small difference in carcinogenic potency between gbp nanomaterials and gbp micromaterials

REVIEW ARTICLE

Small difference in carcinogenic potency between GBPnanomaterials and GBP micromaterials

Thomas Gebel

Received: 28 September 2011 / Accepted: 1 March 2012 / Published online: 15 March 2012

� Springer-Verlag 2012

Abstract Materials that can be described as respirable

granular biodurable particles without known significant spe-

cific toxicity (GBP) show a common mode of toxicological

action that is characterized by inflammation and carcinoge-

nicity in chronic inhalation studies in the rat. This study was

carried out to compare the carcinogenic potency of GBP

nanomaterials (primary particle diameter 1–100 nm) to GBP

micromaterials (primary particle diameter [100 nm) in a

pooled approach. For this purpose, the positive GBP rat

inhalation carcinogenicity studies have been evaluated.

Inhalation studies on diesel engine emissions have also been

included due to the fact that the mode of carcinogenic action is

assumed to be the same. As it is currently not clear which dose

metrics may best explain carcinogenic potency, different

metrics have been considered. Cumulative exposure con-

centrations related to mass, surface area, and primary particle

volume have been included as well as cumulative lung burden

metrics related to mass, surface area, and primary particle

volume. In total, 36 comparisons have been conducted.

Including all dose metrics, GBP nanomaterials were 1.33- to

1.69-fold (mean values) and 1.88- to 3.54-fold (median val-

ues) more potent with respect to carcinogenicity than GBP

micromaterials, respectively. Nine of these 36 comparisons

showed statistical significance (p \ 0.05, U test), all of which

related to dose metrics based on particle mass. The maximum

comparative potency factor obtained for one of these 9 dose

metric comparisons based on particle mass was 4.71. The

studies with diesel engine emissions did not have a major

impact on the potency comparison. The average duration of

the carcinogenicity studies with GBP nanomaterials was

4 months longer (median values 30 vs. 26 months) than the

studies with GBP micromaterials, respectively. Tumor rates

increase with age and lung tumors in the rat induced by GBP

materials are known to appear late, that is, mainly after study

durations longer than 24 months. Taking the different study

durations into account, the real potency differences were

estimated to be twofold lower than the relative potency factors

identified. In conclusion, the chronic rat inhalation studies

with GBP materials indicate that the difference in carcino-

genic potency between GBP nanomaterials and GBP

micromaterials is low can be described by a factor of 2–2.5

referring to the dose metrics mass concentration.

Keywords Nanomaterials � Carcinogenicity � Respirable

granular biodurable particles without known significant

specific toxicity (GBP) � Chronic rat inhalation study

Abbreviations

GBP Respirable granular biodurable particles without

known significant specific toxicity

CKSL Cystic keratinizing squamous lesions

DEE Diesel engine emissions

CB Carbon black

Introduction

Nanomaterials can be characterized as being technically

engineered materials containing primary particles with

This article is published as a part of the Special Issue

‘‘Nanotoxicology II’’ on the ECETOC Satellite workshop, Dresden

2010 (Innovation through Nanotechnology and

Nanomaterials ? Current Aspects of Safety Assessment and

Regulation.

T. Gebel (&)

Federal Institute for Occupational Safety and Health,

Friedrich-Henkel-Weg 1-25, 44149 Dortmund, Germany

e-mail: [email protected]

123

Arch Toxicol (2012) 86:995–1007

DOI 10.1007/s00204-012-0835-1

diameters between 1 and 100 nm (ISO 2008). There is a

current discussion and concern with respect to uncertainties

in the assessment of putative risks to human health and

environment caused by these materials. It is hypothesized

that particulate materials containing or consisting of

nanosized primary particles may not only possess a higher

toxicity in comparison to bulk materials but also cause

additional health hazards.

Nanomaterials are rather diverse with respect to chem-

ical identity. With respect to data generation and evaluation

it would be a tremendous task to perform a risk assessment

for each specific nanomaterial. Thus, it needs to be con-

sidered whether certain nanomaterials may be grouped.

The toxicology of a specific group of dusts can be descri-

bed by a common mode of toxicological action. These

dusts are characterized as respirable, biopersistent and do

not possess a toxicity which is mediated by specific sub-

stances contained in or released from these particles nor by

specific functional chemical groups or significant surface-

related toxicity like, for example, crystalline silica. For

instance, carbon black (CB) and titanium dioxide dusts can

be assigned to this group of dusts. They were termed poorly

soluble particles (PSP) (Oberdorster 2002), respirable

granular biodurable particles without known significant

specific toxicity (GBP) (Roller and Pott 2006), or poorly

soluble, low toxicity particles (PSLT) (Dankovic et al.

2007). In the following the term GBP will be used as it

represents the most detailed description. For the purpose of

this paper, GBP micromaterials are defined as GBP con-

sisting of primary particles bigger than 100 nm in three

dimensions. The term GBP nanomaterials will be used for

GBP materials consisting of primary particles in sizes

between 1 and 100 nm in three dimensions.

In the current German workplace regulation there is an

occupational exposure limit (OEL) established for GBP

dusts covering GBP micromaterials but excluding ultrafine

dusts (AGS 2001). GBP nanomaterials and ultrafine dusts

had been excluded from the German dust OEL as there was

uncertainty whether these materials possess a higher tox-

icity than the GBP dusts in the micromaterial size range.

The reason were respective indications from the scientific

literature available (e.g., see Oberdorster et al. 1992, 1994).

For GBP materials, toxicity is mainly determined by

airway inflammation and carcinogenicity which was

detected in rat inhalation studies. Dermal and systemic

toxicity seems to be of little relevance. It is assumed that

lung inflammation is the driving force leading to cancer

(for review see ILSI 2000; Valberg et al. 2009). The

accepted common mode of action is that the particle is the

likely toxic principle (ILSI 2000; Roller 2009). Prominent

GBP materials like titanium dioxide and CB have been

classified by the International Agency for Research on

Cancer to possess ‘‘sufficient evidence in experimental

animals for carcinogenicity’’ (Baan 2007). CB is a GBP

nanomaterial, titanium dioxide is marketed both in the

nanosized and in the microsized form.

The present analysis was carried out to compare the

carcinogenic potency of GBP nanomaterials to GBP

micromaterials in a pooled approach. For this purpose, the

available positive GBP rat inhalation carcinogenicity stud-

ies have been evaluated. Inhalation studies on diesel engine

emissions (DEE) have also been included due to the fact

that the mode of toxicological action is judged to be the

same, that is, the particle consisting of elemental carbon is

considered to be the toxic principle for DEE (Roller 2009).

This analysis shall help to address the question whether and

how GBP nanomaterials can be included in the regulatory

frameworks dealing with nanomaterials and fine dusts.

Methodical approach

The positive dose groups of the available rat inhalation

carcinogenicity studies on GBP materials have been

included (Table 1), which have been published between

1977 and 2010. In addition to a literature search, three

previous review articles have been screened to assure that

all available and eligible data were included (Nikula 2000;

Dankovic et al. 2007; Roller 2009). Studies with GBP

materials that were negative in all dose groups were not

evaluated. Coal dust, titanium dioxide in the rutile form,

talc, and toner in Table 1 are GBP micromaterials. The

other studies in Table 1 except those on DEE used GBP

nanomaterials. The results from all studies given in Table 1

were included without any correction with two exceptions.

The study by Stinn et al. (2005) had a very thorough but

unusual design of the histopathological analyses. Up to 26

sections had been taken from the lungs covering both lung

lobes. This led the authors to the conclusion that they

detected 5 times more tumors than with a usual study

design. As a consequence, the results of this study have

been included in all evaluations in the present analysis with

the respective correction of the tumor rates by a factor of

1/5. The second exception was that the study from Iwai

et al. (2000) was excluded from the evaluations of car-

cinogenic potency due to an unusual exposure pattern

leading to very high calculated potency values. Iwai et al.

(2000) used short total exposure times for 3, 6, 9, and

12 months for their different exposed study groups.

The carcinogenic potency of GBP nanomaterials was

compared to GBP micromaterials in three different

approaches. The first approach was to compare all available

positive dose groups on GBP nanomaterials with all

available positive dose groups on GBP micromaterials.

Cystic keratinizing squamous lesions (CKSL) are induced

by chronic inhalation exposure to particles in the rat, and

996 Arch Toxicol (2012) 86:995–1007

123

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Arch Toxicol (2012) 86:995–1007 997

123

their relevance has been questioned (Warheit and Frame

2006). As a consequence, in this paper, a differential

evaluation including and excluding CKSL has been per-

formed to check whether this has a relevant impact on the

potency comparison. From the histological perspective,

CKSL are not unique but comprise different specifications

of lesions (Boorman et al. 1996). Some of them may turn

into carcinoma, and others may not. There is no compre-

hensive data for the included rat inhalation carcinogenicity

studies available to be able to subclassify all reported

CKSL accordingly. The consequence for this paper was

that the carcinogenic potency comparison could only be

performed by including (second approach) or excluding

(third approach) all CKSL but not certain subtypes.

As it is currently not clear which dose metrics may best

explain carcinogenic potency, six different metrics have

been considered. Cumulative dose metrics were calculated

from the study data to correct for different exposure pat-

terns (number of hours/day, number of days/week, and

number of months exposed). Cumulative exposure con-

centrations related to mass, surface area, and volume have

been included as well as cumulative lung burden metrics

related to mass, surface area, and volume. Cumulative

volume concentration was derived based on primary par-

ticle volume that does not take into account that the void

space of the primary particles in agglomerated form may be

different for different GBP materials. It was also aimed at

estimating quantitative data for the dose metrics cumula-

tive primary particle number and cumulative particle

agglomerate number. These two approaches are not

described because several estimations that needed to be

made were considered to lead to unreliable data. Cumula-

tive surface area was calculated by using surface area data

from the Brunauer, Emmett, and Teller (BET) analysis

(Brunauer et al. 1938). For most of the studies, BET data

were given with the carcinogenicity studies. These data

were not available in the studies for Elftex-12 (Nikula et al.

1995), talc (NTP 1993), and generally in the diesel studies

and were obtained from other sources.

In all analyses, the tumor rates in the controls were

subtracted from the tumor incidence in all treated groups

and all treated study groups with a remaining positive

tumor incidence were included in the evaluation.

The carcinogenicity potency ratios were obtained by

dividing the tumor rate obtained in a dose group by the

respective cumulative exposure of that dose group. The

second step was to pool and average all these potency

values belonging to the same dose metrics for GBP

nanomaterials and for GBP micromaterials, respectively.

The comparison was performed by calculating the ratio of

this averaged GBP nanomaterial potency value and the

respective average value for GBP micromaterials, that is,

deriving a comparative potency factor.

There is some indication that the second year of expo-

sure does not contribute to a major amount to tumor

induction in the studies evaluated (Roller 2008). Thus, the

cumulative dose metrics could have been corrected for

cumulative external exposure after 12 months as point of

departure for the potency comparison. However, such

correction would have had only small impact on the

potency comparison and was not performed. The reason

was that only few studies used total exposures deviating to

a greater extent from 24 months. Such correction to

cumulative exposure after 12 months may be more relevant

for the dose metrics based on particles deposited in the

lungs, that is, the lung burden dose metrics. Lung clearance

is more strongly retarded the higher the exposures are due

to impaired clearance. This supports the use of the lung

burden after 12 months for calculation of the correspond-

ing cumulative dose metrics. All cumulative dose metrics

were normalized to g lung weight. Normalization was

performed by using a standardized fresh lung weight of

control rats of 1.25 g both for all studies that used Fischer

and Wistar rats. Martin et al. (1977) and Lee et al. (1985)

used SD rats that normally show a higher body and lung

weight, respectively. However, in Martin et al. (1977), the

fresh lung weight of control rats was given as 1.3 g after

12 months. So this value was included. In Lee et al. (1985),

the fresh lung weight of control rats was 3.25 g for male

rats and 2.35 g for female rats. As a consequence, the

normalization to lung weight performed for all included

studies was only relevant to correct for the higher lung

weight in the Lee et al. (1985) study. For the comparison of

all other studies, there is no impact on the results whether

this normalization would have been performed or not.

The statistical calculations were performed with the

software Winstat 2005.1. For the calculation of the Pearson

correlation coefficient, the data were transformed to obtain

a normal distribution of the data.

Results

Study data compilation and survey

Table 1 contains a survey on the included rat inhalation

carcinogenicity studies listing some study characteristics.

It is evident that especially the studies with DEE [mass

median aerodynamic diameter (MMAD) range, 0.1–0.35 lm]

had exposure atmospheres with relatively small particle size

distributions (see Table 1). The studies with GBP nanoma-

terials (MMAD range, 0.64–1.95) had exposure atmospheres

in slightly smaller particle size distributions than the studies

with GBP micromaterials (MMAD range, 1.6–4 lm).

Figures 1 and 2 give an exemplary impression of the

cumulative dose metrics based on mass concentration and

998 Arch Toxicol (2012) 86:995–1007

123

surface area concentration, respectively. The data include

original tumor incidences of the study controls and the

exposed dose groups. In Fig. 1, tumor rates including the

control data are plotted against the dose metrics cumulative

exposure mass concentration. The data from the studies

with GBP micromaterials titanium dioxide (Lee et al.

1985) and coal dust (Martin et al. 1977) seem to indicate a

low potency and seem outlying with respect to all other

data. It has to be noted that these studies had a relatively

short duration of 24 months to terminal section (see

Table 1). In Fig. 2, a lower part of the x-axis given with

Fig. 1 is shown to enhance the graphical resolution for the

majority of the data points but excluding the graphically

outlying results from Lee et al. (1985) and Martin et al.

(1977). The remaining data still seem to be rather scattered.

Dose metrics analyses

These data and further dose metrics were used in a com-

parative evaluation including six different dose metrics in

all (Table 2). The purpose of this analysis was to obtain an

indication that dose metrics may best describe carcinogenic

potency. Cumulative exposure concentrations related to

mass, surface area, and volume were included as well as

cumulative lung burden metrics related to mass, surface

area, and volume. The six chosen dose metrics have been

evaluated in three different sets of evaluations. Firstly, all

studies available were included. The second evaluation

used a subset of data from which cystic keratinizing

squamous lung lesions (CKSL) was included. The third

evaluation was to exclude these lesions from the analysis.

The reason to do so was that there is information that these

lesions may not be generally considered as tumors or early

stages thereof (Boorman et al. 1996; Warheit and Frame

2006). The control group tumor rates in each experiment

were subtracted from all respective positive study groups.

Only positive tumor rates were included in the analysis.

Comparing all dose metrics, the Pearson rank correlation

coefficients for the dose metrics surface area were slightly

higher in the evaluations including all data for the lung

burden metrics and in the evaluation excluding CKSL.

Overall, the differences in the correlation coefficients for

0

10

20

30

40

50

60

0 50000 100000 150000 200000 250000 300000 350000

cumulative mass concentration [mg/m³*h/g lung]

% t

um

ou

rs

TiO2_Lee85Coal_Mart77TiO2_P25_Hein95CB_P90_Hein94CB_P90_Hein95DEE_Hein95DEE_Maud87DEE_Nik95Talc_NTP93Toner_Muhle91DEE_Hein86DEE_Bright89DEE_Iwai86DEE_Ishi86DEE_Stinn05DEE_Iwai00DEE_Kawa93DEE_Iwai97

Fig. 1 Tumor rates and specific

cumulative mass concentration,

all available studies plotted.

Also negative data from the

included studies were plotted

0

10

20

30

40

50

60

0 10000 20000 30000 40000 50000 60000 70000 80000

cumulative mass concentration [mg/m³*h/g lung]

% t

um

ou

rs

TiO2_Lee85Coal_Mart77TiO2_P25_Hein95CB_P90_Hein94CB_P90_Hein95DEE_Hein95DEE_Maud87DEE_Nik95Talc_NTP93Toner_Muhle91DEE_Hein86DEE_Bright89DEE_Iwai86DEE_Ishi86DEE_Stinn05DEE_Iwai00DEE_Kawa93DEE_Iwai97

Fig. 2 Tumor rates and specific

cumulative mass concentration,

all available studies plotted. The

x-axis was cut to obtain a higher

resolution for the lower

cumulative mass concentrations.

Also negative data from the

included studies were plotted

Arch Toxicol (2012) 86:995–1007 999

123

the different dose metrics in each of the three evaluation

sets were small and do not yield clear results. Thus, the

further analysis was continued including all dose metrics.

Analysis on comparative potency

Table 3 presents these results of the carcinogenic potency

comparison for all included dose metrics based on particle

mass in the three data sets analyzed in separate (all studies,

studies including CKSL, and studies excluding CKSL).

Only positive tumor rates were included, and control group

tumor rates in the respective experiment were subtracted

from the corresponding positive study groups. The results

of the diesel studies were once included and once excluded

in parallel evaluations. The reason to do so was to visualize

the impact of the DEE studies on the potency comparison.

The carcinogenic potency of GBP nanomaterials was

compared to GBP micromaterials, and these ratios of the

mean and median carcinogenic potency are given in the

table. These ratios represent the comparative carcinogenic

Table 2 Correlation of tumor rate and dose metrics

Cumulative dose metrics All studies Studies including CKSL Studies excluding CKSL

r n p r n p r n p

External exposure

Mass 0.38 42 0.007 0.61 26 0.0005 0.57 25 0.001

Surface area 0.41 42 0.003 0.59 26 0.0008 0.66 25 0.0001

Volume 0.35 42 0.01 0.61 26 0.0004 0.56 25 0.002

Lung burden

Mass 0.35 26 0.04 0.52 25 0.004 0.50 24 0.007

Surface area 0.54 26 0.002 0.54 25 0.003 0.62 24 0.0006

Volume 0.45 26 0.01 0.49 25 0.007 0.47 24 0.01

Only positive tumor rates were included after subtraction of the tumor rate in the controls

CKSL cystic keratinizing squamous lesions, r Pearson correlation coefficient, n number of data pairs (tumor rate vs. exposure, each positive study

group) included, p value significance one-sided (Pearson)

Table 3 Comparison of carcinogenic potency of GBP nanomaterials and DEE to GBP micromaterials summarizing all results based on dose

metrics related to particle mass

Cumulative external exposure Cumulative lung burden

All studies ?CKSL -CKSL All studies ?CKSL -CKSL

Including the DEE studies

Mean ratio 2.81 2.05 1.93 1.93 1.77 1.75

Median ratio 2.26 3.11 2.95 3.39 3.46 3.75

Max/min 29.97 15.74 15.90 30.88 30.88 28.22

Min/max 0.08 0.14 0.05 0.10 0.10 0.04

U test 0.007 0.013 0.031 0.016 0.04 0.10

n 42 26 25 26 25 24

Excluding the DEE studies

Mean ratio 2.87 2.57 2.23 2.47 2.28 2.30

Median ratio 3.67 3.63 3.58 3.79 3.87 4.71

Max/min 17.02 15.74 14.79 30.88 30.88 28.22

Min/max 0.14 0.14 0.05 0.13 0.13 0.04

U test 0.025 0.046 0.27 0.018 0.032 0.27

n 14 13 11 14 13 11

Only positive tumor rates were included after subtraction of the tumor rate in the controls

U test: significance two-sided

?CKSL including cystic keratinizing squamous lesions, -CKSL excluding cystic keratinizing squamous lesions, max/min maximum GBP

nanomaterial potency estimate divided by minimum GBP micromaterial potency estimate, min/max minimum GBP nanomaterial potency

estimate divided by maximum GBP micromaterial potency estimate

1000 Arch Toxicol (2012) 86:995–1007

123

potency; a factor of, for example, 3 would indicate a

threefold higher carcinogenic potency of GBP nanomate-

rials. In addition, the ratios max/min and min/max were

obtained by calculating the ratio of the maximum GBP

nanomaterial potency value and minimum GBP microma-

terial potency value and vice versa. These values give the

relative maximum and minimum carcinogenic potency

factor by comparing all positive study groups in separate.

Depending on evaluation subset, the relative carcino-

genic potency of GBP nanomaterials compared to GBP

micromaterials was ranging between 1.75 and 2.81 (mean

values) and 2.26–3.75 (median values) when including the

DEE data. Excluding the DEE data, the relative mean and

median carcinogenic potency factors of GBP nanomaterials

compared to GBP micromaterials were found between 2.23

and 2.87 (mean values) and 3.58–3.71 (median values),

respectively. Nine of these 12 comparisons showed statis-

tical significance (U test, two-sided).

Tables 4, 5, and 6 give the results of the carcinogenic

potency comparison of GBP nanomaterials to GBP

micromaterials for all further included dose metrics and the

three data sets analyzed in separate (all studies, studies

including CKSL and studies excluding CKSL). For the

purpose of better comparison, these tables include

the information already given in Table 3 and extend the

description of the results obtained for the further dose

metrics evaluated. The ratios of the different dose metrics

were also averaged over all different dose metrics in

Tables 4, 5, and 6. These values are given in the column

potency average. In addition to these two values like in

Table 3, all ratios max/min and min/max were obtained by

calculating the ratio of the maximum GBP nanomaterial

potency value and minimum GBP micromaterial potency

value and vice versa. Also mean and median values of the

total study duration, that is, times to terminal section from

each respective study are given for the pooled data on GBP

nanomaterials and GBP micromaterials, respectively.

Depending on dose metrics, the relative carcinogenic

potency of GBP nanomaterials compared to GBP

micromaterials was ranging between 0.78 and 2.81 (mean

values) and 0.91–3.39 (median values) when including all

studies and the DEE data (Table 4). Excluding the DEE

data, the relative mean and median carcinogenic potency

factors of GBP nanomaterials compared to GBP

micromaterials were between 0.38 and 2.87 (mean values)

and 0.20–4.76 (median values), respectively. Averaged

over all values and including all dose metrics and the DEE

studies, GBP nanomaterials were 1.69 (mean) and 1.88

(median values) more potent in carcinogenicity. Excluding

the DEE data, GBP nanomaterials were 1.69 (mean) and

3.20 (median values) more potent in carcinogenicity

averaged over all dose metrics. When comparing the total

study durations, the GBP nanomaterial studies were

3.6 months (mean values 25.4 vs. 29.2) and 4 months

Table 4 Comparison of carcinogenic potency of GBP nanomaterials and DEE to GBP micromaterials

Cumulative external exposure Cumulative lung burden Potency factor average Time to terminal section (mth)

Mass Surface area Volume Mass Surface area Volume Nano Micro

Including DEE (n = 35 and 19 positive study groups for cumulative external exposure and cumulative lung burden, respectively).

n = 7 positive GBP micromaterial groups

Mean ratio 2.81 1.38 1.91 1.93 0.78 1.37 Mean 1.69 29.2 25.4

Median ratio 2.26 0.91 1.50 3.39 0.96 2.54 Median 1.88 30.0 26.0

Max/min 29.97 14.98 36.96 30.88 9.88 23.19

Min/max 0.08 0.017 0.05 0.10 0.01 0.07

U test 0.007 0.55 0.061 0.016 0.71 0.09

n 42 42 42 26 26 26

Excluding DEE (n = 7 positive GBP nanomaterial study groups). n = 7 positive GBP micromaterial groups

Mean ratio 2.87 0.38 2.14 2.47 0.41 1.84 Mean 1.69 28.1 25.4


Max/min 17.02 2.83 20.99 30.88 5.75 23.19

Min/max 0.14 0.02 0.09 0.13 0.01 0.09

U test 0.025 0.11 0.085 0.018 0.11 0.085

n 14 14 14 14 14 14

Also studies not including a separate evaluation of cystic keratinizing squamous lesions were included. Only positive tumor rates were included

after subtraction of the tumor rate in the controls


Nano data from studies with GBP nanomaterials, micro data from studies with GBP micromaterials, mth months, max/min maximum GBP



Arch Toxicol (2012) 86:995–1007 1001

123

Table 5 Comparison of carcinogenic potency of GBP nanomaterials and DEE to GBP micromaterials including cystic keratinizing squamous

lesions



Including DEE (n = 20 positive study groups). n = 6 positive GBP micromaterial groups

Mean ratio 2.05 0.79 1.41 1.77 0.73 1.22 Mean 1.33 28.5 25.7


Max/min 15.74 8.39 11.55 30.88 9.88 23.19

Min/max 0.14 0.02 0.09 0.10 0.01 0.07

U test 0.013 1 0.088 0.04 0.70 0.20

n 26 26 26 25 25 25

Excluding DEE (n = 7 positive study groups left). n = 6 positive GBP micromaterial groups

Mean ratio 2.57 0.34 1.88 2.28 0.38 1.64 Mean 1.51 28.1 25.7


Max/min 15.74 2.80 11.55 30.88 5.75 23.19

Min/max 0.14 0.02 0.09 0.13 0.01 0.09

U test 0.046 0.086 0.153 0.032 0.116 0.153

n 13 13 13 13 13 13

Only a subset of the studies could be included. Only positive tumor rates were included after subtraction of the tumor rate in the controls





Table 6 Comparison of carcinogenic potency of GBP nanomaterials and DEE to GBP micromaterials excluding cystic keratinizing squamous

lesions



Including DEE (n = 19 positive study groups). n = 6 positive GBP micromaterial groups

Mean ratio 1.93 0.82 1.36 1.75 0.79 1.22 Mean 1.31 28.4 25.7


Max/min 15.90 11.66 10.51 28.22 13.24 18.64

Min/max 0.05 0.01 0.03 0.04 0.01 0.03

U test 0.031 0.84 0.098 0.10 0.95 0.21

n 25 25 25 24 24 24

Excluding DEE (n = 5 positive study groups left). n = 6 positive GBP micromaterial groups

Mean ratio 2.23 0.35 1.75 2.30 0.42 1.71 Mean 1.46 27.3 25.7


Max/min 14.79 3.78 9.77 28.22 7.22 18.64

Min/max 0.05 0.01 0.03 0.04 0.01 0.03

U test 0.27 0.20 0.36 0.27 0.20 0.36

n 11 11 11 11 11 11

Only a subset of the studies could be included. Only positive tumor rates were included after subtraction of the tumor rate in the controls





1002 Arch Toxicol (2012) 86:995–1007

123

(median values 26.0 vs. 30.0) longer than the GBP

micromaterial studies.

The analogous analyses by evaluating the subset of

studies and taking into account CKSL show similar aver-

age relative carcinogenic potencies (Tables 5, 6). The

maximum potency differences overall were obtained when

excluding the DEE data for cumulative lung mass and lung

volume burden (4.71 and 5.06, respectively). Also for these

data subsets, GBP nanomaterial studies had longer total

study durations than the GBP micromaterial studies.

Excluding the DEE data did not change the relative

potency factors to a greater extent, but by trend the factors

increased except for dose metrics surface area where they

decreased. For the dose metrics surface area, relative

potency factors close to 1 appear in the evaluations

including the DEE data (Tables 4, 5, 6) indicating that this

dose metrics may explain tumor rate best. However, this

does not hold true for the factors obtained for dose metrics

surface area when excluding the DEE data. Nine of all 36

potency comparisons showed statistical significance

(p \ 0.05, U test) all of which were related to dose metrics

based on particle mass (Tables 4, 5, 6). A slower clearance

of GBP nanomaterials could explain the observed differ-

ence in carcinogenic potency. To test this hypothesis, those

studies were identified from which the development of lung

burden mass during the experiment could be followed (Lee

et al. 1985; Heinrich et al. 1995; Nikula et al. 1995;

Bellmann et al. 1991). Thirteen dose groups treated with

GBP nanomaterials could be compared to 12 dose groups

treated with GBP micromaterials (data not shown). When

comparing lung mass burden after 3 and 12 months of

exposure, the increase was 1.14 higher for GBP nanoma-

terials. When comparing lung mass burden after 3 and

24 months of exposure, the increase was 1.41 higher for

GBP nanomaterials. These results indicate a certain support

for higher carcinogenic potency due to a slower clearance

of GBP nanomaterials but a definite conclusion cannot be

drawn.

Discussion

Study characteristics and limitations influencing

the results

It has to be kept in mind that the potency factors derived

were obtained by a comparison of studies performed over

three decades in various laboratories using widely differing

protocols. This may partly explain the variability in car-

cinogenic potency in the results obtained (see Fig. 1).

Depending on data subset, up to seven positive dose groups

could be included both for GBP nanomaterials and also for

GBP micromaterials. For the GBP nanomaterials, it was

possible to additionally complement these data with up to

35 positive DEE dose groups. The analysis is especially

impacted by the limited data on GBP micromaterials.

Besides the fact that only seven positive dose groups could

be included, some of them showed rather small tumor

incidences (Muhle et al. 1991; Martin et al. 1977). As

described below, this is to a relevant part due to the fact

that study termination was early in the GBP micromaterial

studies. It had not been included in the analysis for relative

potency that the median study duration to terminal section

of the positive GBP nanomaterial dose groups was

4 months longer than for GBP micromaterials (26 vs.

30 months). Tumor induction is age dependent. GBP-

induced tumors are known to appear late and to a major

extent later than 24 months after the start of exposure

(Mauderly et al. 1987; Nikula 2000). For instance, only

14–17 % (1/6–1/7) of the tumors were detected at

24 months in the studies with DEE and talc in comparison

to study termination at 28–31 months, respectively (Iwai

et al. 2000; Mauderly et al. 1987; NTP 1993). However,

this observation is not based on analyzing satellite groups

at 24 months. The tumor rates at 24 months were obtained

in these studies by analysis of animals that had died by this

time. There was an unknown portion of animals in these

bioassays that already had lung tumors at 24 months but

were still alive. These tumors could not be identified. Thus,

the real difference in tumor rates comparing 24 and

30 months study duration is lower than 1/6–1/7. Mauderly

et al. (1987) performed a logistic regression analysis to

estimate the age-dependency of DEE-induced tumors.

When referring to these results, a median study duration to

terminal section of 30 months compared to 26 months like

obtained in the present analysis a factor of 2 in tumor rate

difference can be estimated (cf. figure 5 in Mauderly et al.

1987). This means that the study duration-corrected dif-

ference in relative carcinogenic potency obtained in the

present analysis is not described best by a maximum factor

of 4 or 5 but by a factor of 2–2.5.

Lee et al. (1985) and Martin et al. (1977) used higher

exposure concentrations than any other study. The only

dose used by Martin et al. (1977) was 200 mg/m3; the two

higher doses applied by Lee et al. (1985) were 50 and

250 mg/m3. The maximum doses applied in all other

studies were roughly maximally around 10 mg/m3 with the

exception of the talc study (NTP 1993) that used 18 mg/m3

as maximum dose. It could be argued that these very high

and differing exposure levels cannot be used with respect

to a comparison of carcinogenic potency. However, when

correcting for the relatively early terminal section after

24 months in the studies of Lee et al. (1985) and Martin

et al. (1977), the carcinogenic potency derived is close to

the results of the other studies and fits well into the overall

figure.

Arch Toxicol (2012) 86:995–1007 1003

123

Impact of different particle size distributions

As a matter of principle, particle size has got a relevant

influence on the alveolar deposition rate that may influence

carcinogenic potency. The alveolar deposition rate can be

estimated through particle size distribution that is charac-

terized by MMAD and geometric standard deviation (GSD).

Some exemplary calculations were carried out to estimate

the impact of the different particle size distributions in the

studies included. These calculations using the multiple path

particle dosimetry model (MPPD) (RIVM 2002) showed that

the alveolar deposition rates for particles size distributions

with MMADs of 0.2, 1, and 4 lm lie at 15, 7, and 4 % (GSD

1.5 lm, MPPD standard parameters used for the rat, particle

density 2 g/cm3, and clearance included), respectively. The

different test concentrations and particle densities in the

range of the GBP studies did not have a relevant impact on

the calculated deposition rates obtained by MPPD. This

means that for the potency comparison of GBP nanomate-

rials to GBP micromaterials, a different alveolar deposition

rate does not have to be considered due to the small differ-

ences in particle size distributions. Remarkably, in the DEE

studies, the alveolar deposition rates can be estimated to have

been threefold higher than in the studies with GBP nanom-

aterials due to small particle size distributions (MMAD

range, 0.1–0.35 lm). Under the prerequisite that the depos-

ited dose is the relevant point of departure to assess carcin-

ogenic potency, a threefold lower carcinogenic potency for

DEE than described in the results would be to assume.

Specifics of the diesel engine emissions (DEE) studies

Total diesel particulate matter is consisting of elemental

carbon and additionally contains organic carbon (e.g., oils,

fuel, and incomplete combustion products) and inorganic

matter like, example, sulfates. Roughly, 50 % by mass

represent the elemental carbon core (Hebisch et al. 2003;

Mattenklott et al. 2002). On the basis that the particle is the

toxic principle, the potency estimates derived in the present

analysis could have been related to elemental carbon only

and not to total diesel particulate matter. Including this

dose metric transformation into the present analysis would

have had the consequence that the carcinogenic potency of

DEE would have increased by a factor of 2. This factor is

counteracted by a factor of a threefold higher alveolar

deposition rate, that is, threefold lower carcinogenic

potency for DEE. Taken together, no further correction to

estimate potency for DEE needs to be included as these

corrections would not have a relevant impact on the results.

In summary, the results show that there was not a factual

difference in carcinogenic potency between GBP nanom-

aterials and DEE supporting the hypothesis that the particle

is the toxic principle.

Comparison to similar analyses

A similar evaluation had been performed focussing on the

GBP material titanium dioxide (Dankovic et al. 2007). This

analysis came to the result that the carcinogenic potency of

nanosized titanium dioxide was one order of magnitude

higher than the carcinogenic potency for microsized tita-

nium dioxide. This result is in obvious discrepancy to the

results obtained in the present analysis that can be well

explained. Firstly, for the carcinogenic potency comparison

by Dankovic et al. (2007), only the data from the two tita-

nium dioxide studies were used and not for other GBP

materials. It has to be noted that the carcinogenicity study

with microsized titanium dioxide (Lee et al. 1985) showed a

rather low potency in comparison to other GBP microma-

terials like, for example, talc (see Fig. 1). Secondly, the

different study durations have not been corrected by

Dankovic et al. (2007). The study with microsized titanium

dioxide was terminated at 24 months, and the study with

nanosized titanium dioxide was terminated at 30 months

(Heinrich et al. 1995). When taking these parameters into

account the difference in carcinogenic potency in these two

studies accords to the conclusion drawn in the present

analysis, the potency differs only by a factor of 2–3. Overall,

it seems that the Lee et al. (1985) study showed a relatively

low carcinogenic potency for the GBP micromaterial tita-

nium dioxide. When comparing the results obtained with

talc in the NTP study (NTP 1993) to the study with nano-

sized titanium dioxide (Heinrich et al. 1995), a similar car-

cinogenic potency is derived for both materials although the

study duration till terminal section was shorter for talc

(26 months for males and 28 months for females, respec-

tively) than for nanosized titanium dioxide (30 months).

Relevance of the analyzed rat studies for species

extrapolation

Lung tumors induced by GBP materials have been

observed only in rat studies but neither in mice nor in

hamsters. Mice and hamsters are less prone than rats to

developing chronic inflammation and pulmonary fibrosis

(Oberdorster 1995). Lung tumors or fibrosis in the rats

was seen only at very high lung burdens. Thus, the lung

tumors observed in chronic rat studies at high particulate

exposure concentrations were hypothesized not be relevant

for human extrapolation to real-life exposure situations

(Valberg et al. 2009). Several other facts argue against this

hypothesis. On the basis of the studies available, it seems

that the hamster is an insensitive species with respect to

studying lung carcinogenicity due to negative results for

the great majority of carcinogens tested (Mauderly 1997).

The mouse showed negative results for crystalline silica

and equivocal results for DEE in chronic inhalation

1004 Arch Toxicol (2012) 86:995–1007

123

experiments. Crystalline silica (quartz) is classified as

human carcinogen by IARC; DEE were judged to show

sufficient evidence for animal carcinogenicity (category 2

A). The rat also quantitatively well mirrors the carcino-

genic potency of quartz that is a further indicator that the

rat is an adequate species to predict human particulate lung

carcinogenicity (Roller 2009). For DEE, the data indicate

that the rat is even less susceptible than humans (Kuempel

et al. 2010). From these data, it cannot be concluded that

the rat is an inadequate species for the assessment of dust

carcinogenicity.

The hypothesis was expressed that GBP material induced

lung carcinogenicity in the rat is only due to excessive par-

ticulate lung burdens, and the term ‘‘lung overload’’ was

generated to describe these conditions (Morrow 1988, 1992).

The theory is that in the particle-overloaded lung, an impair-

ment of alveolar macrophage-mediated lung clearance leads

to accumulation of high lung dusts burdens, which is associ-

ated with inflammation. Inflammation is thought to be rele-

vant as cause for tumor induction that becomes evident only in

overload conditions. In doses below overload, there should be

no inflammation and no additional tumor risk, that is, there is a

threshold for lung tumor induction. The issue of the overload

hypothesis is controversially discussed. The data can also be

interpreted in a way that there is no detectable threshold below

which lung clearance is not impaired. Already low amounts of

dust deposited in the terminal airways lead to an increase in

clearance half-life (Roller 2009). This could indicate that at

low and realistic exposure situations, inflammation is evident

and that tumor induction is not only relevant in the overload

condition that at least raises questions on a threshold-like

mechanism of GBP carcinogenicity.

Several subchronic inhalation studies are available, which

compared the inflammatory potency of GBP nanomaterials

to GBP micromaterials (e.g., (Oberdorster et al. 1994; Ferin

et al. 1992; Bermudez et al. 2002, 2004; Elder et al. 2005;

Hext et al. 2005). In these studies, it was evident that

inflammation caused by GBP nanomaterials seemed to be far

higher than inflammation caused by GBP micromaterials.

This was the main reason to assume an exceptionally high

toxicity of nanomaterials (Oberdorster et al. 1994). Inter-

estingly, the high inflammation response after GBP nano-

material inhalation or instillation in comparison to the far

lower inflammatory response caused by GBP micromaterials

is not be linearly correlated to the low carcinogenic potency

difference found in the rat inhalation carcinogenicity studies

in the present analysis.

Conclusions

With respect to regulatory consequences, the dose metrics

mass concentration is of special relevance as it is the

standard metrics that is analyzed in practice. The relative

potency factors based on cumulative particle mass resulted

in a 1.75- to 4.76-fold higher potency for GBP nanoma-

terials. A 5.06-fold higher potency was the maximum

factor obtained for the dose metrics cumulative lung vol-

ume burden. Thus, the relative carcinogenic potencies

comparing GBP nanomaterials and GBP micromaterials

indicate that factor 4 or 5 may be a reasonable worst case

estimate which includes a special emphasis on the dose

metrics mass concentration. In addition, the median study

duration to terminal section for the GBP nanomaterial

studies was 4 months longer than for GBP micromaterials.

As tumor incidence is age dependent, these different study

durations have to be taken into account by using a factor of

2. This means that the difference in relative carcinogenic

potency corrected for time to terminal section obtained in

the present analysis is not described best by a factor of 4 or

5 but by a factor of 2–2.5. This means that GBP nanom-

aterials would be 2- to 2.5-fold more potent carcinogens

than GBP micromaterials when referring to the dose met-

rics mass concentration.

Conflict of interest The author has no conflict of interests.

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