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Effects on fertility

Description of key information

There are various published non-guideline intratracheal instillation studies available in which specific reproductive toxicity endpoints were evaluated, usually at one time point or using a single dose level only. It is possible that some of the adverse effects observed in these studies may be the result of non-specific inflammatory effects caused by high exposure doses. The overall weight of evidence strongly indicates that carbon black should not be considered a reproductive toxicant.

Link to relevant study records

Referenceopen allclose all

Endpoint:
reproductive toxicity, other
Remarks:
sexual maturation and neurofunctional development
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
The study assessed whether maternal pulmonary exposure to Printex 90 affects sexual development and neurofunction in the prenatally exposed offspring. Time-mated mice were intr atracheally instilled with Printex 90 dispersed in Millipore water on gestation days (GD) 7, 10, 15 and 18, with total doses of 11, 54 and 268 lg Printex 90⁄ animal
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
other: C57BL/ 6BomTac
Sex:
female
Details on test animals and environmental conditions:
Source: Taconic Europe, Ejby, DK - Age at study initiation: not specified
Received as time-mated pregnant mice on gestation day 3 - Accl
imation period: tests were commenced on gestation day 8:
Route of administration:
intratracheal
Vehicle:
water
Details on mating procedure:
Time-mated nulliparous adult female mice were employed
Duration of treatment / exposure:
Instillations on day 7, 10, 15, 18
Frequency of treatment:
Single instillation per day
Dose / conc.:
11 other: µg/animal
Remarks:
2.7 μg/instillation administered over 4 instillations
Dose / conc.:
54 other: µg/animal
Remarks:
13.4 μg/instillation administered over 4 instillations
Dose / conc.:
268 other: µg/animal
Remarks:
67 μg/instillation administered over 4 instillations
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
After the last exposure on GD 18, the time-mated mice were housed alone and monitored for birth.
The expected day of delivery, GD 20, was assigned as post-natal day zero (PND 0) for the offspring.
At weaning, offspring were randomly distributed into balanced experimental groups with one female
and one male per litter (where possible): a group for collection of organs on PND 23, a group for sexual development data (all dose groups) and a group for behavioural testing (control and 268 μg Printex 90 ⁄ animal). Females and males were housed separately in cages of four
or five (extra animals were added when needed).
Litter observations:
Anogenital distance (AGD) was measured with a slide gauge in all offspring at weaning. Relative AGD was calculated (AGD⁄ cube root of body-weight). Results are reported as litter average separately for female and male offspring. The onset of puberty (vaginal opening in females and pre-putial separation in males) was recorded three times a week, between PND 26 and 40. The offspring were weighed on PND 32, 39 and 47.Behavioural testing was performed on PND 72–75, during the l
ight period with experimenters blinded to exposure status. The same observer was used within tests,
and exposed and control animals were tested alternately. Animals were transferred to the experimen
tal room 1 hr before the first test. Activity was assessed for 3 min in a circular (B = 1 m) Open field.
Total ambulation and ambulation in 1-min time bins (to test for habituation) were calculated. Duration
in each of the Open field zones (central and peripheral) and the number of zone crossings were ext
racted. Acoustic startle reaction (ASR) and pre-pulse inhibition (PPI) were tested. The offspring group
assigned to behavioural testing was weighed on PND 39, 53, 67 and 81
Statistics:
The accepted level of statistical significance was 0.05. Litter was considered the statistical unit, where
relevant. Data were analysed by analyses of variance (ANOVA), with treatment and sex as factors.
When relevant, analysis of repeated measures was applied (days of sampling in PND, trials and time
bins). Significant results from overall analyses were analysed by pairwise comparisons. Analyses
were performed in SYSTAT 9. Puberty start was analysed by log rank testin SAS 9.2.
Clinical signs:
not specified
Body weight and weight changes:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Increase in BAL inflammatory markers
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOEC
Effect level:
54 other: µg/animal
Based on:
test mat.
Sex:
female
Basis for effect level:
other: BAL inflammatory markers
Key result
Dose descriptor:
NOEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
268 other: µg/animal
System:
other: respiratory tract
Organ:
other: respiratory tract
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Neuropathological findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Key result
Dose descriptor:
NOEC
Generation:
F1
Effect level:
268 other: µg/animal
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: sexual maturation
Key result
Dose descriptor:
NOEC
Generation:
F1
Effect level:
54 other: µg/animal
Based on:
test mat.
Sex:
female
Basis for effect level:
developmental neurotoxicity
Key result
Dose descriptor:
NOEC
Generation:
F1
Effect level:
268 other: µg/animal
Based on:
test mat.
Sex:
male
Basis for effect level:
developmental neurotoxicity
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
268 other: µg/animal
System:
central nervous system
Organ:
other: behaviour
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At 268 µg/animal: Only in females, some habituation changes in the Open Field test were noted. These could have been secondary to maternal toxiciy as BAL inflammation markers were increased throughout the study at the highest dose level. No effects were noted at 54 µg/animal
Developmental immunotoxicity:
not specified
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
268 other: µg/animal (total dose)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Despite using very high intratracheal doses, gestational and litter parameters were normal for dams and offspring in a study evaluating the effects of carbon black on the sexual development and neurofunction of mice exposed in-utero to carbon black (NOEL, total dose: 54 µg/animal). The female offspring prenatally exposed to 268 µg/animal displayed altered habituation pattern during the Open Field test, which could have been a secondary effect to marked inflammation recorded at this dose
Executive summary:

Suspended and sonicated Printex 90 was intratracheally administered to ICR mice at total doses of 11, 54, and 268 µg/animal on gestation days 7, 10, 15 and 18. Despite using very high intratracheal doses, gestational and litter parameters were normal for dams and offspring. The female offspring prenatally exposed to 268 µg/animal displayed altered habituation pattern during the Open Field test, which could have been a secondary effect to marked inflammation recorded at this dose (NOEL, total dose: 54 µg/animal).

Endpoint:
reproductive toxicity, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Expsoure route adequate for testing "proof of concept" but not relevant for risk assessment and classification
Qualifier:
no guideline followed
Principles of method if other than guideline:
Time-mated CS7BLI6J mice were exposed by intratracheal instillation with Printex90. In-utero exposed F1 generation were mated with untreated mice of another strain (CBA/J) to produce the F2 generation. In adulthood, testicles were collected in F1 and F2 males for sperm production analysis. Body and testicle weight, sperm content per g testicular parenchyma and daily sperm production (DSP) as well as time-to delivery of the first F2 litters and litter size were assessed. DSP assesses the number of spermatids produced in the testicle per day and served as a measure of the male reproductive function. After homogenisation, counts from the two replicates per sample were averaged and served as basis for calculation of the number of homogenization resistant spermatids per g testicle tissue (sperm content per g testicular parenchyma, 5C/g). The total number of spermatids in the left testicle was then calculated by multiplication with the weight of the left testicle. DSP was then calculated by dividing the total number of elongated spermatids in the testicle with the time divisor value of 4.84. This number corresponds to the time (in days) which developing spermatids spend in stage 14-16 during spermatogenesis in the mouse.
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
other: CS7BLI6J
Sex:
female
Route of administration:
intratracheal
Vehicle:
water
Details on mating procedure:
P- generation: time mated animals were used. Exposed P-generation females from each group gave birth to a litter in the F1 generation.F-1 generation: At 13 weeks of age, F1 male (n=24) and F1 female (n=24) C57BL/6J offspring from control and exposed litters were mated with 10 weeks old naive CBA/J mice (Charles River Wiga, Sulzfeld, Germany), producing the F2 generation, Thus, one F1 C57BL/6J male per litter cohabited with one naive CBA/J female mice to produce F2 litters of the mixed (CBA/J)/(C57BL/6J) strain. Likewise, one F1 C57BL/6J female from each litter were cohabiting with a naive male CBM mice, producing the F2 generation of the mixed (C5761461)/(CBM) strain.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
P: on gestation days 7, 10, 15 and 18 F1: no treatmentF2: no treatment
Frequency of treatment:
P generation: 4 instillations between GD 7 and 18
Details on study schedule:
PARAMETERS MEASUREDOTHER PARAMETERS- Time to delivery of the first F2 litters as well litter sizes- Body weight: recorded on day of kill- Testicle weight: performed on same day as DSP analysis (after removal of adipose tissue)- Relative testicle weight: weight of left testicle/body weight
Dose / conc.:
268 other: µg/animal
Remarks:
(total of 4 instillations @ 67µg/ instillation)
No. of animals per sex per dose:
P: 15 females/dose
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
not reported
Statistics:
STATISTICS-Data were analysed by analysis of variance (ANOVA). Correlation analysis was used to determine Pearson correlation coefficient (Variation between two ha lves from one testicle, left and right testicle, correlation between sperm content and time-to-first F2 litter). In analysis of the effects of nanoparticle exposure in the F2 generation, two ways ANOVA was applied, with the factors of exposure (of the P generation) and gender (of the prenatally exposed F1 offspring). Time to delivery of first F2 litter in the CB study was assessed by log rank test. Data are given as mean +/- SEM. P-values <0.05 were considered statistically significant, 0.05 < P‹ 0.01 indicates a tendency to departure from the null-hypothesis. Statistical calculations were carried out using SYSTAT 9 (log rank test by SAS 9.2).
Clinical signs:
not specified
Key result
Dose descriptor:
LOEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Sex:
female
Basis for effect level:
other: increase of inflammatory markers in BAL (reported in a parallel publication of the same study group)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
268 other: µg/animal
System:
other: respiratory system
Organ:
other: respiratory tract
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Clinical signs:
not specified
Key result
Dose descriptor:
NOEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive function (sperm measures)
Key result
Critical effects observed:
no
Clinical signs:
not specified
Key result
Dose descriptor:
NOEC
Generation:
F1
Effect level:
268 other: µg/animal
Based on:
test mat.
Sex:
male
Basis for effect level:
other: body weight, reproductive parameters (testicle weight, relative testes weight, SC/g and DSP).
Key result
Critical effects observed:
no
Clinical signs:
not specified
Key result
Dose descriptor:
LOEC
Generation:
F2
Effect level:
268 other: µg/animal
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reduced sperm production
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
268 other: µg/animal
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
268 other: µg/animal
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Table 1.

F1
in-utero exposed males mice

F2
males whose mothers were prenatally exposed (C57BL/6J/CBA/J)

F2
(males whose fathers were prenatally exposed (CBA/J/C57BL/6J)

Control

Exposed

Control

Exposed

Control

Exposed

Age (days)

185 ± 20

181 ± 24

80 ± 0

80 ± 0

80 ± 0

80 ± 0

Group size

15

14

14

10

9

12

Body weight (g)

29.1 ± 1.5

29.8 ± 0.4

28.8 ±0.6

28.4 ± 0.1

30.1 ± 1

30.3 ± 0.9

Testicle weight (g)

94.0 ± 2.8

99.0 ± 2.1

78.9 ± 2.5

82.4 ± 3.0

98.7 ± 2.9

97.3 ± 2.2

Relative testes weight (%)

3.2 ± 0.2

3.4 ± 0.1

2.7 ± 0.1

2.8 ± 0.1

3.3 ± 0.1

3.2 ± 0.1

SC/g (x 107)

34.5 ± 1.2

35.9 ± 1.2

25.3 ± 0.8

25.5 ± 0.5

31.4 ± 1.6#, *

27.5 ± 1.1*

DSP/g (x 109)

6.8 ± 0.4

7.4 ± 0.3

4.2 ± 0.2

4.3 ± 0.2

6.5 ± 0.5*

5.5 ± 0.2*

SC/g: sperm content per g testicular parenchyma; DSP: daily sperm production

* P< 0.05 compared to C57BL/6J/CBA/J offspring

# P< 0.05 compared to F2 control offspring

Conclusions:
The study was designed to examine effects on the male reproductive system of maternal airway exposure to particulates during pregnancy. Pregnant mice were exposed to a very high intratracheal dose (268 µg/animal, divided in four instillations on gd 7, 10, 15 and 18). No effects were found in F1 animals. F2 offspring, whose fathers were exposed in utero to carbon black showed statistically significant, but numerically modest lower sperm production. The biological significance of this finding is doubtful. No effects were found on body weights, and testicular weights nor were there any effects in the F1 generation.
Executive summary:

In-utero exposed CB57BL/6J mice, challenged via intratracheal instillation to a total dose of 268 µg/CB over the gestation period of 7 -18 days (instillation on GD 7, 10, 15, 18; 67µg/instillation) did not exhibit any conspicuous effects of the reproduction parameters assessed. Values for testicular weight, relative testicular weight as well as sperm content per gram testicular tissue and daily sperm production were unspectacular in comparison to the concurrent controls. Male offspring crossbred from in-utero exposed C57BL/6J females and naïve CBA/J males displayed unspectacular findings. In contrast, the male progeny (CBA/J/C57BL/6J) of in-utero treated males and untreated CBA/J females displayed slightly reduced daily sperm production in comparison with matched controls. Post hoc, sperm content per gram testis was also reduced.

The investigators applied a non-physiological route of application. In addition, the single dose regimen used in the study precludes any assessment of dose-response relationships. The authors do not present any data on historical controls. Consequently, discussion of the positive findings within the context of historical controls is not possible. This precludes concluding on the biological relevance of the findings reported by Kyjovska and co-workers. This is especially important because the change reported for sperm content, although statistically significant, is numerically modest. Demonstration of statistical significance is not necessarily denotative of biological relevance.

A sustained change (increase/decrease) over multiple evaluation time points could be indicative that observed effect may have biological significance. However, F2 males were analysed at only one time point as young adults (postnatal day 80). This rather limiting aspect in the study design hinders a precise and conclusive determination whether the reported effect is aleatoric or has true biological relevance. The above points limit the present study for use in risk assessment and classification.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
42 mg/m³
Study duration:
subacute
Species:
mouse
Quality of whole database:
NOAEC based on dose conversion made by the study authors of an intratracheal study where at a total dose of 268 µg carbon black/animal no effects on sexual maturation and gestational parameters were found
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for study waiver:

(i)                Carbon black is of low toxicological activity

 Several published studies investigating reproductive parameters are available, all performed in mice and all using very high intratracheally administered doses causing pulmonary inflammation. Even under exaggerated exposure conditions (total carbon black dose: 268 µg/animal), no effects of carbon black were found on sexual development (characterized as anogenital distance in weanlings and as the onset of puberty) (Jackson, Vogel et al. 2011). In-utero exposed mice, did not exhibit adverse effects on the reproduction parameters assessed after intratracheal instillation of a total dose of 267 µg/animal carbon black over the gestation period of 7 -18 days (instillation on GD 7, 10, 15, 18; 67 µg/instillation) (Kyjovska, Boisen et al. 2013; Boisen et al., 2013). The male progeny of in-utero treated males and untreated females displayed marginally reduced daily sperm production in comparison with matched controls.Values for testicular weight, relative testicular weight as well as sperm content per gram testicular tissue and daily sperm production were similar to controls. The dose level employed for the parental generation was 268 µg/animal(Kyjovska, Boisen et al. 2013). In another study, male mice were intratracheally instilled with carbon black (different groups received Printex 90, Printex 25 and Flammruss 101 at a dose of 100 µg given 10 times at weekly intervals (resulting in a total dose of 1000 µg)(Yoshida, Hiyoshi et al. 2009). Observed effects included increased testosterone with exposure to Printex 90 and Printex 25, changes in seminiferous tubules and decreased daily sperm production with all three carbon blacks. The dose used was very high (totaling 1000 µg), therefore it was not possible to determine the relevance of these effects at more reasonable doses or if there is a dose-response for this reported effect. In a subsequent study, pregnant mice were intratracheally instilled with carbon black at 200 µg/mouse on GD 7 and 14, resulting in a total dose of 400 µg(Yoshida, Hiyoshi et al. 2010). In this study, male offspring were evaluated at 5, 10 and 15 weeks after birth. They showed histological changes in the testes, as well as decreases in daily sperm production. There were no changes in body weight, testicle weight, epididymis weight or serum testosterone levels in male offspring.

The employed dose levels in these studies were extremely high and by far exceeded doses typically employed (40 µg/mouse) and considered representative of worker exposures at average facilities(Roberts, Mercer et al. 2016). At a carbon black (Printex 90) dose of 40 µg/mouse, Robertset al. found no or minimally increased indices oflung inflammation and injury. The marginal effects found at extremely high intratracheal doses demonstrate the low toxicological activity of carbon black. Based on the weight of the available in vivo evidence, no toxicological activity of carbon black is expected under conditions that do not induce inflammatory responses.

 

(ii)             Toxicokinetic considerations

Toxicokinetic studies on carbon black and similar carbonaceous materials indicate that industrially-produced carbon black is unlikely to be absorbed or distributed in the body. Therefore, carbon black is unlikely to reach reproductive organs and tissues and have a direct effect on reproductive functions or the developing organism. 

 

(iii)           No significant human exposure

Occupational exposure controls are in place. In consumer products carbon black is tightly bound in a matrix with no release of carbon black nanoparticles. 

Effects on developmental toxicity

Description of key information

There are various published non-guideline tracheal instillation studies and one inhalation study in mice in which specific developmental toxicity endpoints were evaluated, usually at one time point or using a single dose level only. It is possible that some of the effects observed in these studies may be the result of non-specific inflammatory effects caused by high exposure doses. The EU Scientific Committee on Consumer Safety (SCCS) assessed an oral gavage study conducted in accordance with the standard OECD 414 test guideline for developmental toxicity and reported no adverse maternal changes and no effects on embryo-fetal development at the highest dose tested. The overall weight of evidence strongly indicates that carbon black should not be considered a developmental toxicant.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Route of exposure unphysiological - results of limited use for risk assessment and classification
Qualifier:
no guideline available
Principles of method if other than guideline:
The study assessed whether maternal pulmonary exposure to Printex 90 affects sexual development and neurofunction in the prenatally exposed offspring. Time-mated mice were intratracheally instilled with Printex 90 dispersed in Millipore water on gestation days (GD) 7, 10, 15 and 18, with total doses of 11, 54 and 268 lg Printex 90⁄ animal.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Declared Particle size: 14 nm (Degussa Hüls)- Geometric mean size: 65 nm- Morphology: Individual carbon black spheres mainly occurred in open-structured long chain aggregates and fewer large dense aggregates- Surface area: 295 -338 m2/g- Pycnometric particle density: 2.1 g/cm3- Chemical composition 99% C, 0.8% N and 0.01% H2- The total PAH content (Carbon black extract – Soxhlet): 0.0742 µg/g- The total PAH content (DEP extract – NIST SRM 1650): 216 µg
Species:
mouse
Strain:
other: C57BL/ 6BomTac
Details on test animals and environmental conditions:
TEST ANIMALS - Source: Taconic Europe, Ejby, DK - Age at study initiation: not specified - Weight at study initiation: yes; see table 4- Received as time-mated pregnant mice on gestation day 3 - Acclimation period: tests were commenced on gestation day 8:
Route of administration:
intratracheal
Vehicle:
water
Remarks:
g-irradiated Nanopure Diamond UV water (Pyrogens: < 0.001 EU/ml, Total Organic Carbon: < 3.0 ppb),
Details on exposure:
The particle preparation and instillation procedures were described previously. Printex 90 was sonicated for 8 min (10 s pulses and 10 s pauses, total sonication time 4 min) at a concentration of 1.675 mg/mL (67 mg/instillation) in 0.2 mm filtered, g-irradiated Nanopure Diamond UV water (Pyrogens: < 0.001 EU/ml, Total Organic Carbon: < 3.0 ppb), using a 400 W Branson Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT, USA) mounted with a disruptor horn and operated at 10% amplitude. This dispersion was used for the high dose and diluted 1:5 for the medium dose (13.4 mg/ instillation) and diluted futher 1:5 for the low dose (2.7 mg/instillation). Eighty time-mated mice were anesthetized with 3% Isoflurane and instilled with a vehicle or one of the three concentrations of Printex 90 dispersions (40 mL solution followed by 160 mL air) on GD 7, 10, 15 and 18. We chose to instill Printex 90 at times that would cover the major part of the fetal development. We tried to distribute the dose over that period assuming that a fraction of the particles would have been cleared rapidly, but that much of the dose would remain in the lungs for several weeks. Exposure took place between 08:30 and 14:30 h. Time-mated mice were instilled in different order each day, to reduce any variation that might be related to the time of exposure. The total instilled doses were 11, 54 and 268 mg/animal.
Analytical verification of doses or concentrations:
no
Remarks:
not applicable
Details on analytical verification of doses or concentrations:
The particle size distribution in the Printex 90 dispersions was determined with a 633 nm He- Ne Dynamic Laser Scatter (DLS) Zetasizer nano ZS (Malvern Inc., UK).The dispersion of Printex 90 instillation fluid was also analyzed by Scanning Electron Microscopy (QUANTA 200 FEG MKII with EDX).The particle size distribution was similar in the three instilled dispersions with concentrations of 1675, 335 and 67 mg/mL (see Figure 3), and was stable for more than 1 h. The average zeta-size was approximately 140 nm and the hydrodynamic number size-distributions had a peak size between 50 and 60 nm When converted to volume distributions, minor amounts of mm-size particles and two smaller size modes with peak sizes around 50–60 nm and 200–400 nm were identified. The observed DLS sizes were confirmed by TEM and SEM, with a wide size distribution of nm- to mm-size free and agglomerated particles. The agglomerates consisted of spherical to sub-spherical carbonaceous particles as well as minor amounts of free single primary spheres.
Details on mating procedure:
no data. Time-mated nulliparous adult female mice were employed.
Duration of treatment / exposure:
Instillations on day 7, 10, 15, 18
Frequency of treatment:
single instillation per day
Duration of test:
no data
Dose / conc.:
11 other: µg/animal
Remarks:
2.7 µg/instillation administered over 4 instillations
Dose / conc.:
54 other: µg/animal
Remarks:
13.4 µg/instillation administered over 4 instillations
Dose / conc.:
268 other: µg/animal
Remarks:
67 µg/instillation administered over 4 instillations
No. of animals per sex per dose:
Control: 2411& 54 µg/animal: 17268 µg/animal: 22
Control animals:
yes, concurrent vehicle
Details on study design:
After the last exposure on GD 18, the time-mated mice were housed alone and monitored for birth. The expected day of delivery, GD 20, was assigned as post-natal day zero (PND 0) for the offspring. At weaning, offspring were randomly distributed into balanced experimental groups with one female and one male per litter (where possible): a group for collection of organs on PND 23 (in Jackson et al 2012b), a group for sexual development data (all dose groups) and a group for behavioural testing (control and 268 µg Printex 90 ⁄ animal). Females and males were housed separately in cages of four or five (extra animals were added when needed).
Maternal examinations:
Maternal inflammation was assessed by monitoring neutrophil influx in the lungs.
Fetal examinations:
Anogenital distance (AGD) was measured with a slide gauge in all offspring at weaning. Relative AGD was calculated (AGD⁄ cube root of body-weight). Results are reported as litter average separately for female and male offspring. The onset of puberty (vaginal opening in females and pre-putial separation in males) was recorded three times a week, between PND 26 and 40. The offspring were weighed on PND 32, 39 and 47.Behavioural testing was performed on PND 72–75, during the light period with experimenters blinded to exposure status. The same observer was used within tests, and exposed and control animals were tested alternately. Animals were transferred to the experimental room 1 hr before the first test. Activity was assessed for 3 min in a circular (B = 1 m) Open field. Total ambulation and ambulation in 1-min time bins (to test for habituation) were calculated. Duration in each of the Open field zones (central and peripheral) and the number of zone crossings were extracted. Acoustic startle reaction (ASR) and pre-pulse inhibition (PPI) were tested. The offspring group assigned to behavioural testing was weighed on PND 39, 53, 67 and 81.
Statistics:
The accepted level of statistical significance was 0.05. Litter was considered the statistical unit, where relevant. Data were analysed by analyses of variance (ANOVA), with treatment and sex as factors. When relevant, analysis of repeated measures was applied (days of sampling in PND, trials and time bins). Significant results from overall analyses were analysed by pairwise comparisons. Analyses were performed in SYSTAT 9. Puberty start was analysed by log rank testin SAS 9.2.
Historical control data:
no data
Clinical signs:
not specified
Details on results:
Persistent lung inflammation was induced in the exposed time-mated mice (the number of neutrophil cells in bronchoalveolar lavage fluid was increased 28-fold 3 days after exposure and 61-fold 26 days after exposure
Details on maternal toxic effects:
Details on developmental maternal toxicity are described in Jackson et al 2012b. Briefly, no effects on gestation, lactation were observed.
Key result
Dose descriptor:
NOAEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Basis for effect level:
other: Gestational and post gestational parameters
Key result
Dose descriptor:
LOAEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Basis for effect level:
other: Persistent lung inflammation
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: respiratory system
Description (incidence and severity):
persistent increase of inflammatory markers in BAL
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Weight gain after weaning was similar in exposed offspring and their controls, both in the groups observed for sexual maturation and in the group designated for behavioural testing
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Changes in postnatal survival:
no effects observed
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Description (incidence and severity):
Relative AGD measured on all offspring at weaning did not differ between offspring prenatally exposed to Printex 90 and their controls. Overall analysis of onset of puberty (vaginal opening) in females indicated statistically significant differences between groups. Female offspring prenatally exposed to 11 lg Printex 90⁄ animal entered puberty significantly earlier (time of vaginal opening, p = 0.01) compared with controls, while higher-dose groups compared with thecontrols. As data did not indicate dose response this may be a chance finding. Puberty start in the male offspring (time of pre-putial separation) started at similar time in the exposed groups and in the controls.The basal acoustic startle response was of similar magnitude in exposed and control offspring, and the offspring reacted similarly to PPI. For locomotor activity investigated in the Open field, the overall statistical analysis indicated no difference in the total distance moved during the 3-min observation. However, analysis of offspring habituation in minute intervals as repeated measure in the ANOVA indicated that the pattern differed between groups. The females moved differently during the first 2 min, compared with their control; total movement during the first minute was reduced compared with that of control (p = 0.03); and total movement during the second minute was increased compared with that of control (p = 0.03). Thus, the female offspring prenatally exposed to 268 µg Printex 90⁄ animal displayed a different pattern of habituation. There were no differences in Open field activity between the exposed and control male offspring
Key result
Dose descriptor:
NOEC
Effect level:
54 other: µg/animal
Based on:
test mat.
Sex:
female
Basis for effect level:
other: changed habituation pattern
Key result
Dose descriptor:
NOEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Sex:
male
Basis for effect level:
other: neurobehavioural change
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
268 other: µg/animal
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Despite using very high intratracheal doses, gestational and litter parameters were normal for dams and offspring in a study evaluating the effects of carbon black on the sexual development and neurofunction of mice exposed in-utero to carbon black up to a total dose of 54 µg//animal. The female offspring prenatally exposed to 268 µg/animal displayed altered habituation pattern during the Open Field test.
Executive summary:

Time-mated mice were intratracheally instilled with Printex 90 dispersed in Millipore water on gestation days 7, 10, 15 and 18, with total doses of 11, 54 and 268 µg Printex 90/animal. The female offspring prenatally exposed tgo 268 µg/animal displayed altered habituation pattern during the Open Field test.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The study comprised of two parts: An inhalation study and an instillation dose-effect study. Only results of the inhalation part of the study are entered in this study record.The study assessed the effect of maternal gestational exposure of time-mated C57BL/6BomTac mice to ca. 42 mg/m3 Printex 90 for 1 h/day for 11 consecutive gestational days ((GD) 8–18), on the development of the offspring. Shortly before the expected date of delivery, the mother is sacrificed, the uterus removed, and the contents examined for deaths, and live foetuses. Effects on gestation, lactation and DNA strand breaks were evaluated. Traditional gestational and litter parameters were measured. Inflammation was assessed in maternal bronchoalveolar lavage (BAL) after exposure, and at weaning. Levels of DNA strand breaks were assessed in maternal BAL cells and liver, and in offspring liver. Dams were monitored until weaning and some offspring until adolescence.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Printex 90
- Geometric mean size: 65 nm- Morphology: mainly occurred in open-structured long chain aggregates and fewer large dense aggregates- Surface area: 295 -338 m2/g- Pycnometric particle density: 2.1 g/cm3- Chemical composition 99% C, 0.8% N and 0.01% H2- The total PAH content (Carbon black extract – Soxhlet): 0.0742 µg/g- The total PAH content (DEP extract – NIST SRM 1650): 216 µg
Species:
mouse
Strain:
other: C57BL/ 6BomTac
Details on test animals and environmental conditions:
TEST ANIMALS - Source: Taconic Europe, Ejby, DK - Age at study initiation: not specified - Weight at study initiation: yes; see table 4- Received as time-mated pregnant mice on gestation day 3 - Acclimation period: tests were commenced on gestation day 8:
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Remarks on MMAD:
Average size by mass: 310 nm; the mass size distribution was bimodal with one mode around 290 nm and a coarser mode at ca. 1.5 mm
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION - Exposure apparatus: steel-framed pyrex glass exposure chamber - Air flow rate: 20 L/min, 5 bars - Method of particle size determination: The particle concentration-size-distribution was monitored on-line using a GRIMM Sequential (Stepping) Mobility Particle Sizer (Model No. 5.521) connected to a Condensation Particle Counter system (SMPS+C) and a GRIMM Dust Monitor (Model 1.105) for small (9.8–492.2 nm) and coarse particles (0.75–1.00 to >15 mm), respectively.TEST ATMOSPHERE - Brief description of analytical method used: Total aerosolized Printex 90 was sampled periodically from the exposure chamber using Millipore cassettes mounted with Millipore Fluoropore Filters (diameter 2.5 cm, pore size 0.45 mm). Filters were weighed immediately on a Sartorius Microscale (Type M3P). If needed, the airborne mass concentration was adjusted after the control measurement to the target concentration of 40 mg/m3. - Samples taken from breathing zone: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The particle number concentration in the exposure atmosphere was 4.09 ± 0.03 x 10(6)/cm3. The average particle size distribution was multimodal and highly dominated by sub-100 nm particles. The most abundant size number was in the order of 41 nm, which was also the average size. The average size by mass was 310 nm, and the mass size distribution was bimodal with one mode around 290 nm and a coarser mode at ca. 1.5 mm. Only 5% of the mass was below 100 nm, 83% of the particles were in this ultrafine size range by number.The authors modelled the deposition of the test substance in the pulmonary region of the treated animals. 34.8% of the particle mass was expected to deposit in the pulmonary region and 20.1% of particles were expected to deposit in the extra-pulmonary region (11.9% bronchial region, 0.9% trachea, 0.6% larynx, and 6.7% scull). An additional 16.6% was expected to deposit in the gastrointestinal tract. The total inhaled dose was 826 µg Printex 90 (1 h/day x 11 days x 41.7 mg/dm3 x 1.8 dm3/h). Based on deposition estimates, the pulmonary dose would be 287 µg/animal; or 13.1 mg/kg, based on an average body weight of 22 g on GD 4 (287 µg/22 g). The corresponding inhaled particle surface area was at least 0.085 m2/animal (295 m2/g x 0.000287g/animal); or 3.9 m2/kg (0.085 m2/0.022 kg) equal to 243 m2/kg lung (0.085 m2/0.00035 kg lung).
Details on mating procedure:
no data. Time-mated nulliparous adult female mice were employed.
Duration of treatment / exposure:
11 days (8- 18 days of gestation)
Frequency of treatment:
1h/day
Duration of test:
no data
Dose / conc.:
41.7 mg/m³ air (analytical)
Dose / conc.:
826 other: µg/animal
Remarks:
estimated by authors using own deposition model. Value depicts total amount estimated to be ingested via the lungs and orally after inhalation exposure
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
After the last exposure on GD 18, the time-mated mice were housed alone and monitored for birth. The expected day of delivery, GD 20, was assigned as post-natal day zero (PND 0) for the offspring. On PND 1, the offspring were counted and sex determined.
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data DETAILED CLINICAL OBSERVATIONS: No data BODY WEIGHT: Yes - Time schedule for examinations: GD 4, GD 7, and GD 10, 13, 15 (or 16), 19 and post natal days (PND) 2, 8, 12, 17 and 22 (weaning) POST-MORTEM EXAMINATIONS: Yes - Sacrifice on PND 3 (5 days after the last inhalation exposure) and at PND 22-23 (at weaning) - Organs examined: Liver and lungs were weighed, prepared and stored until analysis. Relative organ weight was calculated as (organ weight/bodyweight)*100. OTHER: - After sacrifice, Bronchoalveolar lavage (BAL) fluid from each female was collected.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes Examinations included: - Number of implantations: Yes
Fetal examinations:
Offspring:- On PND 1, the offspring were counted and sex determined.- On PND 2, all except two male and two female offspring in each litter were removed and killed by decapitation. Liver and lungs of newborns were weighted, dissected and stored at -80°C until analysis.- On PND 22, male and female offspring were randomly distributed into balanced experimental groups: A group for collection of organs at weaning PND 22–23, an adolescent group for maturation data and organs at PND 50, and a group for behavioural testing and mating for a 2nd generation (published elsewhere). In the inhalation study all dissected, organs were weighed. This included lungs, liver, kidneys, spleen, heart and brain from the females, dams, offspring at weaning and adolescents. Relative organ weight was calculated as (organ weight/bodyweight)*100.
Statistics:
DATA ANALYSESThe accepted level of statistical significance was 0.05. Litter was considered the statistical unit. Gestational parameters were analyzed by Kruskal-Wallis One-Way Analysis of Variance. Weight data were analyzed by analyses of variance (ANOVA), with treatment as factor, and day of weighing (GD, PND) as repeated measure. Litter size was used as co-variable for weight data during gestation. The number of litters was compared by Fisher’s exact test. Remaining data (BAL results, comet assay results, organ weights) were analyzed by analyses of variance (ANOVA), with treatment, day of sampling (PND) and sex (where relevant) as factors. Significant results from overall analyses were analyzed by pair wise comparisons. Data were analyzed separately for each day of sampling and instillation study results were further analyzed by dose in Fisher’s Least-Significant-Difference Test. Females sampled 3–5 days after exposure and dams sampled after weaning were compared, even though the groups differed by timing of sampling and also by pregnancy status. The “female” group consisted of pregnant mice with small litters and non-pregnant mice. Pregnancy is reported to alter the level of inflammatory response (Fedulov et al. 2008; Lamoureux et al. 2010), thus different background levels had to be accepted. Analyses were performed on SYSTAT Software Package version 9 and Statistical Tables for PC users.
Historical control data:
no data
Clinical signs:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
• 5 days post exposure: increased relative brain weight compared to controls (exposed 2.09 ± 0.04%vs. control 1.86 ± 0.06%, p=0.005)• 24 days post exposure: relative lung weight was higher in exposed compared to control dams (exposed 1.20 ± 0.03% vs. control 1.10 ± 0.02%, p = 0.005). Other organs did not differ.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Implantations: 7.59±0.54 in treatment group versus 7.39±0.56 in controlsImplantation loss (%) 17.01± 4.57 in treatment group versus 14.59±3.88 in controls
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Gestation length (days)- Control: 19.89±0.07- Treatment group: 20.06±0.06
Changes in number of pregnant:
not examined
Description (incidence and severity):
A. DNA Strand Breaks: - BAL: the level of DNA strand breaks in BAL fluid cells 5 and 24 days after exposure were comparable to controls (p=0.2)- Liver: Exposure induced higher levels of DNA strand breaks in the liver 5 and 24 days after exposure in dams compared to their controls (5 days: 1.3-fold increase, p = 0.04; 24 days: 1.6-fold increase, p < 0.001).B. Lung InflammationAnalysis of BAL fluid cell composition by differential cell count indicated the presence of inflammation in the lungs of dams as evidenced in increased levels of neutrophils, lymphocytes and total cell count1) Neutrophils• 5 Days post exposure: increased with statistical significance compared to controls by 11.4-fold (11.47 ± 3.06 vs 1.01 ± 0.61 in controls; p<0.01)• 24 days post exposure: exposure: increased with statistical significance compared to controls by 11.7 fold (4.82 ± 1.07 vs. 0.41 ± 0.07; p<0.001)2). Lymphocytes:• 5 Days post exposure: increased with statistical significance compared to controls by 3.4-fold (2.59 ± 0.63 vs 0.76 ± 0.18 in controls; p=0.02)3). Total count:• 5 Days post exposure: increased with statistical significance compared to controls by 1.5 -fold (96.88 ± 10.72 vs 66.07 ± 6.03 in controls; p=0.032):• 24 Days post exposure: increased without statistical significance compared to controls by 1.2 -fold (54.81 ± 4.26 vs 44.08 ± 3.37 in controls; p=0.057)
Key result
Dose descriptor:
LOAEC
Effect level:
41.7 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: inflammatory markers in BAL increased; persisted until the end of lactation (weaning) as evidenced in the increase in the number of neutrophils and lymphocytes
Key result
Dose descriptor:
LOAEC
Effect level:
41.7 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: DNA strang breaks observed in liver of pregnant dams @ 5 and 24 days samplings
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Lungs:
Description (incidence and severity):
increased and sustained inflammation over the entire gestation period and upto weaning aas evidenced in increased influx of neutrophils (ca. 11.5 fold over controls) and lymphocyte (ca.3 fold over controls) into the lungs. Total lung count was increased upto 1.5 fold over controls.
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Weight gain females PND 1(2)–22 (g) - Control 8.02 ± 0.23- Treatment group: 7.66 ± 0.32Weight gain males PND 1(2)–22 (g) - Control: 8.28 ± 0.18- Treatment group: 7.89 ± 0.38
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Live pups per litter on post natal day Control: 1: 5.09±0.91 Treatment group: 4.95±0.74
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio (females in litter (%)Control: 0.42 ± 0.06 Treatment group: 0.51 ± 0.06
Changes in litter size and weights:
not specified
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Offspring dead during lactation (%) - Control: 4.10 ± 1.98 - Treatment group: 1.72 ± 1.19
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Description (incidence and severity):
DNA Strang breaks: In the offspring, the level of DNA strand breaks was higher in offspring liver at weaning and in adolescents, compared to their controls (weaning: 1.4-fold increase, p = 0.001; adolescents: 1.5-fold increase, p = 0.011). Overall, newborns displayed higher levels of DNA strand breaks in liver tissues compared to tissues from the older offspring at weaning and from adolescents, both in the Printex 90 and the control group (p < 0.001).
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
LOEC
Effect level:
41.7 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: DNA strand breaks observed in liver of offspring
Key result
Dose descriptor:
NOEC
Effect level:
41.7 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: LUNG INFLAMMATION Table of BAL cell composition of females or dams exposed to Printex 90 by inhalation and control mice.

  • Table of. BAL cell composition of females or dams exposed to Printex 90 by inhalation and control mice.

     

     

     

    Control

    Printex 90
    41.7 mg/m3 (ca. 287 µg/animal deposited in lungs

    Females 3–5 days after exposure

    Total count

    66.07 ± 6.03

    96.88 ± 10.72*

    Dead cells

    1.08 ± 0.36

    0. 79 ± 0.32

    Macrophages

    56.35 ± 4.76

    69. 21 ± 6.74

    Neutrophils

    1.01 ± 0.61

    11.47 ± 3.06**

    Lymphocytes

    0.76 ± 0.18

    2.59 ± 0.63*

    Eosinophils

    0.46 ± 0.31

    3.97 ± 2.58

    Epithelial cells

    7.50 ± 1.28

    9.63 ± 1.61

    Dams at weaning 24-27 days after exposure

    Total count

    44.08 ± 3.37

    54. 81 ± 4.26(*)

    Dead cells

    0.68 ± 0.10

    0.45 ± 0.11

    Macrophages

    31.90 ± 3.25

    37.73 ± 3.20

    Neutrophils

    0.41 ± 0.07

    4.82 ± 1.07***

    Lymphocytes

    1.35 ± 0.34

    2.02 ± 0.50

    Eosinophils

    0.37 ± 0.29

    0.67 ± 0.47

    Epithelial cells

    10.03 ± 0.85

    9.56 ± 0.80

    BAL, bronchoalveolar lavage. Data presented as mean cell number_103in BAL±SEM. (*)p~0.05, *p<0.05, **p<0.01, ***p<0.001.

Table 2: DNA STRANG BREAKS; DAMS: Level of DNA strand breaks of dams exposed to Printex 90 by inhalation and control mice.Level of DNA strand breaks of dams exposed to Printex 90 by inhalation and control mice.

  • L evel of DNA strand breaks of dams exposed to Printex 90 by inhalation and control mice.

     

     

    Control

    Printex 90

    41.7 mg/m3ca. 287µg/animal (deposited in lungs)

    3-5 days after exposure

    BAL

    0.55 ± 0.06

    0.07± 0.01

    Liver

    2.57 ± 0.19

    3.25 ± 0.21*

    At weaning 24-27 days after exposure

    BAL

    0.58 ± 0.05

    0.60 ±0.04

    Liver

    1.24 ± 0.07

    1.94 ± 0.11***

    BAL, bronchoalveolar lavage; Data are presented as mean number of lesions per 106base pairs±SEM (calculated from %DNA results). *p<0.05, **p<0.01, ***p<0.001.

Table 3 : DNA STRANG BREAKS; OFFSPRING:  Level of DNA strand breaks liver of offspring exposed in utero to Printex 90 by inhalation and control mice. T

  • Level of DNA strand breaks liver of offspring exposed in utero to Printex 90 by inhalation and control mice.

     

    Control

    Printex 90

    41.7 mg/m3ca. 287µg/animal (deposited in lungs)

    Newborns PND 2

    3.5 ± 0.28

    3.64 ± 0.37

    at weaning PND 22–23

    1.13 ±0.09

    1.56 ± 0.08 ***

    adolescents PND 50 (47)

    1.10 ± 0.16

    1.69 ±0.14 **

    PND, post natal day (days after birth). Offspring data are calculated as litter average, when sibling liver tissues were analyzed at the same collection point. Data are presented as mean number of lesions per 106base pairs ± SEM (calculated from %DNA results). *p < 0.05, **p < 0.01, ***p < 0.001.

Table 4: MATERNAL AND LITTER PARAMETERS

  • Table of gestation, lactation and developmental parameters of dams and offspring exposed to Printex 90 by inhalation and control mice.

     

    Control

    Printex 90, 41.7 mg/m3

    ~ 287 µg/animal

    Time mated/exposure groups

    22

    22

    Dam arrival weight, GD 4 (g)

    22.21±1.47

    21.82±1.27

    Number of litters PND 1

    18

    17

    Dam weight gain, GD 718 (g)

    11.25±0.58

    10.64±0.71

    Dam lactation weoght gain PND 1(2)-17 (g)

    4.31±0.27

    4.34±0.41

    Gestation length (days)

    19.89±0.07

    20.06±0.06

    Implantations

    7.39±0.56

    7.59±0.54

    Implantation loss (%)

    14.59±3.88

    17.01±4.57

    Live pups per litter PND 1

    5.09±0.91

    4.95±0.74

    Offspring dead during lactation (%)

    4.10±1.98

    1.72±1.19

    Birth weight females (g)

    1.40±0.04

    1.41±0.05

    Birth weight males (g)

    1.43±0.03

    1.42±0.05

    Weight gain females PND 1(2)22 (g)

    8.02±0.23

    7.66±0.32

    Weight gain males PND 1(2)22 (g)

    8.28±0.18

    7.89±0.38

    Sex ratio(females in litter (%)

    0.42±0.06

    0.51±0.06

    GD, gestation day (pregnancy day); PND, post natal day (days after birth). Dams were allowed to deliver their offspring on gestation day (GD) 20, equal to post natal day (PND) 0. Weights of dams and individual offspring were recorded on PND 2 (1), and offspring were counted and sex determined. Time mated mice were examined for the number of implantation sites, allowing for calculation of implantation loss. Females that did not give birth or had small litters were killed on PND 3 (12) and the dams on PND 2223 (2425). Data are expressed as mean±SEM, offspring data are calculated as litter average.Weight before exposure.

Conclusions:
Pregnant mice administered Printex 90 by inhalation (42 mg/m3; whole body) from day 8 through day 18 of gestation showed an increase in neutrophils in BAL fluid 5 and 24 days after exposure. No gestational or developmental toxicity in the offspring was observed.
Executive summary:

In an inhalation study, carbon black was administered to 22 time-mated pregnant C57BL/ 6/BomTac mice at a single concentration of ca. 41.7 mg/m3 from days 8 through 18 of gestation. Twenty-two control animals were exposed concomitantly to air. Traditional gestational and post-gestational parameters were assessed in dams and their offspring. In addition, the investigators sought to answer the question whether inhalation exposure to carbon black caused DNA damage in lung (BAL) and liver cells of exposed dams as well as in the liver of their offspring. Lung inflammation in dams was also monitored. Samples for analysis of DNA damage and lung inflammation were collected 5 and 24 days after cessation of exposure in dams and on post-natal days 2, 22-23 and 50 for DNA analysis in offspring.

Gestational and litter parameters in exposed dams and their offspring were similar to controls. Results for weight gain during gestation and lactation, gestation length, offspring weight at birth, during lactation and maturation, litter size, gender ratio, number of implantations, and post-natal viability were unspectacular when compared to controls.

No effects on DNA strand breaks were noted in BAL fluid cell at any investigation time point. Increased level of DNA damage was observed in the livers of exposed dams at both sampling time points of 5 and 24 days after exposure. In the offspring, the level of DNA strand breaks was higher in liver at weaning and in adolescents, compared to their controls (weaning: 1.4-fold increase, p = 0.001; adolescents: 1.5-fold increase p = 0.011). The level of oxidatively generated DNA damage in the liver of offspring was also determined by the level of formamidopyrimidine DNA glycosylase (FPG) enzyme sensitive sites. There was no consistent increase in oxidatively generated DNA damage in the offspring liver cells at any sampling time point i.e. PND 2 (newborn), PND 22-23 (weaning) or PND 50 (adolescent) (newborn; exposed 0.91 ± 0.27 vs. control 0.71 ± 0.20; weaning exposed 1.05 ± 0.12 vs. control 1.28 ± 0.13; adolescents exposed 0.87 ± 0.10 vs. control 1.20 ± 0.11. All data are presented as lesions per 106base pairs).

BAL analysis indicated the presence of persistent inflammation in the lungs of time-mated mice; evidenced in the increased level of neutrophils in BALfluid compared to their controls 5 and 24 days after exposure (5 days: 11.4-fold increase,p=0.008; 24 days: 11.6-fold increase,p<0.001). Also recorded were mildly increased levels of lymphocytes in BALfluid 5 days after exposure (3.4-fold increase,p=0.020) and total cell counts at both time-points (5 days: 1.5-fold increase,p=0.032; 24 days: 1.2-fold increase,p=0.057).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The study comprised of two parts: An inhalation study and an instillation dose-effect study. Only results of the instillation part of the study are entered in this study record.The study assessed the effect of maternal gestational exposure of time-mated C57BL/6BomTac mice on the development of the offspring. Dams were intratacheally exposed to 0, 11, 54 or 268 μg/animal Carbon Black on gestation days 7, 10, 15 an 18 . Shortly before the expected date of delivery, the mother is sacrificed, the uterus removed, and the contents examined for deaths, and live foetuses. Effects on gestation, lactation and DNA strand breaks were evaluated. Traditional gestational and litter parameters were measured. Inflammation was assessed in maternal bronchoalveolar lavage (BAL) after exposure, and at weaning. Levels of DNA strand breaks were assessed in maternal BAL cells and liver, and in offspring liver. Dams were monitored until weaning and some offspring until adolescence.In the second publication (Jackson et al 2012c; Mutation Research 745 (2012) 73– 83), the author report that tissues from the same animals used in the first study were utilised to characterize the molecular and morphological changes associated with the observed pulmonary response in dams exposed to Printex 90 during gestation by applying the followoing methods: histopathology, DNA microarrays, pathway-specific RT-PCR arrays, focussed RT-PCR, and tissue protein analysis. The response of the offspring to prenatal Printex 90 exposure was characterised by global hepatic gene expression profiling of the newborn offspring. Potential relationships and connections between genes and pathways that are perturbed in the dams with those responding in the offspring were examined to gain molecular insight into the effects of maternal particle exposure and pulmonary inflammation on fetal biological responses and development.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Declared Particle size: 14 nm (Degussa Hüls)- Geometric mean size: 65 nm- Morphology: Individual carbon black spheres mainly occurred in open-structured long chain aggregates and fewer large dense aggregates- Surface area: 295 -338 m2/g- Pycnometric particle density: 2.1 g/cm3- Chemical composition 99% C, 0.8% N and 0.01% H2- The total PAH content (Carbon black extract – Soxhlet): 0.0742 µg/g- The total PAH content (DEP extract – NIST SRM 1650): 216 µg
Species:
mouse
Strain:
other: C57BL/ 6BomTac
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Taconic Europe, Ejby, DK- Age at study initiation: not specified- Weight at study initiation: yes; controls (24 mice weighing 23.0 ± 1.3 g), low dose of 11 µg/animal CB (17 mice weighing 22.4 ± 1.2 g), medium dose of 54 µg/animal CB (17 mice 22.6 ± 1.0 g), and high dose of 268 µg/animal CB (22 mice weighing 22.9 ± 0.9 g).- Received as time-mated pregnant mice on gestation day 3- Acclimation period: tests were commenced on gestation day 8: - Mice were housed in white polypropylene (type III) cages with bedding (Jeluxyl HW 300/500, JELU-Werk J. Ehrler GmbH & Co. KG, Rosenberg, Germany). Soft paper wool (Enviro-Dri) was added for nest building and cages were enriched with mini rodent tunnels and small aspen wood blocks (all from Lillico, Betchworth, UK). Enrichment was removed during lactation.- Environmental conditions were controlled (12 h light from 6.00 a.m., temperature 21 ± 2°C, humidity 50 ± 5%, ventilation 13 air changes per hour).- Tap water and food (Altomin1324, Altromin GmbH, Lage, Germany) were available ad libitum.
Route of administration:
intratracheal
Vehicle:
water
Remarks:
g-irradiated Nanopure Diamond UV water (Pyrogens: < 0.001 EU/ml, Total Organic Carbon: < 3.0 ppb),
Details on exposure:
The particle preparation and instillation procedures were described previously. Printex 90 was sonicated for 8 min (10 s pulses and 10 s pauses, total sonication time 4 min) at a concentration of 1.675 mg/mL (67 mg/instillation) in 0.2 mm filtered, g-irradiated Nanopure Diamond UV water (Pyrogens: < 0.001 EU/ml, Total Organic Carbon: < 3.0 ppb), using a 400 W Branson Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT, USA) mounted with a disruptor horn and operated at 10% amplitude. This dispersion was used for the high dose and diluted 1:5 for the medium dose (13.4 mg/ instillation) and diluted futher 1:5 for the low dose (2.7 mg/instillation). Eighty time-mated mice were anesthetized with 3% Isoflurane and instilled with a vehicle or one of the three concentrations of Printex 90 dispersions (40 mL solution followed by 160 mL air) on GD 7, 10, 15 and 18. Exposure took place between 08:30 and 14:30 h. Time-mated mice were instilled in different order each day, to reduce any variation that might be related to the time of exposure. The total instilled doses were 11, 54 and 268 µg/animal.
Analytical verification of doses or concentrations:
no
Remarks:
not applicable
Details on analytical verification of doses or concentrations:
The particle size distribution in the Printex 90 dispersions was determined with a 633 nm He- Ne Dynamic Laser Scatter (DLS) Zetasizer nano ZS (Malvern Inc., UK).The dispersion of Printex 90 instillation fluid was also analyzed by Scanning Electron Microscopy (QUANTA 200 FEG MKII with EDX).The particle size distribution was similar in the three instilled dispersions with concentrations of 1675, 335 and 67 mg/mL (see Figure 3), and was stable for more than 1 h. The average zeta-size was approximately 140 nm and the hydrodynamic number size-distributions had a peak size between 50 and 60 nm When converted to volume distributions, minor amounts of mm-size particles and two smaller size modes with peak sizes around 50–60 nm and 200–400 nm were identified. The observed DLS sizes were confirmed by TEM and SEM, with a wide size distribution of nm- to mm-size free and agglomerated particles. The agglomerates consisted of spherical to sub-spherical carbonaceous particles as well as minor amounts of free single primary spheres.
Details on mating procedure:
no data. Time-mated nulliparous adult female mice were employed.
Duration of treatment / exposure:
Instillations on day 7, 10, 15, 18
Frequency of treatment:
single instillation per day
Duration of test:
no data
Dose / conc.:
11 other: µg/animal
Remarks:
2.7 µg/instillation administered over 4 instillations = 0.5 mg/kg bw
Dose / conc.:
54 other: µg/animal
Remarks:
13.4 µg/instillation administered over 4 instillations = 2.5 mg/kg bw
Dose / conc.:
268 other: µg/animal
Remarks:
67 µg/instillation administered over 4 instillations = 12.2 mg/kg bw
No. of animals per sex per dose:
Control: 2411& 54 µg/animal: 17268 µg/animal: 22
Control animals:
yes, concurrent vehicle
Details on study design:
After the last exposure on GD 18, the time-mated mice were housed alone and monitored for birth. The expected day of delivery, GD 20, was assigned as post-natal day zero (PND 0) for the offspring. On PND 1, the offspring were counted and sex determined.Sampling for DNA analysis and lung inflammation analysis was performed 3-4 days post exposure and at weaning 26-27 days after exposure in dams. Sampling for DNA analysis was conducted on post natal days 2, 23 and 47 in offspring
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data DETAILED CLINICAL OBSERVATIONS: No data BODY WEIGHT: Yes - Time schedule for examinations: on GD 4, GD 7, and GD 10, 13, 15 (or 16), 19 and post natal days (PND) 2, 8, 12, 17 and 22 (weaning) POST-MORTEM EXAMINATIONS: Yes - Sacrifice on PND 1-2 (3-4 days after the last exposure) and at PND 24-25 (at weaning) - Organs examined: Liver and lungs were weighed, prepared and stored until analysis. Relative organ weight was calculated as (organ weight/bodyweight)*100. OTHER: - Lung Inflammation: After sacrifice, Bronchoalveolar lavage (BAL) fluid from each female was collected. Probing for inflammation was performed in animals sacrificed 3 days after end of exposure and on PND 24-25. The number of macrophages, neutrophils, lymphocytes, eosinophils and epithelial cells was determined by counting a total of 200 cells. - Histolopathological determination of lung inflammation: Following the collection of BAL cells, left lung lobes were prepared for histological determination of lung inflammation from animals exposed to all concentration.- Time point of examination: PND 24-25- Preparation of tissue protein extracts and analysis of inflammatory factors: A random section of the frozen left lung lobe from control and treated (low, medium and high doses) dams was used to extract total lung protein. Cytokine and chemokine concentrations were determined- Global microarray analysis: of lung tissue of dams (n=5 dams/group). Dams of all dose groups were sampled at PND 24-25 only
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes Examinations included: - Number of implantations: Yes
Fetal examinations:
Offspring:- On PND 1, the offspring were counted and sex determined.- On PND 2, all except two male and two female offspring in each litter were removed and killed by decapitation. Liver and lungs of newborns were weighted, dissected and stored at -80°C until analysis.- On PND 22, male and female offspring were randomly distributed into balanced experimental groups: A group for collection of organs at weaning PND 24–25, an adolescent group for maturation data and organs at PND (47), and a group for behavioural testing and mating for a 2nd generation (published elsewhere). In the instillation study only thymus of newborns and offspring at weaning was weighed, other organs were rapidly frozen to preserve the tissue quality. Relative organ weight was calculated as (organ weight/bodyweight)*100.OTHER: - DNA Microarray analysis: Performed only for animals of the highest dose group and control animals; total RNA was isolated from random sections of livers of newborns (n=5 males and 5 females/group).
Statistics:
DATA ANALYSESThe accepted level of statistical significance was 0.05. Litter was considered the statistical unit. Gestational parameters were analyzed by Kruskal-Wallis One-Way Analysis of Variance. Weight data were analyzed by analyses of variance (ANOVA), with treatment as factor, and day of weighing (GD, PND) as repeated measure. Litter size was used as co-variable for weight data during gestation. The number of litters was compared by Fisher’s exact test. Remaining data (BAL results, comet assay results, organ weights) were analyzed by analyses of variance (ANOVA), with treatment, day of sampling (PND) and sex (where relevant) as factors. Significant results from overall analyses were analyzed by pair wise comparisons. Data were analyzed separately for each day of sampling and instillation study results were further analyzed by dose in Fisher’s Least-Significant-Difference Test. Females sampled 3–5 days after exposure and dams sampled after weaning were compared, even though the groups differed by timing of sampling and also by pregnancy status. The “female” group consisted of pregnant mice with small litters and non-pregnant mice. Pregnancy is reported to alter the level of inflammatory response (Fedulov et al. 2008; Lamoureux et al. 2010), thus different background levels had to be accepted. Analyses were performed on SYSTAT Software Package version 9 and Statistical Tables for PC users.
Historical control data:
no data
Clinical signs:
not specified
Mortality:
no mortality observed
Description (incidence):
Some time-mated mice were lost during the instillation study apparently due to other causes: One time-mated mouse assigned to the control group died before the start of the exposure; four time-mated mice from the low dose group and two time-mated mice from the medium dose group died during instillation. Data from these mice were excluded from the study. One control, one low dose, three medium dose, and five high dose dams were also lost due to spontaneous acute intestinal pseudo-obstruction, commonly observed in lactating C57B1/6 mice (described in Percy and Barthold 2001). Offspring of these dams were also killed immediately. Since the cause of this disease was related to lactation only, the gestation data and newborns data on PND 2 were included in the study.
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
Lung Inflammation analysis of BAL fluid cell composition by differential cell count indicated the presence of persistent inflammation only in the lungs of time-mated mice exposed to the highest concentration of carbon black. The time-mated mice exposed to Printex 90 by instillation had more neutrophils in the BAL 3 and 26 days after exposure compared to the instilled control animals. This was statistically significant in the high dose group only and at both sampling times. There were twice as many neutrophils observed in dams 26–27 days post-exposure compared to 3–5 days post-exposure (3 days: 28.7-fold increase, p < 0.001; 26 days: 60.9-fold increase, p < 0.001). Also, more lymphocytes were observed 5 and 26 days after exposure in the high dose group (3 days: 7.7-fold increase, p = 0.005; 26 days: 4.4-fold increase, p < 0.001). Printex 90 instilled dams had more total cell counts and more dead cells in BAL fluid 26 days after exposure in the high dose group (total cell count increased 2.6-fold, p < 0.001; dead cells increased 2.8-fold, p < 0.001). No significant changes were observed for macrophages, eosinophils or epithelial cells at any dose. There was no evidence for significant inflammation at the medium or low doses. The increased neutrophils at 3 and 26 days post exposure suggests persistent and prolonged inflammation in response to CB instillation in dams. There was no evidence for significant inflammation at the medium or low doses.Histological changes in lung tissues: Lung morphology and pathology were evaluated in dams sampled 26–27 days post-exposure to the three different doses of Printex 90. The high dose group exhibited significant thickening of the alveolar septa and infiltration of macrophages and neutrophils in the interstitium. Although thickened alveolar septa were also observed in the medium dose group, the effect was sporadic compared to the high dose group. The low dose group was comparable to controls. Histological analysis also revealed significant retention of CB particles in mice exposed to both the medium and the high doses. Collagen deposition was not noted for any dose group. Thus, there was no evidence of increased pulmonary fibrosis in the lungs of exposed animals.DNA damage analysis: Intratracheal instillation of Printex 90 did not affect the level of DNA strand breaks in BAL cells in the females (p = 0.30), while the dams exposed by instillation had significantly less DNA strand breaks in BAL cells in the high dose group 26 days after exposure compared to control dams (20% reduction, p = 0.007). No increase in the level of DNA strand breaks was observed in the liver of time-mated mice exposed to Printex 90 by instillation compared to their controls (two-way ANOVA p = 0.85). Each data point represents an average of duplicate scored in two separate rounds.Maternal gene expression: Major gene expression changes were limited only to the dams of the highest dose (268 µg/animal) with 143 genes being ‘differentially expressed’. ‘Differentially expressed’ was defined by the authors to mean gene expression of fold change greater than 1.5 (i.e. increased or decreased by at least 50% of control values). The top responding genes were identified to be involved in inflammation, acute phase response and immune response. Gene ontology analysis revealed that the major pathways perturbed in dams of the high dose group belonged primarily to functional molecular networks, such as those associated with inflammatory response, cell-to-cell signalling and interaction (activation of normal and inflammatory cells, communication between innate and adaptive immune cells), cell death, and lipid metabolism.When genes were ranked primarily according to fold change (altered by 1.5 folds in either direction), genes expression analysis revealed that lipid metabolism and molcular transport of lipids as pathways associated in altered genes in all dose groups. Also genes involved in carcinogenic processes were similarly affected in all dose groups. These results suggest that stress signalling (lipid metabolism, acute phase response) pathways were mainly induced in the low dose group. However, the number of affected genes involved in processes such as cellular maintenance (phagocytosis of cells) and inflammation were higher in the high dose group. Moreover, at the highest dose level, genes/proteins involved in chronic inflammatory processes were found to be altered suggesting dose dependent transitions in toxic response to CB exposure.Microarray analysis revealed that pulmonary exposure to CB resulted in increased mRNA levels of genes associated with cytokine–cytokine receptor signalling and chemokine signalling pathways in dams 26–27 days post-exposure. The analysis showed significant increases in levels of cxcl5 (15.0-fold), cxcl1 (5.-fold), ccl2 (4.0-fold), ccl22 (2.4-fold), ccl7 (3.4-fold), ccr4 (1.7-fold) and the cytokine tumor necrosis factor (TNF, 1.4-fold) in the lung. Expression of IL-6 and IL1 beta did not change. Ifng was the only inflammation related gene significantly down-regulated (−1.5-fold).
Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Implantations: 7.59±0.54 in treatment group versus 7.39±0.56 in controlsImplantation loss (%) 17.01± 4.57 in treatment group versus 14.59±3.88 in controls
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Gestation length (days)- Control: 19.89±0.07- Treatment group: 20.06±0.06
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
Intratracheal instillation did not affect gestational and lactation parameters
Key result
Dose descriptor:
LOAEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Basis for effect level:
other: Persistent lung inflammation
Key result
Dose descriptor:
NOAEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Basis for effect level:
other: Traditional gestational and post gestational parameters
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Inflammatory markers in BAL increased
Description (incidence and severity):
Persistent inflammation (only seen at the highest dose level tested)
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Changes in postnatal survival:
no effects observed
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Description (incidence and severity):
DNA Strang breaks: In the offspring exposed to Printex 90 by maternal intratracheal instillation, the level of DNA strand breaks in liver cells was comparable to their controls (p = 0.8). Organ weightsThe thymus weight of exposed newborn and weaned offspring was similar to their controls. Other organs were not weighed.Global hepatic response in male and female newborn offspring (PND 2) born to dams exposed to the highest dose of CB was assessed using DNA microarrays. The overall results suggest that females were more responsive to particle-induced maternal effects following in utero exposures than males. Subtle but significant changes in hepatic gene expression were seen in both male and female newborn offspring on PND 2. Generally, male and female offspring showed variable degrees of altered gene expression associated with metabolism, but the specific metabolic processes affected were sex-specific. Hierarchical cluster analysis on differentially expressed genes revealed that samples from treated female offspring clustered separately from matched controls. Treated males clustered on the same branch alongside controls, suggesting that the effects of treatment were clearly seen for female offspring but not for male offspring. Noteworthy: Hepatic gene profiling in offspring: statistical threshold applied to identify significant change was less stringent p=0.1
Key result
Dose descriptor:
LOEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: subtle, but stat. sign. changes in hepatic gene expression
Remarks on result:
other: only investigated at highest dose level
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Executive summary:

Carbon black was administered via intratracheal instillation to three groups of time-mated pregnant C57BL/ 6/BomTac at a total concentrations of 11, 54, 268 µg/m3 carbon black, respectively. Administration of carbon black occurred on gestations days 7, 10, 15 and 18. Carbon black as administered as a suspension in water. Control animals received the vehicle concomitantly. Traditional gestational and post-gestational parameters were assessed in dams and their offspring. In addition, the investigators probed into whether exposure to carbon black caused DNA damage in lung (BAL) and liver cells of exposed dams as well as in the liver of their offspring. Lung inflammation in dams was also examined. Samples for analysis of DNA damage and lung inflammation were collected 3 -4 and 26 -27 days after cessation of exposure in dams and on post-natal days 2, 24-25 and 47 for DNA analysis in offspring.

All gestational and lactation parameters assessed in dams and offspring were comparable in magnitude to the observations in the respective control groups.

Although retention of CB particles was observed in dams from both the medium and the high dose groups, neutrophil-marked persistent inflammation in the lungs that was confined to high dose group only. Histological analysis of the lungs corresponded well with BAL findings: a thickening of the alveolar septa with simultaneous infiltration of macrophages and neutrophils in the interstitium in the high dose animals. However, despite sustained inflammation, collagen deposition, a hallmark of fibrosis, was not evident; neither in the high dose animals nor in otherwise treated animals. The exposure to carbon black did not affect the level of DNA strand breaks in BAL cells (p = 0.30) or liver cells of dams nor did it influence the level of DNA strand breaks in liver cells of offspring (p = 0.8).

Altered expression of several cytokines and chemokines, both at the transcriptional and tissue protein levels, was significant only in the high dose group. Analysis of newborn livers by DNA microarrays revealed that female offspring were more sensitive to maternal exposure than male offspring. Cellular signalling, inflammation, cell cycle and lipid metabolism were among the biological pathways affected in female offspring. Males, however, responded with subtle changes in metabolism-related genes.

Noteworthy: Hepatic gene profiling in offspring: statistical threshold applied to identify significant change was less stringent p=0.1

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January to June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is summarized in a publication by the EU Scientific Committee on Consumer Safety, and not in a peer-reviewed publication.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
The test animals were mated female Wistar rats. The age at start of test was 13 to 14 weeks.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5%
Details on mating procedure:
Pregnant females were used.
Duration of treatment / exposure:
Pregnant female rats were dosed through daily oral gavage at 0, 100, 300 or 1 000 mg/kg/day (24/group) during the sensitive period of organogenesis [day 5 through day 19 of gestation].
Frequency of treatment:
Daily through days 5 to 19 of gestation.
Duration of test:
Following dosing on gestation days 5 through 19, the dams were killed on gestation day 20 and subjected to macroscopic examination. Usual litter parameters were recorded and foetuses were sexed, weighed and submitted to external examination. About one half of the foetuses were also examined for soft tissue anomalies, and remaining foetuses were examined for skeletal anomalies.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 animals per dose
Control animals:
yes, concurrent vehicle
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: discoloration of faeces was not considered an adverse effect
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects reported
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The oral No Observed Adverse Effect Level (NOAEL) for maternal toxicity and the No Observed Effect Level (NOEL) for developmental toxicity were both set at 1000 mg/kg bw/day in a study evaluated by the SCCS
Executive summary:

The SCCS summarized the study and reports that the study authors concluded that oral administration of carbon black to pregnant rats at 100, 300 or 1 000 mg/kg bw/d during the sensitive period of organogenesis was well tolerated. There were no adverse maternal changes or any effects on embryo-fetal development. Accordingly, under the conditions of this study, The No Observed Adverse Effect Level (NOAEL) for maternal toxicity and the No Observed Effect Level (NOEL) for developmental toxicity were both set at 1000 mg/kg bw/d

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
42 mg/m³
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for study waiver:

(i)                Carbon black is of low toxicological activity

 For the developmental toxicity endpoint, there is one oral gavage study that was conducted using a standard OECD protocol (OECD 414; prenatal developmental toxicity study) and assessed by the EU Scientific Committee on Consumer Safety(SCCS 2015). According to the SCCS, the study authors concluded that oral administration of carbon black to pregnant rats at 100, 300 or 1000 mg/kg body weight/day during the sensitive period of organogenesis was well tolerated, and there were no adverse maternal changes or any effects on embryo-fetal development. In the open literature, one inhalation study was identified, where carbon black was administered at a dose of 42 mg/m3for 1 hour/day daily from gestation days 8 through 18 to mice(Jackson, Hougaard et al. 2012). No gestational or developmental toxicity in the offspring were found in this study, even under very high exposure condition which clearly induced persistent lung inflammation as evidenced by inflammatory markers in BAL. Several studies in mice were identified that used intranasal instillation or intratracheal instillation, and evaluated different endpoints in the offspring. Despite using very high intratracheal doses,gestational and litter parameters were normal for dams and offspring in a study evaluating the effects of carbon black on the sexual development and neurofunction of mice exposed in-utero to carbon black (NOEL, total dose: 54 µg/animal). The female offspring prenatally exposed to 268 µg/animal displayed altered habituation pattern during the Open Field test, which could have been a secondary effect to marked inflammation recorded at this dose(Jackson, Vogel et al. 2011).

 

(ii)             Toxicokinetic considerations

Toxicokinetic studies on carbon black and similar carbonaceous materials indicate that industrially-produced carbon black is unlikely to be absorbed or distributed in the body. Therefore, carbon black is unlikely to reach reproductive organs and tissues and have a direct effect on reproductive functions or the developing organism. 

 

(iii)           No significant human exposure

Occupational exposure controls are in place. In consumer products carbon black is tightly bound in a matrix with no release of carbon black nanoparticles 

Justification for classification or non-classification

There are various published non-guideline tracheal instillation studies and one inhalation study in mice in which specific reproductive and developmental toxicity endpoints were evaluated, usually at one time point or using a single dose level only. It is possible that some of the effects observed in these studies may be the result of non-specific inflammatory effects caused by high exposure doses. No gestational or developmental effects were reported in a whole-body mouse inhalation study at 42 mg/m3. The EU Scientific Committee on Consumer Safety (SCCS) assessed an oral gavage study conducted in accordance with the standard OECD 414 test guideline for developmental toxicity and reported no adverse maternal changes and no effects on embryo-fetal development at the highest dose tested. The overall weight of evidence strongly indicates that carbon black should not be considered a reproductive toxicant. The criteria for classification according to the EU CLP Regulation are not met.