h o l o g y t es t w a s u s e d to a s s e s s t h e e f f ec t s o f 154 o f t h e 182
chemical agents covered. 4 other murine sperm tests were also used: the induction of acrosomal abnormalities (4 agents), reduction in sperm counts (6 agents), motility (5 agents), and F 1 sperm morphology (7 agents). In addition, sperm tests for the spermatogenic effects of 35 agents were done in 9 nonmurine mammalian species; these included analyses for sperm count, motility, and morphology, using a large variety of study designs. For the mouse sperm morphology test, 41 agents were judged by the reviewing committee to be positive inducers of sperm-head shape abnormalities, 103 were negative, and 10 were inconclusive.
To evaluate the relationship between changes in sperm morphology and germ cell mutagenicity, the effects of 41 agents on mouse sperm shape were compared to available data from 3 different mammalian germ-cell mutational tests (specific locus, heritable translocation, and dominant |ethal). The mouse sperm morphology test was found to be highly sensitive to germ-cell mutagens; 100% of the known mutagens were correctly identified as positives in the sperm morphology test. Data are insufficient at present to access the rate of false positives. Although it is biologically unclear why one might expect changes in sperm morphology to be related to carcinogenesis, we found that (a) a positive response in the mouse sperm morphol- ogy test is highly specific for carcinogenic potential (100% for the agents surveyed), but (b) overall, only 50% of carcinogens were positive in the test (i.e., sensitivity --- 50%). Since many carcinogens do not produce abnormally shaped sperm even at lethal doses, negative findings with the sperm test cannot be used to classify agents as noncarcinogens. We conclude that the mouse sperm morphology test has potential use for identifying chemicals that induce spermatogenic dysfunction and perhaps heritable mutations. Positive test results may also be useful in assessing carcinogenic potential. Insufficient numbers of chemicals agents have been studied by the other sperm tests to permit similar comparisons.
A comparison of 25 chemicals tested with sperm counts, motility, and morphol- ogy in at least 2 species (including man, mouse and 9 other mammals) demonstrated good agreement in response among species. With further study, interspecies com- parisons of chemically induced sperm changes may be useful for predicting and evaluating human effects.
I. Introduction
This paper reviews the use of sperm tests in nonhuman mammals as measures of spermatogenic dysfunction, germ-cell mutagenicity, and carcinogenicity. The mam- malian sperm tests developed historically from infertility studies in domestic animals and man. The effectiveness of human sperm tests in assessing chemically induced spermatogenic dysfunction (see the accompanying paper on human sperm tests in this volume) has emphasized the need for developing animal models to help understand the human response.
Most of the chemical effects reviewed were observed using the mouse sperm morphology test. Effects observed using other mouse sperm tests were also included:
the induction of acrosome abnormalities, the reduction in sperm counts and motility, and the F~ sperm morphology test for abnormally shaped sperm in the offspring of treated mice. The latter test is reviewed and evaluated in Appendix I.
Sperm studies were also reviewed in nonhuman mammalian species other than the mouse. The studies involved the effects of chemical exposure on traditional sperm parameters: sperm counts, motility and morphology.
All of the papers reviewed were supplied by the Environmental Mutagen Infor- mation Center (EMIC) at Oak Ridge National Laboratory, TN, or by the members of the reviewing committee. 71 papers were accepted for review (29 papers on the mouse, including 4 F 1 sperm morphology papers; and 42 nonmouse papers). This is a first attempt to gather such a data base and, with advance apologies to the authors, some studies may have been inadvertently missed.
Papers were included for evaluation based on the following criteria: (a) primary data were presented (review articles and papers repeating data published elsewhere were excluded), (b) whole animals were exposed in vivo (exposure of sperm to chemical agents in vitro was excluded), (c) sperm measurements included counts, motility, morphology or acrosome abnormalities on sperm from ejaculate, vas deferens, or epididymis (chemical effects on testicular histology or fertility were excluded), (d) only chemical agents were tested (radiation, heat, trauma, disease, altitude and dietary deficiencies were excluded), (e) non-English papers were in- cluded only when translations into English, German, French or Spanish were available, and (f) sufficient quantitative data or detailed descriptions of effects were available for evaluation by the reviewing committee (abstracts were usually ex- cluded).
II. Description of sperm tests
A. Description of sperm test methods
1. Studies in mice 5 murine sperm tests were reviewed: sperm morphology, acrosomal abnormality,
counts, motility and F 1 sperm morphology. In the sperm morphology test, assessment of chemical effects on exposed mice is
based on visually scoring for the percentage of sperm with abnormal head forms in smears of sperm from the epididymis or vas deferens (Wyrobek and Bruce, 1975). Animals are typically sampled 5 weeks after exposures up to lethal doses (see Section II D for further details). The test for acrosomal abnormalities is based on visually scoring for defects in the morphology of the acrosome in stained smears of sperm from exposed mice (Moutschen and Colizzi, 1975).
Sperm counts and motility tests measure the number and swimming ability of the mature epididymal sperm (Cattanach and Edwards, 1958; Ficsor apd Ginsberg, 1980). Studies in mice have generally avoided the use of ejaculated sperm because of the extreme difficulty of obtaining sperm free of solid coagulum.
In the F~ sperm morphology test the proportion of male offspring with elevated
sperm abnormalities is determined after exposure of male or female mice before mating. This test is further described in Appendix I.
2. Studies in other mammals Several sperm tests have been used to study chemically induced spermatogenic
dysfunction in mammalian species other than man or mouse: sperm counts (number of sperm per ml of ejaculate or a similar quantitative evaluation of sperm number), motility (some measure of sperm movement), and morphology (an assessment of sperm head shape or tail abnormalities). These studies encompass a broader range of design than the murine studies. Semen was often collected at various times from individual animals after they were exposed, using a variety of exposure routes and regimens (see Table 6).
B. The reproductive and genetic implications of chemically induced sperm anomafies
For many years, semen analysis has been routinely performed to diagnose testicular damage and infertility in humans and domestic animals. Though it is generally agreed that large reductions in sperm number or motility, or large increases in sperm with abnormal shapes are associated with reduced fertility (Amelar, 1966), it is not clear whether smaller changes are also associated with fertility changes.
Sperm tests provide a direct measure of the quality of sperm production in chemically treated animals. However, the genetic consequences of fertilization by sperm affected by chemical exposure during spermatogenesis remain unclear; em- bryonic death or the transmission of genetic aberrations to live-born progeny are also possibilities.
Studies evaluating the genetic consequences of chemically induced sperm changes have focused mainly on understanding the genetic basis of induced shape abnormali- ties in mice. A number of lines of evidence suggest that induced changes in sperm morphology reflect genetic damage in the male germ cell. First, considerable evidence indicates that sperm shaping is polygenetically controlled by numerous autosomal and sex-linked genes, including T-locus alleles (Bennett, 1975), hop-sterile (Johnson and Hunt, 1971), pink-eye sterile (Hunt and Johnson, 1971), quaking (Bennett et al., 1971), factors on chromosome 17 (Forejt, 1976), factors on the Y chromosome (Brozek, 1970; Krzanowska, 1972, 1976), and factors on the X chro- mosome (Hugenholtz and Bruce, 1977b). Spontaneous levels and types of abnorm- ally shaped sperm are remarkably consistent within and are characteristic of strains, even though the levels may vary widely (1-65%) among strains (Wyrobek, 1979). Furthermore, the heritability for sperm-head dimensions in mice is remarkably high: h 2= 0.8-0.9 (Illisson, 1969; Beatty, 1970, 1972).
Second, virtually all murine germ-cell mutagens tested induce sperm-shape abnormalities in mice (Wyrobek and Bruce, 1978; Bruce and Heddle, 1979; Topham, 1980a,c). The evaluation of agents reviewed in this paper supports this high correlation (see Table 7 and Section IV B for details). Third, agents that show sperm-shape abnormality in offspring of treated males also induce abnormalities in the exposed males (Staub and Matter, 1977; Wyrobek and Bruce, 1978; Hugenholtz
and Bruce, 1979; Topham, 1980b; Sotomayor, 1979). (See Appendix I for a descrip- tion of the F~ sperm morphology test and further details on the transmission of sperm shape abnormalities to the offspring of treated mice.)
Although elevated levels of sperm abnormalities are seen under certain presuma- bly nonmutagenic circumstances, such as in very young age (Krzanowska et al., 1972; Krzanowska, 1981), dietary restriction (Komatsu et al., 1982), and increased body temperature (Cairnie and Leach, 1980), the appropriate tests for possible heritable consequences associated with these factors have not been done in mice or other mammals.
C. Past uses o f sperm tests
The reviewed studies were conducted for several purposes. For the mouse sperm morphology test, most exposures were to single purified agents selected to assess relationships with mutagenicity and carcinogenicity (Wyrobek and Bruce, 1975; Bruce and Heddle, 1979; Topham, 1980a, c; Wyrobek et al., 1981). The sperm morphology test has also been used as a short-term bioassay for spermatotoxic effects of agents responsible for significant human exposures (e.g., anesthetic gases: Land et al., 1981; and diesel exhaust: Pereira et al., 1980). Studies have also shown that the sperm morphology test can be used to evaluate commercial agents for which large potential human exposures may be involved at various phases of their manufacture (e.g., anilines: Topham, 1980a).
The major uses of nonmurine sperm tests were to evaluate the ability of agents to modulate fertility (either by improving fertility or by acting as male contraceptives), to study side effects of drug therapy, and to develop animal models of chemically induced spermatogenic damage. Animal sperm tests (especially in studies where repeated sperm samples were taken before, during, and after exposure) have pro- vided important data on dose dependence, spermatogenic stage specificity, as well as the reversibility of induced sperm effects.
D. Protocol guidelines
1. Mouse sperm morphology test
Based on the available hterature and the opinion of the reviewing committee, the following are provided as guidelines for the use of this test. Since the test is still undergoing development and validation, this should not be taken as a standard protocol.
a. Animals
F~ hybrid mice are recommended, as they tend to have a lower and more stable spontaneous incidence of abnormal sperm than outbred or pure inbred strains. The (C57BL/6 x C3H)F) and (CBA × BALB/c)F 1 have been extensively used. Several studies reviewed also used inbred mice (C3H/HeJ , CBA/CaJ, DBA/2J , Q-strain, and C-strain: see Tables 1, 2, and 3 for refs.); more work is needed for selecting optimal genotypes for this test. It is desirable that animals be acclimatized to their environment for several weeks (at least one week) before administration of the
compound. A specific-pathogen-free environment is desirable, as infection, allergic reactions, and changes in body temperature and in blood flow to the testes may affect this test. Male mice are usually acquired at 6 -8 weeks of age and kept in quarantine until tested at 11-15 weeks of age. This schedule avoids the transitory increase of abnormal sperm seen at the onset of mouse spermatogenesis in younger animals. Mice should be randomly allocated to the test groups.
b. Exposure For each test compound, solubility and toxicity data should be obtained. Corn
oil, saline, water, dimethyl sulfoxide (DMSO), tricapryline, and diluted Tween 80 have been used as solvent vehicles. To test a chemical, single intraperitoneal injections on 5 consecutive days are recommended, using a minimum of 3 but preferably 4 -6 dose levels in addition to negative and positive controls. There should be at least 5 surviving animals at each dose level. The highest total 5-day dose should be lethal to some animals or be the highest concentration that can be satisfactorily formulated. If the LDs0 has been determined for the 5-day dosing schedule, this should be used as the highest total dose. Since the available LDs0 data tend to be limited to single exposures, a useful rule of thumb is to set the highest total 5-day dose to 2 - 4 times the single-exposure LDs0. The other dose levels are then, in turn, lower by a factor of 2 or less. It is recommended that daily injection volumes be limited to approximately 0.1 ml if distilled water or DMSO is used as the solvent vehicle, and to approximately 0.3 ml for less toxic vehicles if solubility permits. Freshly prepared (daily) materials should be used, unless the stability of the compound is well known.
Intraperitoneal injection has been the most frequently used route for routine testing, because the dose can be carefully controlled. However, in designing a future study, consideration should be given to exposure by other routes more similar to those likely to be encountered by man (e.g., inhalation, ingestion, skin contact). Studies are also needed of possible interactive effects between solvent (e.g., DMSO) and test compound and of effects of simultaneous exposure to more than one test agent. Studies are also needed to determine the opt imum number of injections. 5 injections were typically used because this approach was found to result in less variability in response and more sensitivity than single injections.
c. Controls Positive and negative controls should be included in every study. Negative control
animals should receive the vehicle in the same volume that the exposed animals received. It is important that the vehicle be without detectable effect on the mice (e.g., large volumes of DMSO should be avoided because of toxic effects on the testes, liver, and other organs). Daily, intraperitoneal doses of 20 m g / k g cyclophosphamide monohydrate or 75 m g / k g methyl methanesulfonate (MMS) for 5 days have been found to be effective positive controls.
d. Sperm sampling and evaluation Historically, sperm have been sampled from the cauda epididymides at 35 days (5
weeks) from the first injection. For 25 chemicals, this sampling time was found to be as effective or more effective than sampling at 1 or 10 weeks after exposure (Wyrobek and Bruce, 1975). Unfortunately, little further work has been done to determine the most effective sampling time for chemicals. Sperm sampling at time points other than 5 weeks might yield valuable data on which stages of spermatogen- esis are most sensitive to the agent, and the persistence or reversibiilty of induced damage.
Mice are sacrificed by cervical dislocation, and the epididymides are excised. Both epididymides from each mouse are usually minced together with small scissors in isotonic medium (e.g., 4 ml of phosphate-buffered saline) and filtered to exclude large tissue fragments. Smears are prepared on slides before or after staining the cells with approximately 0.05% Eosin Y (aqueous). The slides from the animals of each study are coded and evaluated for sperm head abnormalities. Epididymal sperm count is determined by hemocytometer or automated methods. Usually, testes weights are also recorded.
Sperm smears are usually examined by light microscopy at 400-800 x magnifica- tion, with a green or blue filter. For each animal, 200-500 sperm (usually a minimum of 1000 sperm per dose point) are assessed for morphological abnormali- ties of the sperm head according to the criteria of Wyrobek and Bruce, 1975. Heads without tails or heads that are in contact or overlaid by other sperm or debris are excluded.
e. S ta t i s t i ca l cri teria
Statistical evaluation is based on comparison of data for negative control, positive control and the individual dosage groups. The incidence of abnormal sperm in the negative control group should be similar to historical negative control values for the laboratory. Individual animals with an unexplained high incidence of abnormal sperm in the negative control groups may occur at frequencies from less than 1 in 100 to 1 in 500 animals. The increase for the positive control group should be within the expected range for the laboratory and be a statistically significant elevation from the negative control ( p < 0.01). If these criteria are not met, the experiment should be reevaluated because the scoring, or the procedure for the administration of the test compound, or some other technical aspect of the study is in question.
Each dosage group should be individually compared with the concomitant negative control group with nonparamet r i c statistical procedures, e.g., Kolmogorov-Smirnov, Mann-Whitney, or Wilcoxon Rank Sum Test. A t-test may be used if the data are normally distributed, although this is usually not the case.
The recommended criteria for a positive response at any individual dose level are that the increase in abnormal heads be significant at the p < 0.05 level, and preferably be double the negative control level. For a test compound to be judged a positive inducer of abnormal sperm, the response should yield statistically significant increases at a minimum of 2 consecutive dose levels and be reproducible. It is judged negative if no increase in sperm abnormalities is seen up to doses that cause whole animal lethality. Compounds should be retested if (a) dose response is erratic, (b) an increase in abnormal sperm is seen at only one dose level, or (c) if negative results
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were obtained but whole animal lethality is not achieved. With compounds of very low toxicity, such as some essential amino acids, a presumptive negative classifica- tion should be made noting the maximum dose tested.
Dose-response curves and cumulative frequency plots may be useful for visual presentation. For summary purposes, group means may be plotted either before or after negative control values are subtracted. The negative control data should always be presented.
2. Other sperm tests
None of the mouse sperm tests other than the sperm morphology test have been sufficiently tested to prescribe a protocol at this time. The studies reviewed in the 9 other mammalian species encompass an assortment of experimental designs and include studies that use single sperm samples per animal when comparing groups of exposed and control animals (cross-sectional design); studies that use repeat samples from individual animals before, during, and after exposure (longitudinal design); and studies that combine these formats. Both chronic and acute exposures adminis- tered by varying routes are reported.
III. Interpretation of the reviewed studies
A. Presentation o f data
1. Sperm tests in mice
A summary of the mouse sperm morphology test data is presented in Tables 1, 2 and 3, with the following information recorded for each chemical agent: name of agent, carcinogenicity ( + , - , or not available (NA) as judged by a literature search by the reviewing committee and by a list provided by EPA), CAS Registry number, genotype of mouse used in the study, source of sperm (cauda epididymis, vas deferens, etc.), vehicle in which test agent was delivered, exposure route and regimen, number of animals tested at each dose, number of doses tested (excluding negative controls), highest total dose tested, lowest effective dose (the lowest dose at which a statistically significant response was observed, Table 1 only), whether lethality was achieved at the highest dose tested (Tables 2 and 3 only), original author 's classifica- tion of response (Table 3 only), the degree of statistical evaluation (Table 1 only), and the reference. Agents appearing in Table 1 were judged positive inducers of abnormal sperm, agents in Table 2 were judged negative, and agents in Table 3 were judged inconclusive because of insufficient or inconsistent data (for statistical criteria used, see Section I I I C, below).
All the chemical agents were studied with the mouse sperm morphology test by very similar procedures (i.e., based on the method of Wyrobek and Bruce, 1975). There were two reports on the effects of four chemicals on acrosomal abnormalities, and these results appear in Table 4. There were 6 chemicals evaluated in the mouse for their effects on sperm counts and 5 for motility (Table 5).
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2. Sperm tests in other mammals Table 6 summarizes the nonmurine animal sperm test data by species in the
following format: agent and CAS Registry number, study design (e.g., cross-sec- tional or longitudinal), source of sperm (ejaculate, epididymis, or vas deferens), vehicle, route of exposure and dosage regimen, number of animals tested per dose, number of doses tested, highest total dose tested, test system studied (counts, motility or morphology), the response observed (e.g., increase or decrease of sperm count, percent motile sperm, percent normal forms), lowest effective dose, whether the induced effect was reversible, the degree of statistical evaluation, and the reference.
B. Criteria for acceptability of published data
Data from the papers dealing with sperm tests in mice accepted under the criteria listed in Section I were considered for analyses and are included in Tables 1-5. Similar criteria were applied in the preparation of Table 6 which deals with sperm tests in other mammals. However, due to the lack of uniformity in the non-mouse studies, and the desire of the reviewing committee to include as broad a spectrum of non-mouse studies as possible, the criteria used with the latter papers were less stringent than those for the murine studies.
C. Statistical evaluations provided by original authors
Since raw data were not usually presented, the reviewing committee relied heavily on the interpretation given by the original authors of each study. In general, the ability of agents to induce sperm-shape abnormalities, especially in the mouse studies, was expressed as changes in percentage of abnormally shaped sperm. The majority of data from treated groups was compared with historical controls or simultaneous data from untreated mice. Several criteria were applied by the original authors to evaluate the response. Positive classification of compounds was usually based on an indication of a dose response (i.e., with more than one dose point statistically different from control at p < 0.05 by a non-parametric test such as the Wilcoxon Rank Sum test, or dose points exceeding the control level by 1%). In a few cases other criteria, such as doubling of the control level or p < 0.05 by single-sided t-test, were applied. The statistical evaluation applied for each agent determined to be positive on the mouse morphology test is listed in the statistics column in Table 1 and explained in the legend. The statistical methods utilized in non-mouse tests were varied and not always clearly described; the degree of statistical evaluation provided by the authors of each paper reviewed is noted in the statistics column and footnotes of Table 6.
D. Criteria used by the reviewers to classify agents as positive, negative, or inconclusive inducers of sperm anomalies
1. Sperm tests in mice For the mouse sperm morphology test, agents were classed as positive (Table 1) if
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the original authors considered the results as positive and if there was a statistically significant increase in the percent of abnormall~¢ shaped sperm for at least one dose level. If the increase was statistically significant but small (4% or less over back- ground), then a response was judged positive only if evidence of reproducibility was available (acriflavin, aminopterin and iododeoxyuridine). These criteria for a posi- tive response are more lenient than the ones recommended for future studies (see Section II-D-l-e). Agents were classed inconclusive (Table 3) when: (a) there was a conflict in results by different laboratories, (b) the agents gave small, apparently significant increases in sperm abnormalities (less than 4% over background) but were not tested for reproducibility, (c) they showed an atypical response with technical grade material, or (d) they were classed presumptive positives by the original authors.
Conflicting results between laboratories may have been due to different genetic strains, different exposure regimens or different criteria for positive responses. In one apparent conflict, hexamethylphosphoramide (HMPA), the positive response was found by the laboratory testing at the higher doses; HMPA was therefore classed as a positive (Table 1).
Compounds were classified as negative (Table 2) if according to the original authors they gave no significant increase in sperm abnormalities above control levels across the range of administered doses, or resulted in small, nonreproducible effects. Some compounds were classified as negative even though lethality was not noted at any dose tested (see Table 2, lethality heading). In Section IV-C-4 we discuss the importance of these latter agents on the calculation of the sensitivity of the mouse sperm morphology test to carcinogens.
The reviewing committee accepted author classifications of positive or negative for agents tested for the induction of acrosomal abnormalities (Table 4), reduction in sperm counts, and sperm motility in mice (Table 5).
2. Sperm tests in other mammals The conclusions of the authors of the original studies are generally accepted by
the reviewing committee (Table 6). In some cases, the reviewing committee recorded the results as marginal when statistics or design were questionable.
E. Usefulness of sperm tests in hazard evaluation
Although the endpoints used in sperm tests are not direct measurements of transmitted genetic damage, sperm data may be useful in hazard assessment for several reasons: (1) chemically induced sperm changes yield strong evidence of an agent's ability to interfere with normal sperm development and production, (2) several agents that induce transmissible sperm damage also interfere with sperm-head morphology in exposed males (see Appendix I and Table AI), and (3) the com- pounds that induce increases in sperm-head damage in mice (Table 1) are highly correlated with known germ-cell mutational activity (see Section IV and Table 7). This suggests that chemicals yielding positive results in the mouse sperm morphology test (Table 1) should be regarded as suspect germ-cell mutagens in mammals, particularly if supportive data are available in other mutational test systems. Agents
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positive in these sperm tests should be considered with high priority as candidates for study in human populations if suitable numbers of exposed men can be found (see the paper on human sperm tests in this volume).
Agents appearing in Table 2, although classed as negative, may induce sperm- shape abnormalities under different experimental conditions or in different geno- types/species. Often these studies did not include high doses. Agents appearing in Table 3 (inconclusives) also need further testing.
IV. Test performances
A. Overall performance," number of chemicals and chemical classes tested in the various sperm tests
1. Mouse sperm morphology test 154 chemicals have been tested in the mouse sperm morphology test. 41 com-
pounds were judged positive (Table 1), 103 negative (Table 2), and 10 inconclusive (Table 3). Of the negatives, 67 were known to be tested up to lethal doses. For the remainder of the negatives, lethality was not reached, lethality data were not presented, or such data could not be obtained.
The 154 chemicals represent 50 of the EPA Gene-Tox chemical classes. An agent may appear in more than one classification. In 9 classes, 10 or more compounds were tested, and in another 11 classes, 5-9 compounds were tested. The remaining 30 classes were represented by 4 or fewer compounds each. In only 2 of the 20 classes in which 5 or more members were tested was there a consistent response by all members of that class: (1) the class 'halogenated ethers and halohydrins', in which 7 agents were classed as negative and 1 as inconclusive; and (2) 'phosphoric acid esters and phosphamides', in which 6 agents were classed as positive and 1 as inconclusive. Other examples, in which positive responses were obtained with all tested members of a class with 2 -4 members are: alkyl sulfonates (4 tested positive); aziridines (4 positive); and nitrogen and sulphur mustards (3 positive). Examples where negative responses were obtained by all tested members of a class with 2-4 members are: amine-N-oxides (4 negative); azoxy, hydrazo compounds (2 negative); nitriles, azides (2 negative); nitroaromatics (4 negative); nitroquinolines (2 negative); and nitrosamines (3 negative). Chemicals that gave positive responses included antimetabolites, alkylating agents, spindle poisons and polycyclic aromatic hydro- carbons. This evaluation suggests that the sperm morphology test can be applied to test agents from a wide variety of chemical classes.
2. Acrosome abnormality test in mice 4 chemicals were studied with this test (Table 4) showing 3 positive and 1 negative
responses.
3. Sperm counts in mice 6 chemicals were studied with this test (Table 5) showing uniformly positive
results. Lu and Meistrich (1979) reported dose-related reductions in testicular sperm
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counts in mice exposed to adriamycin, cytosine arabinoside, cyclophosphamide, hydroxyurea, vinblastine, and vincristine (because this study used testicular sperm, it was not included in the present review).
4. Sperm motility in mice 5 chemicals were studied with this test (Table 5), showing 4 positive and 1
negative result.
5. Sperm tests in other mammals Sperm tests have been used to study the effects of at least 35 chemicals in 9 other
species of mammal. As shown in Table 6, 16 chemicals have been tested in rabbits; 14 chemicals in rats; 3 chemicals each in dogs, hamsters, and monkeys; 2 chemicals each in cattle, guinea pigs, and sheep; and 1 chemical in pigs. T h e tests included sperm counts, morphology, and motility.
For tests other than the sperm morphology test in mice, there were generally too few compounds tested in any particular chemical class to permit any meaningful generalization.
B. Correlation with mammalian mutagenicity
In an attempt to study the relationship between the induction of sperm-shape abnormalities and male germ cell mutational damage, we compared the sperm data collected in this review with the available literature on chemically induced germ-cell mutations in mice (specific locus mutations, heritable translocations and dominant lethals).
1. Specific locus mutation (SLM) test 14 chemicals were studied by both the SLM test (Russell et al., 1981) and the
mouse sperm morphology test. 11 of these chemicals were studied for SLM in postspermatogonial (usually spermatid) stages of spermatogenesis and 12 for SLM in spermatogonia.
Since it is difficult to compare the postspermatogonial and spermatogonial responses of the SLM test with the 5-week response of the sperm morphology test, the SLM results were grouped as positive at some stage, inconclusive at some stage, or negative at all stages (see Table 7). Only 7 of the 14 agents gave conclusive results (positive or negative) in both tests. Though the number of agents compared is small, the two tests show a 100% concordance (positives: cyclophosphamide, ethyl methanesulfonate, methyl methanesulfonate, mitomycin C, triethylenemelamine and procarbazine; negative: caffeine).
2. Heritable translocation (HT) test 9 chemicals were studied by both the HT test (Generoso et al., 1980) and the
mouse sperm morphology test. 8 of these chemicals were studied for HT in the postspermatogonial stages, and 3 chemicals for HT in spermatogonia. Grouping the postspermatogonial and spermatogonial HT results as positive at some stage, incon-
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clusive at some stage, or negative in all stages tested (Table 7), we again see a 100% concordance for the positive results on both tests (positives: cyclophosphamide, ethyl methanesulfonate, methyl methanesulfonate, mitomycin C, procarbazine, thiotepa, and triethylenemelamine). Unfortunately, there are no examples of the agents that tested negative on both tests.
3. Dominant lethal (DL) test Topham (1983) compared the results for 36 chemicals for the DL and sperm
morphology tests. As shown in Table 7, 15 agents were positive by both tests (actinomycin D, azathioprine, busulfan, colchicine, cyclophosphamide, ethyl methanesulfonate, ICI 42 464, lead acetate, metepa, methyl methanesulfonate, mitomycin C, procarbazine, thiotepa, triethylenemelamine, and trimethylphosphate). 9 agents were negative by both tests (2-AAF, caffeine, calcium cyclamate, 1,1-di- methylhydrazine, epichlorohydrin, MNNG, fl-propiolactone, thalidomide, and urethane). Although 8 agents were negative by DL yet positive by the sperm morphology test (acriflavine, aminopterin, 5-bromodeoxyuridine, ethionine, griseo- fulvin, hydroxyurea, 5-iododeoxyuridine and vinblastine sulfate), there are no exam- ples of agents positive by DL and negative by sperm morphology. This comparison between sperm morphology and the DL test (Table 7) should be reevaluated when the Gene-Tox report on the DL test becomes available.
4. The sensitivity and specificity of the mouse sperm morphology test for germ-cell mutagens
Using the above data, the sensitivity of the sperm morphology test to germ-cell mutagens (i.e., the proportion of mutagens correctly identified as positive by the sperm morphology test) was 100% for all 3 germ-cell mutational tests (6/6 for specific locus, 7/7 for heritable translocation, and 15/15 for dominant lethal). The specificity of the sperm morphology test (i.e., the proportion of nonmutagens correctly identified as negative by the sperm test) ranged from 53 to 100 % depending on the tests compared (1/1 for specific locus, undefined (0/0) for heritable translo- cation, 9/17 for dominant lethal). These comparisons suggest that the sperm morphology test properly identified all germ-cell mutagens thus far studied, i.e., it appears to be highly sensitive to germ-cell mutagens. The specificity of the test remains unclear; too few negatives were studied in the SLM and HT tests. The 8 apparent false positives when compared to the DL results need further study to determine whether the sperm morphology test is more sensitive than the DL test or whether there is a mismatching of spermatogenic stages. The inferences based on these comparisons should be considered preliminary until data on more agents become available.
C. Correlation with mammalian carcinogenicity
It is biologically unclear how changes in sperm morphology may be related to carcinogenesis, especially since different organs are usually involved. However, it has been suggested that a correlation may exist and we present the following evaluation.
The results of the mouse sperm morphology test were compared to carcinogenic-
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ity data derived from 2 sources: (1) a merged carcinogen list provided by the EPA Gene-Tox Steering Committee, and (2) a list generated by the reviewing committee. These lists classified each agent as (a) carcinogen, (b) noncarcinogen, (c) variable or (d) unclassified. Data from both lists were incorporated into columns 2 and 3 on Tables 1, 2 and 3.
1. Carcinogenicity list provided by EPA Of the 154 compounds evaluated in the mouse sperm morphology test, only 55
(36%) had an oncogenic classification on the EPA Merged Carcinogen List (see column 3 of Tables 1, 2, and 3), and only 3 compounds (2%) were classed as noncarcinogens.
As shown in Table 8 (row 1), of the 41 compounds positive in the mouse sperm morphology test, 20 were classed as carcinogens by the EPA list and 21 were unclassified. Of the 103 compounds found to be negative in the sperm morphology test (Table 8, row 5), 26 agents were classed as carcinogens, 2 were noncarcinogens, 1 was classed as an agent with variable carcinogenicity, and 75 were unclassified. The 10 compounds tested as inconclusive in the sperm morphology test (Table 8, row 2) included 6 carcinogens, 1 noncarcinogen, 1 having variable carcinogenicity, and 2 unclassified.
2. Carcinogenicity list developed by reviewing committee The above mentioned EPA Merged Carcinogen List has several important omis-
sions: (a) agents known to be carcinogens, e.g., procarbazine (Griesemer and Cueto, 1980) and (b) numerous agents generally considered noncarcinogens (the EPA list contained only 3 noncarcinogens relevant to our review). Therefore, the reviewing committee developed a separate listing of carcinogens and noncarcinogens as shown in column 2 of Tables 1-3. This list was based both on the EPA Merged Carcinogen List and on additional reference literature as showfi in Table 8.
Using the reviewing committee list, 102 of the 154 compounds evaluated in the mouse sperm morphology test (i.e., 66% of agents tested) were given an oncogenic classification by the reviewing committee. Of these, 76 (i.e., 75%) were carcinogens and 26 (i.e., 25%) were noncarcinogens. The remaining 52 were unclassified. It should be noted that when compared to the EPA list, the reviewing committee list contains (a) a larger proportion of agents with a positive or negative classification of oncogenicity (66 vs. 36% for the EPA list) and (b) a much larger proportion of agents classed as negatives (25 vs. 2% for the EPA list).
As shown on Table 8, row 1, under the headings of reviewing committee fist, the 41 compounds that were positive in the mouse sperm morphology test included 30 carcinogens, 0 noncarcinogens, and 11 agents whose carcinogenic activity is un- known. The, 11 compounds of undefined carcinogenicity that induce sperm shape abnormalities are acriflavine, 4-aminophenol, aminopterin, 5-bromodeoxyuridine, cannabinol, colchicine, cycloheximide, 6-9-tetrahydrocannabinol, gossypol, hydroxy- urea and vinblastine.
As shown on Table 8 (row 5), of the 103 compounds classed as negative on the sperm morphology test, 39 were classed as carcinogens by the reviewing committee,
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24 as noncarcinogens, and 40 of unknown carcinogenicity. Of the 10 compounds judged inconclusive in the sperm morphology assay (Table 8, row 2), 7 were carcinogens and 2 were noncarcinogens and 1 showed variable carcinogenicity.
3. The sensitivity and specificity of the mouse sperm morphology test for carcinogens Using the sperm morphology data gathered in this review and EPA Merged
Carcinogen List and excluding all inconclusives, the sensitivity of the sperm abnormality test to carcinogens (i.e., the proportion of carcinogens correctly identi- fied as positive by the sperm test) was 20 of 46 (43%) and the specificity (the proportion of noncarcinogens correctly identified as negatives by the sperm test) was 2 of 2 (100%).
Using the reviewing committee carcinogenicity list, we calculate the sensitivity and specificity of the sperm morphology test for carcinogens to be 43% (30 of 69) and 100% (24 of 24) respectively. Even though these percentages are based on larger numbers of chemicals, they are the same as those calculated from the EPA Merged Carcinogen List.
4. Importance of testing to lethality As listed in Table 2 and summarized in Table 8 (rows 3 and 4), only 65% of
agents that were negative by the sperm morphology test were tested at doses up to the lethal range. In assessing the relationship to carcinogenicity, it is of interest to correct the estimated sensitivity (43%) by considering only those compounds that were tested to sufficiently high doses. Using only those agents that were tested to lethal doses, the carcinogenic sensitivity of the test improved to 57% for the EPA Merged Carcinogen List, and to 54% for the reviewing committee list.
Although the above improvement in sensitivity reflects the importance of testing agents at high doses, these calculations emphasize the observations that many carcinogens are negative by the sperm morphology test even when tested at lethal doses.
5. Summary of correlations with carcinogenicity 3 inferences can be drawn from the above calculations. First, using the limited
data set available, agents positive in the mouse sperm morphology test appear to be carcinogens as well (100%, 20 of 20 from EPA Merged Carcinogen List, 30 of 30 from the reviewing committee's list, not including agents positive by the mouse sperm morphology test for which carcinogenicity is presently unknown). Second, of all agents known to be carcinogens, only 43% (sensitivity) were classed as positive in the sperm morphology test. This increased to approximately 55% when the analysis was limited to carcinogens tested to lethal doses. Third, 100% of the established noncarcinogens are negative in the sperm morphology test (2 of 2 from EPA Merged Carcinogen List and 24 of 24 from reviewing committee list). These comparisons suggest that a positive response in the mouse sperm morphology test may be helpful in assessing carcinogenic potential, but no conclusion as to carcinogenicity can be drawn from a negative result even when the agent is tested to a lethal dose. Tissue-specific access and the inability of the testes to metabolically activate certain
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chemicals to their toxic forms may explain these negative results for certain carcinogens.
Since these comparisons are based on the currently available chemical data, which are heavily biased towards carcinogens, these inferences should be reevaluated as more data become available.
D. Comparison of sperm data from man, mouse, and other mammals
25 compounds or mixtures were evaluated in more than one mammalian species. Table 9 lists the results of sperm tests in man (see the paper on human sperm tests in this volume), mouse (Tables 1-5), and other mammalian species (Table 6). In the case of conflicting studies within a species, the result from the study with the highest total dose is indicated; if the dose was approximately the same in 2 studies with conflicting results, both results are indicated.
Though the data are fragmentary, and there is little uniformity in study design, there is general agreement among species. Of 16 agents in which sperm morphology was assessed in 2 or more species, 11 show complete agreement in results. Sperm motility was studied in multiple species for only 4 compounds, too few for adequate comparison. Results for 6 of the 9 compounds tested for changes in sperm count in multiple species were in agreement.
Detailed examinations of individual studies are required when comparing results from different species. For example, diethylstilbestrol yields differing results in both mouse and man, which may reflect species differences in sensitivity, the unique sensitivity of the embryo, or other unknowns. Ethanol exposure does not induce sperm-shape abnormalities in mice, but human alcoholics do show sperm defects in morphology, motility, and counts. It is possible that the human defects may be due to poor nutrition, liver disease, or estrogen effects caused by the inability of damaged liver to detoxify endogenously produced estrogens. Agents which were part of complex human exposures were sometimes tested individually in other species (e.g., marijuana and the cancer chemotherapy regimen MVPP).
The general uniformity of response and the small numbers of agents for which data are available underscore the need for further studies of interspecies compari- sons. A promising direction for research may be to compare responses in mouse and man. Although 154 agents have been studied with the mouse sperm morphology test and 89 with human sperm tests, only 6 agents have been studied in both species. More work is clearly needed before any meaningful assessment can be made for using animal sperm tests to predict or evaluate human reproductive risks.
V. Conclusions
The following summarizes the strengths and weaknesses of the sperm tests, evaluates their roles in testing chemicals for mutagenic and carcinogenic potentials, and gives recommendations for future research.
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A. Strengths and weaknesses of sperm tests
Far more compounds encompassing diverse classes of chemical and biological activities have been evaluated by the mouse sperm morphology test than by any other sperm test in animals or man. Its major advantages are:
(a) Germ cells are exposed in vivo; a positive result in the test demonstrates an agent's ability to damage spermatogenesis. As such, it is a valuable tool in safety evaluation for assessing an agent's potential adverse effects on sperm production.
(b) The induction of abnormally shaped sperm in mice appears to be very sensitive to mammalian germ-cell mutagens; this test may therefore be a valuable tool for identifying germ-cell mutagens.
(c) Positive results in the mouse sperm morphology test appear to be highly specific for carcinogenicity; however, many carcinogens produce negative responses in the test.
(d) In the opinion of the reviewing committee the mouse sperm morphology test is simple, inexpensive, and relatively rapid when compared with other in vivo short-term tests.
(e) The sperm morphology protocol is adaptable to different species, dosage regimens, sampling times, routes of exposure, etc. This versatility makes it a useful animal model for human exposure.
The major disadvantages of the mouse sperm morphology test are: (a) The mutational consequences of induced sperm-shape abnormalities are not
clearly understood. (b) Current protocols may 'miss' certain agents (e.g., agents that exert only
transient effects at time points other than those sampled, agents that require different exposure regimens, or agents that require different routes of exposures). Also, different strains of mice may be insensitive to particular agents, as seen with aryl-hydrocarbon-hydroxylase responsiveness and hydrocarbon exposure (unpub- lished results).
(c) Certain factors such as ischemia, infection, changes in body temperature, may cause false positive responses. However, careful study design and good animal husbandry should minimize the occurrence of these effects.
B. The role of animal sperm tests in assessing mutagenic potential
One aim of a mutational test is to accurately assess the effect of environmental agents on the incidence of transmissible mutations in man. A bioassay that tests for spermatotoxic effects is of particular importance, as the activity of an agent in bacterial or somatic cells is often a poor predictor of its activity in the testes after in vivo exposure (de Serres and Ashby, 1981). The mouse sperm morphology is a test for agents that affect spermatogenic development. As suggested by its very high sensitivity for known germ-cell mutagens, it may be useful in identifying agents that induce mutations in germ cells in vivo.
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Sperm studies in other mammals may greatly assist in extrapolating the possible reproductive risk to man. Furthermore, the comparison of animal and human sperm data for the same agents may be a useful approach for reproductive risk assessment (see the paper on human sperm tests in this volume).
C. The role of animal sperm tests in assessing carcinogenic potential
With the understanding that the list of chemicals used in this review is heavily biased in favor of carcinogens and that the classification of the chemicals as carcinogens may not be completely accurate, we have shown that the mouse sperm morphology test has a moderate sensitivity (40-55%) and very high specificity (100%) with respect to carcinogenic potential. In a battery of short-term tests for oncogenic potential, these characteristics may be used to advantage.
D. Recommendations for further research, development and validation of sperm tests
This review presents evidence that sperm tests, particularly the mouse sperm morphology test, may be useful for identifying chemical agents that induce sperma- togenic dysfunction, and perhaps heritable mutations. Positive test results may also be important when assessing carcinogenic potential. The following areas are in need of further development:
(1) The mechanisms involved in the chemical induction of morphological changes in sperm need to be better understood.
(2) Agents that are known to affect semen quality a n d / o r fertility in humans should be evaluated in the mouse sperm morphology test and other animal sperm tests to increase our understanding of the extrapolation of animal data to humans. Many agents for which some positive human semen data are available appear never to have been studied in any animal sperm system (see the paper on human sperm tests in this volume). Particular care should be taken to ensure that routes of exposure, doses of agents, and semen sampling times are monitored so that the animal studies are as similar as possible to the human exposure conditions.
(3) An enlarged data base and better understanding are needed of the transmis- sion of chemically induced abnormal sperm morphology to the offspring of treated mice. Compounds should be chosen for the F~ sperm morphology test (see Appen- dix) that have already been evaluated for sperm morphology in the exposed male or were tested in other in vivo mammalian tests for mutational activity. Compounds evaluated should include a wide range of mutagens considered to act through different mechanisms, as well as compounds not expected to be mutagenic. An at tempt should be made to establish a protocol for studies of this type. Furthermore, these studies of the inheritance of abnormal sperm shapes can be used to get a better description of the genetic loci involved in sperm-shape control. Studies of the transmission of heritable mutations from treated mothers to sons should also be made.
(4) Further investigations are needed to evaluate the effects of prolonged treat- ment with agents and use of different sperm sampling times in the mouse sperm
21
morphology test to establish whether the procedures can be integrated into conven- tional protocols for general or reproductive safety evaluation.
VI. Acknowledgments
We thank Mr. Rudy Furrer for kindly supplying the lethality data for the murine sperm morphology studies done at the Ontario Cancer Institute in Toronto, Canada. We thank M. Mendelsohn, B. Gledhill, L. Dobson, B. Strack, J. Cherniak, G. Watchmaker and E. Rogers for their incisive comments and help in the preparation of this manuscript. We also thank A. Riggs, J. Adams and L. Corell for their assistance in typing and formatting this review.
22
TABLE 1
MOUSE SPERM MORPHOLOGY TEST: POSITIVE COMPOUNDS
Agent Carcinogenicity CAS No. Genotype
Reviewing EPA committee
Source of sperm
Vehicle
Acriflavine NA
Actinomycin D +
2-Aminofluorene +
4-Aminophenol NA
Aminopterin J +
Azathioprine 2 NA (Imuran)
Benzene +
3,4-Benzopyrene 3 +
5-Bromodeoxy- NA uridine (BUDR)
Busulfan 3 + (Myleran)
Cannabinol NA
Colchicine 4 NA
NA 8048-52-0 B6C3F1 Cauda epid.
+ 50-76-0 B6C3F1 Cauda epid.
B6C3F 1 Cauda epid.
NA 153-78-6 B6C3F1 Cauda epid.
NA 123-30-8 CBCF1/ICI Cauda epid.
NA 54-62-6 B6C3FI Cauda epid.
B6C3 F 1 Cauda epid.
NA 446-86-6 B6C3FI Cauda epid.
+ 71-43-2 CBCF1/ICI Cauda epid.
+ 50-32-8 B6C3F1 Cauda epid.
B6C3F 1 Cauda epid.
C B C F I / I C I Cauda epid.
B6C3F1/Crl Cauda epid.
NA 59-14-3 B6C3F1 Cauda epid.
+ 55-98-1 B6C3F 1 Cauda epid.
B6C3F 1 Cauda epid.
NA 521-35-7 B6C3FI/Bio Cauda epid.
NA 64-86-8 B6C3FI Cauda epid.
B6C3F1 Cauda epid.
I C R / C u m Cauda & caput epid.
H20
H20
H20
DMSO
Corn oil
H20
DMSO
H20
Corn oil
Trica- pryline DMSO
Corn oil
DMSO
DMSO
Trica-
pryline DMSO
DMSO
H20
H20
PBS
23
Exposure Number Number Highest Lowest of of doses total effective
Route Regi- animals tested dose dose men per dose (mg/kg) (mg/kg)
Statis- t ics
Ref.
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
IT
qd×5
q d x 5
q d x 5
qdx5
qdx5
q d x 5
qdx5
q d x 5
qd×5
qdx5
q d x 5
qd×5
q d x 5
q d x 5
qdx5
qdx5
qdx5
qdx5
qdx5
Mul- tiple
3
4
3
3
5
4
3
4
5
4
3
5
4
3
4
3
27-31
4
3
6
50
0.8
1.0
500
2 000
3.5
3.5
750
5 ml/kg
500
500
1 250
464
10000
125
125
125
5
5
l x10 -4 M
25
0.8
0.8
500
625
1
2
375
2.5 ml/kg
100
100
250
232
500
25
12.5
50
2.5
2.5
1×10-5 M
Bruce and Heddle, 1979
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Bruce and Heddle, 1979
Topham, 1980a
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Wyrobek and Bruce, 1975
Topham, 1980c
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Topham, 1980c
Wyrobek et al., 1981
Bruce and Heddle, 1979
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Zimmerman et al., 1979
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Handel, 1979
24
TABLE 1 (continued)
Agent Carcinogenicity
Reviewing EPA committee
CAS No. Genotype Source of sperm
Vehicle
Cycloheximide
Cyclophosphamide
Cyclophosphamide (-monohydrate)
9,10-Dimethyl- 1,2-benz- anthracene
/3-Estradiol
NA NA 66-81-9 B6C3F1
+ + 50-18-0 B6C3FI
B6C3FI
C57BL
CBB6F1
6055-19-2 CBCFI/ICI
+ NA 57-97-6 B6C3FI
+ + 50-28-2
Ethionine + NA
Ethyl methane- sulfonate 5
FNT 6
Gossypol acetic acid
Griseofulvin i
Hexamethyl phosphoramide 7 (HMPA)
13073-35-3
+ + 62-50-0
+ +
NA NA
+ +
+ +
3570-75-0
303-45-7
126-07-8
680-31-9
CBCF1/ICI
CBCFI/ICI
B6C3FI/Crl
B6C3F1
B6C3FI
CBCF1/ICI
B6C3FI
B6C3FI
B6C3F1
B6C3F1
CBCF1/ICI
B6C3FI/Crl
Cauda epid.
Cauda epid. Cauda epid. Epid.
Epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid. Cauda epid. Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid. Cauda epid.
Cauda epid.
Cauda epid.
H20
H~O
H20
H20
H20
H20
DMSO
0.5% Tween 80 in water
0.5% Tween 80 in water Saline
H20
H20
0.5% Tween 80 in water
DMSO
Unk
H20
DMSO
Saline
DMSO
25
Exposure Number Number Highest Lowest of of doses total effective
Route Regi- animals tested dose dose men per dose (mg/kg) (mg/kg)
Statis- tics
Ref.
IP qd x 5 3 2
IP qd × 5 4 2
IP qd x 5 3 4
IP Sin~e 5 3 dose
IP Sin~e 13-15 3 dose
IP qd x 5 5 7
IP qd x 5 3 3
IP qd × 5 5
250 100 a
250 100 b
500 100 a
300 100 d
300 100 d
800 100 c
250 100 a
6 625 375 c
Bruce and Heddle, 1979
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Pomerantseva et al., 1981
Pomerantseva et al. 1981
Topham, 1980a
Bruce and Heddle, 1979
Topham, 1980c
IP qd x 5 5 8 7 500 1250 c Topham, 1980a
IP qd × 5 3 -4 3
IP qd x 5 4 4
IP qd × 5 3 7
IP qd × 5 5 2
38000 9500 d
1000 625 b
1000 500 a
1250 1250 c
Wyrobek et al., 1981
W y r o b e k a n d Bruce, 1975
Bruce andHeddle , 1979
Topham, 1980a
IP q d x 5 3 3
IP q d x 5 5 3
IP q d × 5 4 3
IP q d × 5 3 4
IP q d × 5 5 8
1 500
40
7500
7500
5 m l / k g
500
40
'4000
2000
3.75 m l / k g
Bruce and Heddle, 1979
DePeyster, 1981
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Topham, 1980c
IP q d x 5 4 5 2.56 m g / k g NA b Wyrobek et al., 1981
26
TABLE 1 (continued)
Agent Carcinogenicity
Reviewing EPA committee
CAS No. Genotype Source of sperm
Vehicle
Hycanthone + methanesulfonate
Hydroxyurea 5 NA
ICI 42464 s +
5-Iodo-2'-deoxy- uridine l (IUDR)
Lead acetate 9 q._
Mechlorethamine + (Nitrogen mustard)
Metepa 2.3,10 +
+
NA
NA
NA
NA
NA
3-Methyl + NA cholanthrene 3
Methyl methane- + + sulfonate t
Mitomycin C 2.5 + +
23255-93-8
127-07-1
20794-96-1
54-42-2
15347-57-6
51-75-2
57-39-6
56-49-5
66-27-3
50-07-7
B6C3F1
B6C3F1
B6C3F1
C B C F I / I C I
B6C3FI
B6C3F1
Balb/c ÷
B6C3FI
B6C3F1
B6C3F1
C B C F I / I C I
B6C3F1
B6C3F1
CBCF1/ICI
B6C3FI
B6C3F 1
CBCFI / ICI
B6C3F1
B6C3FI
B6C3F 1/Bio
Cauda epid.
Cauda epid. Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid. Cauda epid.
Cauda epid.
Cauda epid. Cauda epid.
Cauda epid. Cauda epid. Cauda epid.
Cauda epid. Cauda epid. Cauda epid. Cauda epid.
Cauda epid. Cauda epid.
H20
H20
H20
0.5% Tween 80 in water
H20
DMSO
Feed
H20
H20
H20
0.5% Tween 80 in water Trica- pryline DMSO
Corn oil
H20
H20
Saline
Saline
H20
DMSO
27
Exposure Number Number Highest Lowest of of doses total effective
Route Regi- animals tested dose dose men per dose (mg/kg) (mg/kg)
Statis- tics
Ref.
IP
IP
IP
IP
q d x 5
qdx5
qd×5
q d x 5
500
2500
2000
500
500
250
250
375
Bruce and Heddle, 1979
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Topham, 1980c
IP
IP
Oral
IP
IP
IP
IP
q d x 5
qd×5
Chronic
q d x 5
q d x 5
q d x 5
qd×5
4
3
5
3
3
4
5
1250
1250
1.0%
900
5
125
62.5
125
125
1.0%
100
1
25
50
b
a
a
a
a
b
c
Wyrobekand Bruce, 1975
Bruce and Heddle, 1979
Eyden et al., 1978
Bruce and Heddle, 1979
Bruce and Heddle, 1979
Wyrobek and Bruce, 1975
Topham, 1980a
IP
IP
IP
IP
IP
IP
IP
IP
IP
qd ×5
qd x5
qd×5
q d x 5
q d x 5
q d x 5
qd×5
q d x 5
q d x 5
4
3
5
4
3
5
7
4
20
125
125
250
500
500
250
389
12
3,
125
62.5
125
250
250
125
389
3
3
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Topham, 1980a
Wyrobek and Bruce, 1975
Bruce andHeddle, 1979
Topham, 1980a
DePeyster, 1981
Wyrobekand Bruce, 1975
Zimmermanet ~., 1979
28
TABLE 1 (continued)
Agent Carcinogenicity CAS No. Genotype Source Vehicle of
Reviewing EPA sperm committee
Mitomycin C CBCF1/ICI Cauda Saline (continued) epid.
N-Nitroso-N- + + 684-93-5 CBB6F 1 Epid. H 2 ° methylurea
Procarbazine N + NA 366-70-1 B6C3F1 Cauda DMSO (Natulan) epid.
DBA/2J Vas Saline def.
TEM 12:3 + + 5 1 - 1 8 - 3 C3H/HeJ Vas Hank's CBA/CaJ def. buffered DBA/2J salt
solution
A-9-Tetra- NA NA 1972-08-3 B6C3FI/Bio Cauda DMSO hydrocannabinol epid.
Thiotepa s:4 + + 52-24-4 B6C3FI/Cum Cauda HzO epid.
Trichloroethylene + + 79-01-6 B6C3FI Cauda Air epid.
Trimethyl + + 512-56-1 B6C3F 1 Cauda H 20 phosphate l~ epid.
B6C3F1 Cauda H20 epid.
Tris-BP 16 + + 1 2 6 - 7 2 - 7 B6C3F1/Bio Cauda Corn oil epid.
Vinblastine NA NA 8 6 5 - 2 1 - 4 B6C3F1 Cauda H20 sulfate epid.
B6C3Fl Cauda H20 epid.
Legend Agent, name of agent tested. Full chemical names referenced in footnotes where necessary. Carcinogenicity, Classification of an agent's carcinogenic potential determined by the reviewing committee (see Table 8) or the EPA Merged Carcinogen List. +, carcinogenic; - , non-carcinogenic: ?. variable response; NA, classification not available. CAS No., Chemical Abstracts Registry Service number. Genotype B63CFI, (C57BL/6 ~ × C3H/He 8 )F1 hybrids. CBCFI, (CBA ~ × BALB/c ~ )FI hybrids. CBB6F1, (CBA ~ × C57BL/6 8 )F1 hybrids. Suppliers are indicated with the following symbols if information is supplied in paper: Bio, Bio Breeders, Ottawa, Canada; Crl, Charles River Laboratories, MA; Cum, Cumberland View Farms, TN; ICI, Imperial Chemical Industries, U.K.; J, Jackson Laboratory, ME. Mice used by Wyrobek and Bruce, 1975 and Bruce and Heddle, 1979, were supplied both by Bio Breeding Laboratories and Cumberland View Farms.
29
Exposure Number Number Highest Lowest Statis- of of doses total effective tics
Route Regi- animals tested dose dose men per dose (mg/kg) (mg/kg)
Ref.
IP q d x 5 5 3 10 1 c Topham, 1980a
IP Single 5 1 50 50 d Pomerantseva dose et al., 1981
IP qd × 5 3 4 1000 125 a Bruce and Heddle, 1979
IP Single 2 - 4 2 400 400 e Sharma et al., dose 1979
IP Single l0 1 0.25 0.25 e Soares et al., dose 1979
IP qd x 5 24-30 2 50 25 d Z immerman et al., 1979
IP qd x 5 4 4 25 3 b Wyrobek and Bruce, 1975
INH 4 h / d 5 2 0.20% 0.20% d Land et al., 1981 x 5 d
IP qd x 5 4 4 5 000 2 500 b Wyrobek and Bruce, 1975
IP q d x 5 3 5 10000 7500 a Bruce and Heddle, 1979
IP qd x 5 4 6 5 4 d Salamone and Katz, 1981
IP qd x 5 4 4 5 5 b Wyrobek and Bruce, 1975
IP qd × 5 3 5 5 3.5 a Bruce and Heddle, 1979
Source of sperm. Caput epid., caput epididymidis; Cauda epid., Cauda epididymidis; Vas def., Vas
deferens. Vehicle, solvent in which agent was delivered: dimethyl sulfoxide (DMSO); phosphate-buffered saline
(PBS). Route of exposure: GAV, gavage; INH, inhalation; IP, iqtraperitoneal; IT, intratesticular; Oral, in feed. Regimen: qd, every day. Number of animals per dose, number of animals initially exposed per dose. Number doses tested, number of doses tested (excluding negative control) at which at least one animal survived and was assessed for sperm head abnormalities. Highest total dose, highest total dose tested at which at least one animal survived and sperm-head abnormalities were assessed. Units are m g / k g unless otherwise stated. Lowest effective dose, lowest dose tested which indicated a statistically significant increase in sperm-head abnormalities by the author 's criteria. Units are m g / k g unless otherwise stated.
30
TABLE 1 (continued)
Statistics
a Author 's criteria for positive response: Value at any dose point exceeded control group by 1% (approx. double the control). Control distributions were provided.
b Author 's criteria for positive response: Value at any dose point exceeded negative control range. Control distributions were provided.
c Author 's criteria for positive response: Value at dose point at least double negative control level and p < 0.05 Rank-Wilcoxon test.
d Author 's criteria for positive response: Value at dose point exceeded control, p < 0,05, t-test. c Author 's criteria for positive response: Value at dose point exceeded control, p < 0.05, M a n n - W h i t -
ney U-test. Footnotes
i Also + when sampled 1 week postinjection (Wyrobek and Bruce, 1975). 2 Wyrobek and Bruce, 1975 data also presented by Bruce and Heddle, 1979. 3 Also + when sampled at 10 weeks postinjection (Wyrobek and Bruce, 1975).
TABLE 2
MOUSE SPERM M O R P H O L O G Y TEST: NEGATIVE C O M P O U N D S
Agent Carcinogenicity CAS No. Genotype
Reviewing EPA committee
Source of sperm
Acetamide + + 60-35-5 C B C F I / I C I Cauda epid.
2-Acetylamino- + NA 53-96-3 C B C F I / I C I Cauda fluorine epid.
Acetylsalicylic - NA 50-78-2 B6C3F1 Cauda acid epid.
AF-2 i + NA 3688-53-7 B6C3FI Cauda epid.
Aflatoxin BI + + 1162-65-8 B6C3F1 Cauda epid.
4-Aminobenzonitrile NA NA 873-74-5 C B C F I / I C I Cauda epid.
4-Aminothiophenol NA N A 1193-02-8 C B C F 1 / I C I Cauda epid.
3-Aminotriazole + + 61-82-5 CBCF 1.ICI Cauda epid.
Androsterone NA NA 53-41-8 C B C F l / I C I Cauda epid.
31
4 Increase in sperm abnormalities in Handel (1979) observed in caput epididymal sperm sampled at 5
and 9 days after injection, cauda epididymal sperm sampled at 9 days. 5 Also + at 1 and 10 weeks postinjection (Wyrobek and Bruce, 1975). 6 FNT, 2-(2-formylhydrazino)-4-(5-nitro-2-furyl)thiazole. 7 Wyrobek et al. (1981) study of HM P A yielded negative results. However Topham achieved a +
response at a higher dose. 8 ICI 42464, 2-(1-chloro-2-isopropylaminoethyl)naphthylene. 9 Increase in sperm abnormalities in Eyden et al., 1978 was at 8 and 11 weeks after start of treatment;
increase not seen at 4 weeks. io Metepa, Tris(2-methyl-l-aziridinyl)phosphine oxide. II Sampling time in Sharma et al. (1979) was 52 days postinjection. 12 TEM, triethylenemelamine. 13 TEM + at 4, 6 and 12 weeks postinjection. ~4 Thiotepa, triethylene thiophosphoramide; also tris(l-aziridyl)phosphine sulfate. 15 Positive samphng time was 7 days postinjection for Wyrobek and Bruce (1975). ~6 Tris-BP, tris(2,3-bromopropyl)phosphate.
Vehicle Exposure Number Number Highest Lethality Ref. of of total
Route Regimen animals doses dose per dose tested tested
(mg/kg)
Saline
0.5% Tween 80 in water
DMSO
DMSO
DMSO
0.5% Tween 80 in water
Corn oil
Saline
0.5% Tween 80 in water
IP qd × 5 5 4 5000 No Topham, 1980c
IP qd × 5 5 6 7500 Yes Topham, 1980c
PO qd x 5 3 4 1000 Yes Bruce and Heddle, 1979
IP qd × 5 3 4 2 500 Yes Bruce and Heddle, 1979
IP qd × 5 3 4 100 No Bruce and Heddle, 1979
IP qd × 5 5 5 2 500 Yes Topham, 1980a
1P qd x 5 5 4 250 Yes Topham, 1980a
IP qd × 5 5 4 2 500 No Topham, 1980c
IP qd x 5 5 4 2 500 Yes Topham, 1980c
32
TABLE 2 (continued)
Agent Carcinogenicity
Reviewing EPA committee
CAS No. Genotype Source of sperm
Aniline hydrochloride
Anthracene
+ NA 142-04-1
Aroclor 1254 ? 9
Ascorbic acid
Azoxybenzene
Benzidine
4-Bromoaniline
2-Bromoethanol
y-Butyrolactone
Caffeine 2
Cannabidiol
Carbon tetrachloride
Chloral hydrate
+
NA
NA
+
NA
+
NA
NA
NA
NA
NA
NA
+
NA
NA
+
NA
120-12-7
11097-69-1
50-81-7
495-48-7
92-87-5
106-40-1
540-51-2
96-48-0
58-08-2
13956-29-1
56-23-5
302-17-0
CBCF1/ICI
CBCF1/ICI
B6C3FI
CBCF1/ICI
B6C3F1
CBCFI/ICI
CBCF1/ICI
CBCF1/ICI
B6C3FI/Crl
CBCF1/ICI
B6C3F1
CBCFI/ICI
B6C3F1
B6C3FI/Bio
CBCF1/ICI
B6C3FI
Cauda epid~
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
33
Vehicle Exposure Number Number Highest of of total
Route Regimen animals doses dose per dose tested tested
(mg/kg)
Lethality Ref.
0.5% IP qd x 5 5 Tween 80 in water
0.5 % IP qd X 5 5 Tween 80 in water
EtOH IP qd × 5 3
7 1000 Yes Topham, 1980a
6 5 000 Yes Topham, 1980c
3 5000 Yes Bruce and Heddle, 1979
Corn oil IP qd x 5 5 4 5 000 No Topham, 1980c
H 20 IP qd x 5 3 4 25 000 Yes Bruce and Heddle, 1979
Saline I P qd x 5 5 4 10 000 Yes Topham, 1980a
0.5% IP qd x 5 5 4 1250 No Topham, Tween 80 1980a in water
0.5% IP qd × 5 5 5 1250 Yes Topham, Tween 80 1980c in water
DMSO I P qd × 5 4 5 440 No Wyrobek, et al., 1981
Corn oil IP qd x 5 5 4 1000 Yes Topham, 1980a
Saline IP qd × 5 5-7 3 460 Yes DePeyster, 1979
Saline IP qd x 5 5 4 5 ml Yes Topham, 1980c
H 2 0 IP qd x 5 4 2 1050 No Wyrobek and Bruce, 1975
DMSO IP qd × 5 21-23 2 125 Yes Zimmerman, et al., 1979
Corn oil 1P qd x 5 5 5 7.5 ml Yes Topham, 1980c
H 20 IP qd × 5 3 4 2 500 Yes Bruce and Heddle, 1979
34
TABLE 2 (continued)
Agent Carcinogenicity
Reviewing EPA committee
CAS No. Genotype Source of sperm
2-Chloroaniline NA NA
3-Chloroaniline NA NA
2-Chloroethanol NA NA
5-Chloro-2- + + methylaniline
Chloromycetin NA NA succinate
Chlorpropham 3 _ NA
Codeine phosphate NA
Cyclamate, Calcium 2 NA
Cyclamate, Sodium NA
2,4-Diamino NA anisole
2,4-Diaminotoluene +
2,4-Dichloroaniline NA
DDT 2,4
NA
NA
NA
NA
+
NA
Diesel exhaust NA NA
95-51-2
108-42-9
107-07-3
95-79-4
3544-94-3
101-21-3
52-28-8
139-06-0
139-05-9
615-05-4
95-80-7
554-00-7
50-29-3
CBCF1/ICI
CBCF1/ICI
B6C3F1
CBCF1/ICI
B6C3FI
CBCFI/ICI
B6C3F1
B6C3F1
CBCF1/ICI
B6C3F1
CBCF1/ICI
CBCF1/ICI
B6C3FI
CBCF1/ICI
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Complex A/Strong Cauda mixture epid.
35
Vehicle Exposure Number Number of of
Route Regimen animals doses per dose tested
Highest total dose tested (mg/kg)
Lethality Ref.
Corn oil IP qd x 5 5 5
Corn oil IP qd x 5 5 5
Saline IP qd × 5 5-7 3
Corn oil IP qd x 5 5 4
H20 IP qdX5 3 3
0.5% Tween 80 in water
H20
H20
0.5% Tween 80 in water
DMSO
0.5% Tween 80 in water
Corn oil
Trica- prylin
0.5% Tween 80 in water
Air
IP
IP
IP
IP
IP
IP
IP
1P
IP
INH
qd×5
q d x 5
q d x 5
q d x 5
q d x 5
q d x 5
q d x 5
q d x 5
q d x 5
8 h / d × 31 or 39 wks
30
2500
2 000
440
2 500
5 000
5000
500
2500
5000
500
2500
2500
125
2500
6 m g / m 3
No
Yes
Yes
Yes
Yes
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
No
Topham, 1980a
Topham, 1980a
DePeyster, 1979
Topham, 1980c
Bruce and Heddle, 1979
Topham, 1980c
Bruce and Heddle, 1979
Wyrobek and Bruce, 1975
Topham, 1980c
Bruce and Heddle, 1979
Topham, 1980c
Topham, 1980a
Wyrobek and Bruce, 1975
Topham, 1980c
Pereira et al., 1980
36
TABLE 2 (continued)
Agent Carcinogenicity
Reviewing EPA committee
CAS No. Genotype Soulce of sperm
Diethyl ether N A NA
9,10-Dimethyl anthracene
Dimethyl formamide
1, l - D i m e t h y l -
hydrazine
N-Dimethyl nitrosamine
Diphenyl nitrosamine
Epichlorohydrin
Epinephrine
Ethanol
60-29-7
+ NA 781-43-1
- NA 68-12-2
+ + 57-14-7
B6C3F1/Cum
CBCFI/ICI
CBCFI/ICI
B6C3F1
B6C3F1
+ + 62-75-9 B6C3F1
+ + 86-30-6
106-89-8
51-43-4
+
NA
NA 64-17-5
Ethylene thiourea + + 96-45-7
51-21-8 5-Fluorouracil NA NA
B6C3FI
CBCF1/ICI
CBCF1/ICI
B6C3FI
C B C F I / I C I
CBCF1/ICI
CBCFI/ICI
B6C3 F 1/Cr I
CBCFI/ ICI
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
37
Vehicle Exposure Number Number of of
Route Regimen animals doses per dose tested
Highest total dose tested (mg/kg)
Lethality Ref.
Air INH 4 h / d 5 2 x 5 d
Corn oil IP qd x 5 5 4
Corn oil IP qd x 5 5 5
H 20 IP qd × 5 4 4
H 20 IP qd × 5 3 5
H20 IP q d x 5 4 2
DMSO I P qd x 5 3 5
0.5% IP qd x 5 5 4 Tween 80 in water
Corn oil IP qd x 5 5 4
H20 IP q d x 5 3 3
0.5% Tween 80 in water
0.5% Tween 80 in water
0.5% Tween 80 in water
DMSO
0.5% Tween 80 in water
IP
GAV
IP
IP
GAV
q d x 5
q d x 5
q d x 5
q d x 5
q d x 5
1.6%
2 500
7.5 ml
400
500
30
30
5000
1 ml
10
80
20 ml
10000
2655
400
No
No
Yes
No
Yes
Yes
Yes
No
No
Yes
Yes
No
No
No
Yes
Land et al., 1981
Topham, 1980c
Topham, 1980c
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Wyrobek and Bruce, 1975
Bruce and Heddle, 1979
Topham, 1980c
Topham, 1980c
Bruce and Heddle, 1979
Topham, 1980a
Topham, 1980a
Topham, 1980c
Wyrobek, et al., 1981
Topham, 1980a
38
TABLE 2 (continued)
Agent Carcinogenicity
Reviewing EPA committee
CAS No. Genotype Source of sperm
4-Fluoroaniline
Glucose
Halothane
NA NA
NA
NA
Hydrazine sulfate + NA
4-Iodoaniline NA NA
2-lodoethanol NA NA
Isoflurane - NA
Isonicotinic + + acid hydrazide (Isoniazid)
Methionine NA NA
Methoxyflurane NA
2-Methylaniline
Methylazomethanol acetate
Methyl mercury acetate
371-40-4
50-99-7
151-67-7
10034-93-2
540-37-4
624-76-0
26675-46-7
54-85-3
63-68-3
76-38-0
+ NA 95-53-4
+ + 592-62-1
- NA 108-07-6
CBCF1/ICI
B6C3FI
CBCF1/ICI
B6C3FI /Cum
CBCFI / ICI
B6C3F1/Crl
CBCF1/ICI
B6C3F1
B6C3F1/Cum
CBCFI / ICI
CBCFI / ICI
B6C3FI /Cr l
B6C3F1/Cum
CBCF1/ICI
CBCF1/ICI
B6C3F1
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
39
Vehicle Exposure Number Number of of
Route Regimen animals doses per dose tested
Highest total dose tested (mg/kg)
Lethality Ref.
Corn oil
H / O
0.5% Tween 80 in water
Air
0.5% Tween 80 in water
H 2 0
Corn oil
Saline
Air
Saline
IP
IP
IP
INH
IP
IP
IP
IP
INH
IP
q d × 5
q d × 5
q d x 5
1 500
50000
40 000
Yes
Yes
No
4 h / d 5 2 0.8% No × 5 d
qd × 5 5 8 500 Yes
qd X 5 4 8 400
5 5 2 500
5 -7 3 460
5 2 1.0%
q d x 5
q d x 5
4 h / d x 5 d
q d x 5 5 4 1 250
Yes
Yes
Yes
No
Yes
0.5% IP qd x 5 5 6 7 500 No Tween 80 in water
Saline IP qd x 5 4 3 40000 No
Air INH 4 h / d 5 2 0.1% No x 5 d
Corn oil IP qd x 5 5 7 2.5 ml Yes
Saline IP qd x 5 5 6 125 Yes
DMSO IP qd x 5 3 3 15 Yes
Topham, 1980a
Bruce and Heddle, 1979
Topham, 1980a
Land et al., 1981
Topham, 1980c
Wyrobek et al., 1981
Topham, 1980a
DePeyster, 1979
Land et al., 1981
Topham, 1980c
Topham, 1980a
Wyrobek et al., 1981
Land et al., 1981
Topham, 1980c
Topham, 1980c
Bruce and Heddle, 1979
40
TABLE 2 (continued)
Agent Carcinogenicity
Reviewing EPA committee
CAS No. Genotype Source of sperm
Methyl orange 5
M N N G 6
NA 547-58-0
+ + 70-25-7
3-Methyl-4-NQO 7 - - NA
Monosodium NA NA glutamate
2-Naphthylamine + +
4-Nitroaniline NA NA
2-Nitrofluorene + NA
NA Nitrofurantoin
Nitrofurazone + NA
4-Nitro-o-phenylene diamine
14073-00-8
142-47-2
91-59-8
100-01-6
607-57-8
67-20-9
59-87-0
99-56-9
CBCF1/ICI
B6C3FI /Cr l
B6C3F1
B6C3FI /Cr l
B6C3F1
B6C3FI
CBCFI / ICI
B6C3F1/Crl
CBCF1/ICI
CBCF1/ICI
CBCF1/ ICI
B6C3FI
B6C3 F 1
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Nitrosodibutylamine + + 924-16-3 B6C3F1 Cauda epid.
41
Vehicle Exposure Number Number of of
Route Regimen animals doses per dose tested
Highest total dose tested (mg/kg)
Lethality Ref.
0.5% Tween 80 in water
DMSO
H20
DMSO
H20
DMSO
0.5% Tween 80 in water
DMSO
0.5% Tween 80 in water
0.5% Tween 80 in water
0.5% Tween 80 in water
DMSO
H20
H20
IP qd × 5 5 6 1250 Yes Topham, 1980c
IP qd x 5 4 9 480 Yes Wyrobek et al., 1981
IP qd x 5 3 3 100 Yes Bruce and Heddle, 1979
IP qd x 5 4 8 800 Yes Wyrobek et al., 1981
IP qd x 5 3 3 10000 Yes Bruce and Heddle, 1979
IP qd x 5 3 3 500 Yes Bruce and Heddle, 1979
IP qd x 5 5 6 1250 Yes Topham, 1980c
IP
IP
IP
IP
IP
IP
q d x 5
q d x 5
qd×5
qd×5
qd×5
q d x 5
q d x 5
720
2500
7500
1250
150
2500
150
Yes
Yes
Yes
Yes
No
Yes
Yes IP
Wyrobek et al., 1981
Topham, 1980a
Topham, 1980c
Topham, 1980a
Bruce and Heddle, 1979
Bruce and Heddle, 1979
Bruce and Heddle, 1979
42
TABLE 2 (continued)
Agent Carcinogenicity
Reviewing EPA committee
CAS No. Genotype Source of sperm
N-Nitrosomorpholine +
Nitrous oxide
4-NQO 8 +
+
NA
NA
Penicillin NA NA
Phenobarbitone + NA
Phenobarbitone, + + Sodium
p-Phenylene NA NA diamine
Practolol - NA
Progesterone +
Pronethalol + NA
fl-Propiolactone + +
Pyrene NA NA
NA Quinacrine dihydrochloride
NA
59-89-2
10024-97-2
56-57-5
1406-05-9
50-06-6
57-30-7
106-50-3
6673-35-4
57-83-0
54-80-8
57-57-8
129-00-0
69-05-6
CBB6F1
B6C3F1/Cum
B6C3F1
B6C3F1/Crl
CBCF1/ICI
B6C3F1
CBCF1/ICI
CBCF 1/ICI
CBCFI/ICI
CBCFl / IC |
CBCF1/ICI
CBCF1/ICI
CBCFI/ICI
B6C3FI/Crl
B6C3FI
Epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
43
Vehicle Exposure Number Number of of
Route Regimen animals doses per dose tested
Highest total dose tested (mg/kg)
Lethality Ref.
H 2 0 IP Single 5-8 dose
Air INH 4 h / d 5 × 5 d
DMSO IP qd x 5 3
DMSO IP qd × 5 4 -8
0.5% IP qd x 5 5 Tween 80 in water
DMSO IP qd × 5 3
Saline IP qd × 5 5
Saline IP qd x 5 5
0.5% IP q d × 5 5 Tween 80 in water
0.5% Tween 80 in water
0.5% Tween 80 in water
Saline
0.5% Tween 80 in w a t e r
DMSO
H 2 0
IP
IP
IP
IP
IP
IP
q d × 5
q d × 5
q d × 5
q d x 5
q d × 5
q d × 5
150
80%
25
48
8000
500
2 500
500
2500
2500
375
0.5 ml
2500
1542
500
NA
No
Yes
Yes
No
Yes
Yes
Yes
No
No
Yes
No
No
Yes
Yes
Pomerantseva et al., 1981
Land et al., 1981
Bruce and Heddle, 1979
Wyrobek et al., 1981
Topham, 1980a
Bruce and Heddle, 1979
Topham, 1980c
Topham, 1980a
Topham, 1980c
Topham, 1980c
Topham, 1980c
Topham, 1980c
Topham, 1980a
Wyrobek et al., 1981
Bruce and Heddle, 1979
44
TABLE 2 (continued)
Agent Carcinogenicity
Reviewing EPA committee
CAS No. Genotype Source of sperm
Quinoxaline
Quinoxaline di-N-oxide
Quinoxaline mono-N-oxide
NA
+ NA
+ NA
Saccharine, Sodium NA NA
Sodium azide NA NA
Sodium chloride NA NA
Testosterone + +
Thalidomide NA NA
3,3',5,5'- Tetramethyl benzidine
NA
Thioacetamide + +
Thiourea + +
Toluene NA NA
1,2,4-Triamino NA NA benzene
1,1,1-Trichloro- - NA ethane
Urea NA NA
91-19-0
2423-66-7
6935-29-1
128-44-9
26628-22-8
7647-14-5
58-22-0
50-35-1
54827-17-7
62-55-5
62-56-6
108-88-3
615-71-4
71-55-6
57-13-6
CBCFI/ICI
CBCF1/IC!
CBCFI/ICI
CBCF1/ICI
CBCF1/ICI
B6C3F1
CBCFI/IC!
CBCFI/ICI
CBCFI/ICI
B6C3FI/Crl
CBCF1/ICI
CBCF1/ICI
CBCF1/ICI
CBCFI/ICI
CBCFI/ IC |
CBCFI/ICI
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Cauda epid.
Canda epid.
45
Vehicle Exposure Number Number of of
Route Regimen animals doses per dose tested
Highest total dose tested (mg/kg)
Lethality Ref.
0.5% IP q d x 5 Tween 80 in water
0.5% IP qd x 5 Tween 80 in water
0.5% IP qd x 5 Tween 80 in water
0.5% IP qd x 5 Tween 80 in water
Saline IP qd × 5
H 20 IP qd x 5
0.5% Tween 80 in water
0.5% Tween 80 in water
0.5% Tween 80 in water
DMSO
Saline
Saline
Corn oil
Corn oil
Corn oil
Saline
IP
IP
IP
IP
IP
IP
IP
IP
IP
IP
q d x 5
q d x 5
q d x 5
qd×5
q d x 5
q d x 5
qd×5
q d x 5
qd×5
q d x 5
2500
2500
2500
2500
100
10000
2500
500
2500
2255
250
2500
7.5 ml
125
10 ml
10000
Yes
Yes
Yes
No
Yes
No
No
No
Yes
Yes
Yes
No
Yes
No
Yes
No
Topham, 1980c
Topham, 1980c
Topham, 1980c
Topham, 1980c
Topham, 1980c
Bruce and Heddle, 1979
Topham, 1980c
Topham, 1980c
Topham, 1980a
Wyrobek et aL, 1981
Topham, 1980c
Topham, 1980c
Topham, 1980c
Topham, 1980a
Topham, 1980c
Topham, 1980c
46
TABLE 2 (continued)
Agent Carcinogenicity CAS No. Genotype
Reviewing EPA committee
Source of sperm
Urethane 2
Vitamin A
+ + 51-79-6 B6C3F 1 Cauda epid.
B6C3F 1 Cauda epid.
CBCF1 / ICI Cauda epid.
NA NA 68-26-8 CBCF I / I C I Cauda epid.
Zinc chloride NA NA 7646-85-7 CBB6FI Epid.
Code No. 9A0 NA NA 67110-84-3 CD-I Cauda 20-438 epid.
Legend, see Table 1 except: Number of doses tested, number of doses tested (excluding negative control). This number may or may not include doses at which some or all animals died. Highest total dose tested, this number represents the highest total dose reported by the author and in some cases may represent a dose at which all animals died. Lethality," yes, at least one animal died at the highest dose tested; No, no death at highest dose tested; NA, data not available. Lethality data for Bruce and Heddle (1979) were obtained by personal communica- tion; in some cases the lethal dose was higher than the'dose reported in the paper.
TABLE 3
MOUSE SPERM M O R P H O L O G Y TEST: INCONCLUSIVE C O M P O U N D S
Agent Carcinogenicity CAS No. Genotype Source Vehicle of
Reviewing EPA sperm committee
Butylated ? ? 128-37-0 B6C3FI Cauda DMSO hydroxytoluene I epid.
Cadmium + + 10108-64-2 B6C3FI Cauda H 2 0 chloride 2 epid.
CBB6F 1 Epid. H 20
47
Vehicle Exposure Number Number Highest of of total
Route Regimen animals doses dose per dose tested tested
(mg/kg)
Lethality Ref.
H 2 0 IP q d × 5 4 2 5000
H 20 IP qd × 5 3 3 5 000
Saline
0.5% Tween 80 in water
H20
2% gelatin in water
Yes Wyrobek and Bruce, 1975
Yes Bruce and Heddle, 1979
IP qd × 5 5 3 5 000 No Topham, 1980c
IP qd × 5 5 3 1250 Yes Topham, (M.I.U.) 1980c
IP Single 5 1 15 NA Pomerantseva dose et al., 1981
GAV Single 10 1 300 No Matter dose et al., 1979
Footnotes 1 AF-2, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide. 2 Also negative at 1 and 10 weeks post injection (Wyrobek and Bruce, 1975). 3 Chlorpropham, o-isopropyl-N-(3-chlorophenyl)carbamate. 4 DDT, l,l-bis(p-chlorophenyl)-2,2,2-trichloroethane. 5 Methyl orange, 4-dimethylaminoazobenzene-4-sulfonic acid. 6 MNNG, N-methyl-N-nitro-N'-nitrosoguanidine. 7 3-methyl-4-NQO, 3-methyl-4-nitroquinoline-N-oxide. s 4--NQO, 4-nitroquinoline-N-oxide. 9 Code No. 20-438, (4aRS, 5SR, 9bRS)-2-ethyl-l,3,4,4a,5,9b-hexahydro-7-methyl-5-p-tolyl-2H-
indeno[ 1,2-c]pyridine hydrochloride. io Sperm sampled at 1, 7, 21, 42 and 84 days post-treatment.
Exposure Number Number Highest of of total
Route Regi- animals doses dose men per dose tested (mgJkg)
Lethality Response Ref.
IP qd × 5 3 4 1000 Yes +
IP q d × 5 3 3 15 Yes -
IP Single 4 -6 3 4 Unk + dose
Bruce and Heddle, 1979
Bruce and Heddle, 1979
Pomerantseva et al., 1981
48
TABLE 3 (continued)
Agent Carcinogenicity CAS No. Genotype Source Vehicle of
Reviewing EPA sperm committee
Chloroform 3 + + 67-66-3 B 6 C 3 F I / C u m Cauda Air epid.
C B C F I / I C I Cauda Corn oil epid.
Dichlorvos 4 _ - 62-73-7 B6C3F 1 Cauda Trica- epid. prylin
Diethylstil- + + 56-53-1 C B C F I / I C I Cauda 0.5% besterol 5 epid. Tween 80
in water B 6 C 3 F I / C r l Cauda DMSO
epid.
4-Dimethyl 2 + + 60-11-7 CBCF1 / ICI Cauda 0.5% aminoazobenzene epid Tween 80
(Butter Yellow) in water B 6 C 3 F I / C r l Cauda DMSO
epid.
Enflurane 6 - - NA 13838-16-9 B 6 C 3 F I / C u m Cauda Air epid.
F A N F T 1.7 + NA 24554-26-5 B6C3F1 Cauda DMSO epid.
1-Naphthyl- + + 134-32-7 CBCF1/ IC1 Cauda 0.5% amine 2 epid. Tween 80
in water B6C3F1 Cauda H 2 0
epid. B6C3F1/Cr l Cauda DMSO
epid.
Safrol 8 + + 94-59-7 CBCF1 / ICI Cauda Corn oil epid.
B6C3F1/Cr l Cauda DMSO epid.
Legend See Table 1 for Agent, Carcinogenicity, CAS No., Genotype, Source of sperm, Vehicle, Route, Regimen, Number of animals per dose. See Table 2 for Number of doses tested, Highest total dose, Lethality. Response, Author 's classification of response. + , positive; - , negative; Pres. + , presumptive positive. Footnotes
Author indicates + response, but agent did not fulfill Reviewing Committee 's criteria for inclusion on Table 1; elevated points are < 4% over background, with no indication of reproducibility in a repeat experiment.
2 Conflict in results between laboratories; positive classification from study using lower doses.
49
Exposure Number Number Highest of of total
Route Regi- animals doses dose men per dose tested (mg/kg)
Lethality Response Ref.
INH 4 h / d 4 2 0.08% No + Land et al., x 5 d 1981
IP qd X 5 5 5 1.25 ml Yes - Topham, 1980c
IP qd X 5 4 4 55 Yes + Wyrobek and Bruce, 1975
IP qd × 5 5 7 1250 Yes + Topham, 1980a
IP qd × 5 4 5 1 125 Yes - Wyrobek et al.. 1981
IP qd × 5 5 4 1 250 Yes - Topham, 1980c
IP qd × 5 4-8 7 600 Yes + Wyrobek et al., 1981
INH 4 h / d 5 2 1.2% No + Land et al., x 5 d 1981
IP qd × 5 3 4 1 500 No + Bruce and Heddle, 1979
IP qd x 5 5 8 500 Yes - Topham, 1980c
IP qd x 5 3 5 300 NA + Bruce and Heddle, 1979
IP qd x 5 4 4 400 Yes - Wyrobek et al., 1981
IP qd x 5 5 4 0.25 ml /kg No - Topham, 1980c
IP qd x 5 4 7 200 Yes Pres. Wyrobek et + al., 1981
3 Conflict in results between laboratories; difference in response possibly due to different exposure regimen, dose levels, and / o r strain.
4 Atypical response with technical grade material. 5 Conflict in results between laboratories; positive response ( - 14% over background) seen at dose
showing no effect in a different strain. 6 Author indicates a statistically significant response, but agent did not fulfill Reviewing Committee's
criteria for inclusion on Table 1; elevated point represents < 1% increase over control values. 7 FANFT, 2-formylamino-4-(5-nitro-2-furyl)thiazole. 8 Original author classed this agent as 'presumptive positive;' an increase of 14% over background was
seen at highest dose with only one surviving animal. Also, conflict in results between laboratories.
50
TABLE 4
AGENTS TESTED FOR I N D U C T I O N OF A C R O S O M A L ABNORMALITIES IN MICE
Agent Carcinogenicity CAS No. Genotype Souce Vehicle of
Reviewing EPA sperm committee
Carbaryl 1,2
Ethyl methane- sulfonate
Isopropyl methane- sulfonate 3
Methyl methane- sulfonate 3
+ NA 63-25-2 Q strain Vas H 2 0 def.
+ + 62-50-0 Q strain Vas Unk def.
NA NA 926-06-7 Q strain Vas. Unk def.
+ + 66-27-3 Q strain Vas. Unk def.
Legend See Table 1 for Agent, Carcinogenicity, CAS No., Genotype, Source of Sperm, Vehicle, Route, Regimen, Number of animals per dose. See Table 2 for Number of doses tested, Highest total dose. Response: + , agent induced acrosomal abnormalities; - , no induction of acrosomal abnormalities.
TABLE 5
AGENTS TESTED FOR C HANGE S IN SPERM C O U N T S A N D MOTILITY IN MICE
A g e n t / C A S No. Study Source of Vehicle Exposure Animals Number design sperm per of
Route Regimen dose doses tested
Adriamycin X-sect. Caput Saline IV Single 12 i 2 (23214-92-8) and and dose
Longit. cauda epid.
Gossypol X-sect. Epid. Corn oil SC qd × 10 6 -8 4 acetic acid
(303-45-7)
Hydroxyurea X-sect. Cauda Saline IP qd × 5 3 -4 4 (127-07-1) epid.
Mitomycin C X-sect. Cauda Saline IP qd × 5 4 -5 6 (50-07-7) epid. and
vas def.
Cauda Saline IP Single 9-15 2 epid. and dose vas deL
Streptozotocin X-sect. Cauda Saline IP Single (18883-66-4) epid. and dose
vas def.
X-sect.
4 2
51
Exposure Number of Number of Highest animals doses total
Route Regimen per dose tested dose (mg/kg)
Response Ref.
GAV qd × 7 Unk 1 IP
IP Single Unk 1 dose
IP Single Unk 1 dose
IP Single Unk 1 25 dose
0.5 ml I × I 0 - 3 M
25
25
+
4-
+
Degraeve et al, 1976
Moutschen and Colizzi, 1975
Moutschen and Colizzi, 1975
Moutschen and Colizzi, 1975
Footnotes l Carbaryl, N-methyl-l-naphthyl-carbamate. 2 10-fold increase over controls. 3 Author's criteria for positive response, t-test at p < 0.05.
Highest Assay Response Lowest total effective dose dose (mg/kg) (mg/kg)
Rever- sible
Statis- tics
Ref.
12 Count J, 3 Motil. ,L 3
Ptl 2
Ptl Au and Hsu, 1980
250 Count $ 125
5 000 Count ,L 625 Motil. J, 2 500
12.5 Count $ 5.0 Motil. $ 2.5
5.0 Count J, Motil. J,
Yes
Unk Unk
Unk Unk
2.5 Unk a 2.5 Unk a
Coulson et al., 1980
Ficsor and Ginsberg, 1980
Ficsor and Ginsberg, 1980
Ficsor and Elansari, 1981
26.5 Count $ 26.5 Unk a Ficsor and Elansari, Motil. + 2.65 Unk a 1981
52
TABLE 5 (continued)
Agent /CAS No. Study Source of Vehicle Exposure Animals design sperm per
Route Regimen dose
Number of doses tested
TEM 3 Longit. Ejac. Saline IP qd x 7, 5 1 (51-18-3) then
every 3 - 4 d x 28-45 d
Longit. Ejac. Saline IP qd x 2 5 1
X-Sect. Cauda Saline IP qd x 5 10 1 epid. and 44 vas def.
Legend (Tables 5 and 6) Agent/CAS No., Name of agent tested and its Chemical Abstracts Registry Service number. Study design." X-sect., cross-sectional; samples collected from animal or groups of animals after treatment and compared with control group; Longit., Longitudinal; samples collected from individual animals before, during, a n d / o r after treatment. Source of sperm: Ejac., ejaculate; Epid., epididymis; Vas def., Vas deferens. Vehicle, solvent in which agent was delivered. Route of exposure: ID, intradermal; IM, intramuscular; IMP, implant; INH, inhalation; IP, in- traperitoneal; IV, intravenous; PO, oral (garage or feed); SC, subcutaneous; SKC, skin contact. Regimen: bid, twice daily; qd, every day; q2d, every other day. Animals per dose, number of animals exposed per dose. Number of doses tested, number of doses tested excluding negative control. Highest total dose, highest total dose tested in mg/kg unless otherwise indicated. Assay: Morph., morphology; Motil., motility.
TABLE 6
SPERM TESTS IN OTHER MAMMALIAN SPECIES
Agent /CAS No. Study Source of Vehicle Exposure Animals design sperm per
Route Regimen dose
CA TTLE Ethylene Longit. Ejac. Ethanol PO qd for 12 mo
dibromide and soybean then q2d 106-93-4 oil, then
gelatin
Longit. Ejac. and Olive PO q 2 d x 10 and X-sect. Epid. oil
Fenchlorfos i X-sect. Ejac. NS SKC Single dose, 299-84-3 or 1 0 x / d
× 2 d
3
2-4
53
Highest Assay Response Lowest Rever- Statis- Ref. total effective sible tics
dose dose (mg/kg) (mg/kg)
> 2.8 Count J, NA Yes c Cattanach and Edwards, 1958
4.0 Count ~, NA Yes c Cattanach and Edwards, 1958
1.0 Count ~ NA NA c Sherman and Motil. --* NA NA c Steinberger, 1960
Response: ~, decrease in sperm count, sperm motility, and percent morphologically normal sperm; ---,, no change; marg. J,, marginal decrease. Lowest effective dose, lowest total dose tested at which a significant change in the semen parameter was noted; in mg /kg unless otherwise stated. NA (not applicable) - - when only 1 or 2 doses were tested, or when there was no response at any dose tested; NS (not stated) - - data not provided by author. Reversibility: yes, if paper indicates that semen parameters returned to pretreatment or control levels with time; Unk (unknown), if the data presented where insufficient to evaluate reversibility; NA (not applicable), when there was no response. Statistics: a, statistics done in ref.; b, statistics not done but data sufficient; c, insufficient data for statistical analysis; d, descriptive study, data not presented or statistics not applicable. Footnotes
A mouse was sacrificed every 10th day for 120 days. 2 Sperm counts returned to normal in the 3 mg/kg group, but animals treated with 12 mg /kg remained
azoospermic up to 120 days. 3 TEM, triethylenemelamine.
Number Highest Assay Response Lowest Rever- Statis- of total effective sible tics
doses dose dose tested (mg/kg) (mg/kg)
Ref.
1 2 m g / kg /d Count $ NA Yes c then Motil. J, NA Yes c 4 m g / Morph. $ NA Yes d
kg/2d
1 40 Count $ NA Unk a Morph. ~, NA Yes a
2 200 Count -~ NA NA a Motil. $ NS Unk a Morph. J, NS Yes a
Amir and Volcani, 1965
Amir et al., 1977
Jaskowski et al., 1973
54
TABLE 6 (continued)
Agent /CAS No. Study design
Source of sperm
Vehicle Exposure
Route Regimen
Animals per dose
DOG Cadmium Longit. Ejac.
chloride 10108-64-2
Cogazocine Longit. Ej ac. lactate 2
75639-72-4
Diethyl- Longit. Ejac. carbamazine
90-89- l
GUINEA PIG Busulphan Longit. Electro- 55-98-1 ejac.
Chlorambucil Longit. Electro- 305-03-3 ejac.
H A M S T E R
Dimethyl- X-sect. Epid. nitrosamine and Longit.
62-75-9
Gossypol X-sect. Epid. and acetic acid Vas def.
303-45-7
Methyl X-sect. Epid. methane- and Longit. sulfonate
66-27-3
M O N K E Y a-Chloro- X-sect. Epid. and
hydrin 4 Vas def. 96-24-2
DICA 6 Longit. Electro- 50264-69-2 ejac.
Ovine FSH X-sect. Electro- 9046-70-2 ejac.
PIG a-Chloro-
hydrin 4 96-24-2
Longit. ~ac
Saline
5% Dextrose
Tablets
Peanut oil
Peanut oil
Water
Steroid suspending vehicle
Water
Distilled water
NS
Freund's adjuvant
Feed
IT Single dose
IM q d x 9 wk
PO qd x 6 mo
IP Single dose
IP Single dose
IP qd × 5
PO qd × 6-14 wk
2-5
13
13
15-18
5-8
IP q d × 5 15-18
PO qd × 15 bid x 7 day, then qd ×42 d
PO q d × 1, 3, 5; 5 mo chronic
ID, Single dose, IM 7 boosters
over 18 mo
PO qd x 5
1-4
1-3
4
55
Number of doses tested
Highest total dose (mg/kg)
Assay Response Lowest effective dose (mg/kg)
Rever- sible
Statis- tics
Ref.
0.2
63 m g / k g
2.3 g / k g
Count
Count Motil. Morph.
Count Motil. Morph.
Count
Count
$ $3
NA
NA NA NA
NA NA NA
NA
NA
Unk
Yes
Yes Yes
NA NA NA
Yes
Yes
b b b
a a
a
Donnelley and Monty, 1977
James et al., 1980
Courtney and Nachreiner, 1976
Freund, 1968
Freund, 1968
25
700
Count Morph.
C o u n t
Motil.
__+
NA NA
N A
420
NA NA
NA Unk
Wyrobek et al., 1978
Chang et al., 1980
250 C o u n t
Morph. marg. ~, NA
15 Unk Yes
Wyrobek et al., 1978
5 7
1
1 120
2500
700 / ,g
Count Mot i l . Morph.
Count Morph. s
Count Motil. Morph.
marg. $ 5 NA NA NA
NA NA
NA NA NA
NA NA NA
Unk Unk
Unk Unk Unk
Setty et al., 1970
Lobl and Mathews, 1978
Wickings and Nieschlag, 1980
Motil. J, NA Yes a Crabo et al., Morph. 9 __. NA NA a 1975
56
TABLE 6 (continued)
Agent/CAS No. Study Source of design sperm
Vehicle Exposure
Route Regimen
Animal., per dose
R A B B I T
6-Azauridine 54-25-1
Longit. Ejac. NS
Busulfan Longit. Ejac. 55-98-1
Cadmium Longit. Ejac. chloride
10108-64-2
Cyclophos- Longit. Ejac. phamide
50-18-0
Gossypol Longit. Ejac. acetic acid
303-45-7
Arachis oil
NS
NS
Steroid suspending vehicle
Hexamethyl Longit. Ejac. NS phosphoramide
680-31-9
Isoniazid Longit. and Ejac. Buffered 54-85-3 X-sect. saline
lsopropyl Longit. Ejac. Buffered methane- saline sulfonate
926-06-7
Levonorgestrel X-sect. Ejac. Sesame 797-64-8 oil
Methyl Longit. Ejac. Saline ethane- sulfonate
1912-28-3
Methyl Longit. Ejac. Saline methane- sulfonate
66-27-3
Nafoxidine Longlt. Ejac. Water + hydro- 0.25% chloride n methyl
1847-63-8 cellulose
Procarbazine Longlt. and Ejac. Buffered hydro- X-sect. saline chloride
366-70-1
SC bid × 18 wk
IP Single dose
SC Single dose
SC qd x 4
PO 5 x / w k × 5-25 wk
PO qd× 10
IV Single dose
IV Single dose
PO qd × 8 wks
IV Single dose
NS Single dose
SC qd × 45
IV Single dose
1-2
57
Number of doses tested
Highest total dose (mg /kg)
Assay Response Lowest effective dose (mg/kg)
Rever- sible
Statis- tics
Ref.
Var 158.8 g
40
0.05 m M / k g
96
940
Count Motil. Morph.
Count
Count Motil. Morph.
Count Motil. Morph.
Count Motil.
marg.
marg. ~ to
NS NS NS
NA
NA NA NA
96 32 NA
NA NA
Unk Unk Unk
Yes
Unk Unk NA
Yes Unk NA
NA NA
M r o u e h a n d McDonNd, 1969
Fox et al., 1963
Paufler and Foote, 1969
Cranz et al., 1976
Chang et al., 1980
1000
125
100
Count
Count
Count Morph.
NA
NA
NA NA
Yes
NA
Yes Unk
Jackson and Craig, 1966
Bfirg, in et al., 1979
Fox et al., 1963
2 8 0 m g
50
Count Motil.
Count Morph.
NA NA
NA NA
NA NA
NA Unk
Freund et al., 1980
Fox et al., 1963
30 Count Morph.
NA N A
NA Unk
Fox et al., 1963
225
50
Count Motil. Morph.
Count
,22.5 225 225
NA
Yes Yes Yes
Yes
Ericsson, 1966
Biirgin et al., 1979
58
TABLE 6 (continued)
Agent /CAS No. Study design
Source of sperm
Vehicle Exposure
Route Regimen
Animals per dose
R A B B I T (continued) Prosta- Longit.
glandin F2a 551-11-1
STS 557 12 X-sect, 65929-58-7
Triethylene- Longit. melamine ~3
51-18-3
Longit.
Ejac., Epid., Vas def,
Ejac.
Ejac.
Ejac.
Silastic polyvinyl pyrroli- dine tube
Sesame oil
Saline
NS
IMP Single dose
PO qd × 8 wks
IV Single dose
IV Single dose
11
6-8
1
2-5
Longit. Ejac. NS IV 1/wk × 5
RAT Aflatoxin B1 X-sect. Epid. 1162-65-8
5 -Aminoin- N S Epid. dazole
64309-76-8
Cadmium X-sect. Epid. chloride
10108-64-2
1% albumin
1% gum tragacanth
Water
a-Chloro- X-sect. Vas Propylene hydrin 4 def. glycol
96-24-2
Longit. Epid.
X-sect. NS
X-sect. Vas def.
X-sect. 15 Epid.
Chloroprene 126-99-8
CL 88236 t4 34839-13-9
Cyclohexyl- amine hydrochloride
4998-76-9
0.25% methyl cellulose in H20
Air
Propylene glycol
Feed
IP
PO
In utero, then PO
PO
PO
INH
PO
PO
q2d x 3, 6
4 doses in 2 wks
Chronic
q d × 14
Single dose
Chronic 4.5 mo
q d × 14
Chronic 90 d
10
3-5
7-10
10
NS
8
11-16
12
59
Number of doses tested
Highest total dose (mg/kg)
Assay Response Lowest effective dose (mg/kg)
Rever- sible
Statis- tics
Ref.
3 mg
1 120 mg
0.2
0.4
20
Count Motil. Morph.
Count Motil.
Count Morph.
Count Motil. Morph. Count Motil. Morph.
2 300 # g / k g Count Morph. ---,
1 400 Count J, Motil. $
3 5 ppm Count $
NA NA NA
560mg 280mg
NA NA
Y e s
Yes Yes
Unk Unk
Yes Unk
NA Yes a NA NA a NA NA a NA Yes a NA Yes a NA Unk a
NA NA NA NA
NA Yes NA Unk
5.0 ppm Unk
Saksena and Lau, 1979
Freund et al., 1980
Fox et al., 1963
Paufler and Foote, 1969
Paufler and Foote, 1969
a Egbunike, 1979 a
d Lobl and d Porteus, 1977
a Laskey et al., 1980
112 Count ---, Motil. $
100 Count $, MotH. Morph.
1.69 m g / m 3 Motil. ,L
1 120 Count J,
6000 ppm Count ,[
Motil. marg. $
Morph. marg. ~,
NA NA NA Unk
NA Unk NA NA NA NA
0.15 m g / m 3 Unk
0
1120 Unk
6000 ppm Unk
NA NA
NA NA
a Coppola, 1969 a
d Kalla and d Chohan, 1976 d
a Sanotskii, 1976
a Coppola and Saldarini, 1974
a Mason and Thompson, 1977
a
a
60
TABLE 6 (continued)
Agent/CAS No. Study design
Source of sperm
Vehicle Exposure
Route Regimen
Animals per dose
RA T (continued) Cyclophos-
phamide 50-18-0
/3-Estradiol ~6
50-28-2
Estradiol propionate
113-38-2
Estriol 3- methyl ether
1474-53-9
Gossypol acetic acid
303-45-7
Neomycin sulfate
1405-10-3
Propylene dimethane- sulfonate
15886-84-7
SHEEP a-Chloro-
hydrin 4 96-24-2
X-sect. Epid. NS IP qd x 60
Longit. NS NS IP Single dose
X-sect. Epid. Olive oil IM qd x 14 and Vas def.
Longit. Epid. NS IM qd x 12-18
X-sect. Epid. and Olive oil I M qd x 15 Vas def. or 30
X-sect. Epid. and Olive oil IM qdx 14 Vas def.
X-sect. Epid. NS PO qd x 2-4 wks
X-sect. Epid. and Steroid PO qd x Vas def. suspending 8-14 wks
vehicle IMP Single dose
X-sect. Vas def. Aqueous PO qd x 4 wks
X-sect. Epid. Saline I M qd × 20
Longit. Epid. Arachis oil IP Single dose
Longit.
Longit
Ejac.
Ejac.
Feed
Saline
PO qd x 25
IM qd x 5
NS
1
4-6
3-6
1 1 - 2 2
4-6
NS
3-6
3
8
NS
NS
Cyclophos- pharnide
50-18-0
Longit. Ejac. Water PO Single dose 3-4
61
Number of doses tested
Highest total dose (mg/kg)
Assay Response Lowest Rever- effective sible dose (mg/kg)
Statis- tics
Ref.
2 360 Count ,L NA Unk a
1 50 Count J, NA Yes b
1 1.68/tg Count ~, NA Unk a Motil. J, NA Unk d
3 27.0 mg Count J, NS Unk d Motil. J, 22.5 mg Unk b
10 3000 pg Count J, 5/,tg/d Unk a Motil. $ 5 ~ g / d Unk d
l 1 400/.tg Count ], NA Unk a Motil. J, NA Unk d
NS l 120 Count $ NS Yes d Morph. J, NS Unk d
3 980 Count marg. J, 17 NA NA a Motil. $ 280 Unk c
l 10 mg Count. marg. J, 17 NA NA a Motil. marg. J, NA NA c
l 140 Motil. J, NA Unk c
2 2000000 Count J, NA Unk a uni ts /kg Motil. ], NA Unk a
Morph. J, NA Unk a
1 50 Count ], NA Unk d
Sykora et al., 1979
Mauss, 1972
Das, 1977
Kaur and Mangat, 1978
Singh et al., 1970
Das, 1977
National Coordinating Group, 1978
Chang et al., 1980
Kalla et al., 1981
Yunda and Kushniruk, 1973
Cooper and Jackson, 1970
6 2 5
125
40
Count - o , N A N A a
Motil. $ NA Yes a Morph. J, NA Unk a
Motil. J, NA Yes a Morph. ], 19 NA Yes a
Count Motil. Morph.
NA Yes a NA Unk a NA Unk a
Kreider and Dutt, 1969 18
Brown-Wood- man and White, 1976
Inskeep et al., 1971
62
TABLE 6 (continued)
Legend: see after Table 5 for Legend. Footnotes
i Fenchlorfos, o-dimethyl 2,4,5-trichlorophenyl phosphate. 2 Cogazocine lactate, 2-cyclobutyl methyl-5-ethyl-2'-hydroxy-9,9-dimethyl-6,7-benzmorphan lactate. 3 The principal abnormalities were coiled tails and loose heads. 4 a-Chlorohydrin, 3-chloro-l,2-propanediol. 5 Motility affected only in caput epididymis. 6 DICA, 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid. 7 5 regimens, each with a unique total dose. s Tail morphology only, 9 Includes head and tail morphology.
~0 Inconsistent decreases in motility during treatment. i1 Nafoxidine hydrochloride, 1-(2-(p-(3,4-dihydro-6-methoxy-2-phenyl-l-naphthyl)phenoxy)-ethyl)-pyr-
rolidine hydrochloride. 12 STS 557, 17a-cyanomethyl- 17j3-hydroxy-estra-4,9(10)diene-3-one. 13 Triethylenemelamine, N,N' ,N'- t r ie thylenemelamine, also tretamine or TEM. 14 CL 88 239, X-l-amino-3-chloro-2-propanol hydrochloride. t5 Study of 2 strains, Wistar and Sprague-Dawley. 16 Das refers to this compound as 'estradiol 178', Kaur and Mangat use the synonym 'ovocyclin'. 17 Decrease in counts seen in epididymis only. is Abstract with statistics.
TABLE 7
GERM CELL MUTATIONAL ACTIVITY OF AGENTS TESTED MORPHOLOGY TEST
63
IN THE MOUSE SPERM
Germ cell test Number of Germ cell agents test result studied
Mouse sperm morphology test results
Positive Negative Inconclusive
Specific locus a 14 Positive 6 0 0 Negative 0 1 1 Inconclusive 4 2 0
Heritable translocation b 9 Positive 7 0 0 Negative 0 0 0 Inconclusive 1 1 0
Dominant lethal c 36 Positive 15 0 0 Negative 8 9 2 Inconclusive 2 0 0
a Russell et al. (1981). b Generoso et al. (1980). c Topham (1983).
TABLE 8
CARCINOGENIC ACTIVITY OF AGENTS EVALUATED IN THE MOUSE SPERM MORPHOL- OGY TEST
Sperm test Total Carcinogens Noncarcinogens Unknown classification carcinogenicity c
Committee a EPA b Committee EPA Committee EPA
Positive 41 30 20 0 0 11 21
Inconclusive 10 7 6 2 1 1 3
Negatives Tested to lethality 67 26 15 15 2 26 50 Not tested to lethality d 36 13 11 9 0 14 25
Total negatives 103 39 26 24 2 40 75
a Reviewing Committee carcinogen list was based on evaluation of the following literature: (1) EPA Merged Carcinogen List April 1980; (2) Griesem~r and Cueto, 1980; (3) International program for the evaluation of short-term tests for carcinogenicity, de Serres and Ashby (Eds.), 1981; (4) Bruce and Heddle, 1979; (5) Jenssen and Ramel, 1980; (6) Baden and Simmon, 1980; (7) Purchase et al., 1978; (8) Imperial Chemical Industries, unpublished observations, 1970-1980.
b EPA Merged Carcinogen List only. c Unclassified or variable carcinogenicity. d Includes 2 agents for which lethality data were not available.
6 4
T A B L E 9
C O M P A R I S O N O F S P E R M T E S T R E S U L T S F O R A G E N T S T E S T E D IN M O R E T H A N O N E
M AMMALIAN S P E C I E S INCLUDING M A N
C o m p o u n d Species M o r p h o l o g y Mot i l i ty Coun t
Af la tox in B 1 M o u s e - 0 0
Rat - 0 -
Alcohol i sm, chronic a or H u m a n + + +
Ethanol M o u s e - 0 0
Anes the t i c gases b or H u m a n - 0 -
N i t r o u s oxide M o u s e - 0 0
H a l o t h a n e M o u s e - 0 0
Busu l fan G u i n e a pig 0 0 +
M o u s e + 0 0
R a b b i t 0 0 +
C a d m i u m chlor ide D o g 0 0 +
Mouse M 0 0
R a b b i t M + +
Rat 0 0 +
C a r b a r y l H u m a n M 0 -
M o u s e + ~" 0 0
Ch lo r amb u c i l G u i n e a pig 0 0 +
H u m a n - - +
a - C h l o r o h y d r i n M o n k e y - M -
Pig - + 0 Ra t - + / _ d + / _ d
Sheep + + -
Colchic ine H u m a n 0 _ + / _ e
Mouse + 0 0
C y c l o p h o s p h a m i d e H u m a n 0 M +
M o u s e + t 0 0
R a b b i t - + +
Ra t 0 0 +
Sheep + + +
Die thyls t i lbes t ro l H u m a n + / _ s M / - s + / _ s
M o u s e + / _ h 0 0
D i m e t h y l n i t r o s a m i n e H a m s t e r - 0 -
Mouse - 0 0
Ep ich lo rohydr in H u m a n 0 0 -
M o u s e - 0 0
f l -Est radiol M o u s e + 0 0
(Ovccyc l in , Ra t 0 + +
Est radiol - 17fl)
G o s s y p o l or
Gossypo l acet ic acid
H e x a m e t h y l -
p h o s p h o r a m i d e
H u m a n +
H a m s t e r 0
M o u s e +
R a b b i t 0
Rat +
Mouse +
R a b b i t 0
+ +
+
0 +
M + +
0 0
0 +
T A B L E 9 (cont inued)
C o m p o u n d Species Morpho logy Mot i l i ty Coun t
65
I son iaz id Mouse - 0 0 Rabb i t 0 0 -
Lead or H u m a n + + +
Lead aceta teJ Mouse + f 0 0
Mar i j uana k or H u m a n + + +
Cannab ino l Mouse + 0 0
C a n n a b i d i o l Mouse - 0 0
8-9-Tetrahydro- Mouse + 0 0
cannab ino l
Methy l me thanesu l fona te Hams te r + 0 M Mouse + 1 0 O
R a b b i t + 0 -
M V P P m or H u m a n 0 + +
Mech lo re thamine Mouse + 0 0
Vinblas t ine Mouse + 0 0
Procarbaz ine Mouse + 0 0 Procarbaz ine R a b b i t 0 0 +
N i t ro fu r an to in H u m a n 0 0 M
Mouse - 0 0
Proges te rone H u m a n 0 + +
Mouse - 0 0
Tes tos te rone H u m a n + / _ n + / _ n + / _ .
Mouse - 0 0
T r i e thy l eneme lamine Mouse + f _ + i Rabb i t + + i + i
+ , posi t ive effect (decrease in % normal forms, % mot i le forms, or sperm counts).
M, marg ina l or inconclus ive effect.
- , no effect observed.
0, not tested.
a H u m a n exposure repor ted for complex mixture , i.e., for alcoholics.
b H u m a n exposure to anes the t ic gases in occupa t iona l setting, mouse exposure repor ted for 2 mos t
c o m m o n gases used.
c Carba ry l posi t ive for induc t ion of ac rosomal abnormal i t i e s in the mouse (see Table 4).
a Conf l ic t ing results in 2 s tudies at app rox ima te ly equal total doses, bu t wi th di f ferent dosage regimens. c Unreso lved conf l ic t in studies.
f Posi t ive effect also seen on mouse F~ sperm morpho logy assay.
g Conf l i c t ing s tudies; posi t ive and negat ive results (marg ina l for mot i l i ty) are repor ted in s tudies of
t r ansp lacen ta l exposure of sons in t rea ted mothers. Nega t ive results were ob ta ined for sperm counts when s tudied in adu l t males.
h Posi t ive wi th C B C F I mice, negat ive wi th B6C3FI mice; s imi lar doses.
i Conf l i c t ing s tudies; s tudy repor ted for which highest total dose was given.
J H u m a n exposure to lead in occupa t iona l se t t ing (complex mixture) ; mouse exposure to lead acetate. k H u m a n exposure to mar i juana ; mouse exposure repor ted for 3 agents k n o w n to be present in
mar i juana .
i Also pos i t ive for the induc t ion of ac rosomal abnormal i ty ; marg ina l ly posi t ive effect seen on mouse F 1 assay.
m M.V.P.P. is a comb ina t i on chemothe rapeu t i c course of mech lo re thamine (ni t rogen mustard) , vinblas- tine, procarbazine , and prednisone. Tes t ing of ind iv idua l agents carr ied out in mouse and rabbi t .
n Posi t ive and negat ive results repor ted in different s tudies of tes tos terone and several der ivat ives of testosterone.
66
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IX. Appendix I
Transmission of chemically induced sperm defects to the F l progeny of treated mice: The F 1 sperm morphology test
A. Introduction
Under s t and ing the na ture of the muta t ions affecting sperm shaping is very impor tan t in evaluat ing the genetic implicat ions of chemically induced sperm
abnormali t ies . In man, very few data exist on the genetic basis of sperm abnormal i - ties, but there have been some suggestions, though controversial, that defects such as reduced sperm counts, increased sperm abnormali t ies , may be associated with increased frequencies of spontaneous abor t ion (Furuh je lm et al., 1962). Some h u m a n data suggest that sperm shaping is genetically determined, as it is in the mouse; the inher i tance of defects in h u m a n sperm tail morphology has been demonst ra ted (Bisson et al,, 1979) and some twins have shown identical pat terns of sperm morphology (Kul lander and Rausing, 1975).
Several studies have demonst ra ted the t ransmiss ion of sperm-shape defects in mice. These studies were done by exposing sexually mature mice of either sex to a test compound, mat ing the treated animals to untreated partners, and scoring for abnormal sperm morphology in male progeny when they mature. The papers on the
69
mouse F~ sperm morphology test were supplied by the Environmental Mutagen Information Center or by the members of the reviewing committee. Lenient criteria of acceptability were used by the reviewing committee to include all chemicals; data for two of the agents appeared in abstracts.
B. Test description
1. Genetic basis of the method In mice, sperm-head shape has a high heritability (h 2 -- 0.9) and the spontaneous
level of sperm abnormalities has been shown to be characteristic of genotype (for refs. see Section IIB). A large number of autosomal as well as sex-linked genes appear to influence sperm-head shape. Autosomal genes may be associated with the structural aspects of the abnormalities, while the genes on the Y chromosome may be mainly responsible for their expression. Hugenholtz and Bruce (1977a) have presented evidence that an increased incidence of sperm abnormalities seen in the offspring of X-ray-treated mice represents inherited genetic damage. Though certain chemicals produced similar effects, it is unknown whether induced sperm changes seen in the progeny of chemically treated mice are genetically based and if so, whether or not it is the same type of damage that leads to transmission of abnormal sperm shapes to the progeny of X-ray treated mice.
2. Protocol used in studies reviewed So few studies have been published with the F l sperm morphology test that it is
premature to recommend a standard protocol. The following is a brief description of the study designs that have been used, with some recommendations for future studies. In general, mature male or female mice were exposed to the test agent and mated to unexposed animals at various periods after exposure. The most effective mating time may depend on chemical classes and remains to be determined. Male progeny were sacrificed or hemiorchiectomized at maturity (11-15 weeks of age), and the sperm from the testis, epididymis or vas deferens analyzed for percentage of sperm with shape abnormalities. To determine whether the chemical has induced transmissible sperm defects, it is recommended that statistical evaluation of the proportion of F~ males with high levels of abnormally shaped sperm be based on a comparison of means or the proportion of outliers (e.g., using Z test, Snedecor and Cochran, 1967) or some nonparametric evaluation. Genetic verification of trans- mitted sperm defects may be carried out by mating hemiorchiectomized F~ males who showed higher levels of sperm abnormalities, and establishing the pattern of transmission of sperm defects to their progeny. No studies to date have reported this for offspring of chemically treated mice.
The number or parent mice exposed (usually < 30) varied with the chemical. The number of F~ males analyzed varied according to efficacy of the chemical to produce transmissible genetic effects and ranged between 30 and 500 males. Clearly, the sample sizes of some of the reported studies would preclude the detection of small effects.
70
TABLE AI
Fj SPERM M O R P H O L O G Y TEST: RESULTS WITH CHEMICALS
Agent Car- CAS No. Treated Mated Mating cin- F o F o days post ogen genotype genotype treatment
Vehicle
Cyclophos- NA 50-18-0 C B A / C a phamide females monohydrate
B a l b / c / 0 L a 0 -7
(C3H (C3H 18-21 × 101)F I x 101)F 1 males
Ethyl methane- + 62-50-0 C B A / C a B a l b / c / 0 L a 35-42 sulfonate males 56-63
(C3H (C3H 5.5-8.5 × 101)F I × 101)F~ males
Lead acetate + 15347-57-6 C57BL/6J C 3 H / H e Pre- males sterile
period
Methyl methane + 66-27-3 C57BL/6J C 3 H / H e Pre- sulfonate males sterile
period
0 -7 Mitomycin C + 50-07-7 C B A / C a B a l b / c / 0 L a females
Metepa g + 57-39-6 C B A / C a B a l b / c / 0 L a 0 7 females
Triethylene + 51-18-3 CD males CD 8-14 melamine
H 2 0
Saline
0.5% Tween 80 in water
Saline
NA
NA
Saline
Saline
Hank's BSS
Inadequate number of F I animals. b F~ sperm collected from cauda epididymus.
F I spermatids examined. a Small dose-related increase in abnormalities but concomitant controls very low. e Statistics done. I Statistical analysis not possible from data presented. g Tris(2-methyl-1 aziridinyl)-phosphine oxide.
C. Results
T h e r e s u l t s o f t h e r e v i e w e d s t u d i e s o n t h e t r a n s m i s s i o n s o f i n d u c e d s p e r m d e f e c t s
a r e s u m m a r i z e d i n T a b l e A I . S e v e n a g e n t s h a v e b e e n t e s t e d : e t h y l m e t h a n e s u l f o n a t e
71
Exposure Number Number of Highest Number Re- of F o of dose of F I sponse
Route Regi- animals doses tested mice men per dose
Ref. Com- ments
IP qd × 5 10 2 400 39
IP Single NS 1 350 100 +
dose
IP qd × 5 10 1 625 30 +
IP Single NS 1 200 100 + dose
IP qd X 5 25-30 1 50 188
IP qd × 5 25-30 1 125 43
IP qd x 5 10 1 5.0 17
IP qd × 5 10 2 62.5 53
IP Single 40-50 1 0.3 152 dose
Topham, a, b , e 1980b
Sotomayor, 1979
c, e
Topham a , b , e 1980b
Sotomayor, 1979
c ,e
+ Wyrobek b, f and Bruce, 1975
? Wyrobek b, f and Bruce, 1975
? Topham, a, b, e 1980b
? Topham, b, d, e 1 9 8 0 b
+ Staub and b, e Matter, 1977
(EMS), methyl methanesulfonate (MMS), triethylenemelamine (TEM), cyclo- phosphamide, mitomycin C, metepa and lead acetate. 4 agents were classed positive and 3 as inconclusive inducers of heritable sperm abnormalities.
Treatment of male mice. After treatment of males, some evidence of transmission of induced sperm defects to the F~ progeny was obtained with TEM, EMS, cyclophosphamide and lead acetate. However, the results with MMS were inconclu- sive (perhaps because of small sample sizes, a n d / o r too low a dosage).
Treatment of female mice. Female mice were treated with metepa, mitomycin C and cyclophosphamide. These experiments studied small numbers of F~ progeny males and the results were classed as inconclusive (mitomycin C and metepa) or negative (cyclophosphamide).
72
D. Discussion
Very few studies have been reported on the transmission of induced sperm defects to the progeny of animals exposed to mutagens, and the lack of a common protocol makes comparison difficult. The results with cyclophosphamide, lead acetate, TEM and EMS suggest that exposure of males to any of these agents results in heritable damage leading to abnormal sperm morphology in the progeny. The evidence for a similar effect following treatment of F 0 females is equivocal.
All the agents that produce a positive response in this test also are positive in other in vivo mammalian mutagen tests (e.g., specific locus test and dominant lethal test in mice) and are positive inducers of sperm abnormalities in the exposed male parent.
E. Conclusions
Strengths and weaknesses of the test The major advantages of this method are that it measures heritable genetic
damage in a mammal in vivo. Because a large number of genes (perhaps hundreds) may be involved in controlling sperm shape, this test may be a means of assessing heritability using small numbers of male offspring per study.
The major disadvantages of the method are: (1) very few compounds (seven) have been evaluated, (2) the method is still under development and there is no standard protocol, and (3) genetic proof that the induced changes seen in the F~ generation are due to mutation (which has been demonstrated for irradiation-induced damage) is not yet available for chemically induced damage.