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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Prenatal Exposure to Carbon Black (Printex 90): Effects on Sexual Development and Neurofunction
Author:
Jackson P., Vogel U., Wallin H., Hougard KS
Year:
2011
Bibliographic source:
Nanotoxicology, August 2012; 6(5): 486–500

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

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:

Administration / exposure

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
Doses / concentrationsopen allclose all
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).

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

Neuropathological findings:
effects observed, treatment-related

Results: F1 generation

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, treatment-related

Effect levels (F1)

open allclose all
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

Target system / organ toxicity (F1)

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

Results: F2 generation

Developmental neurotoxicity (F2)

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 (F2)

Developmental immunotoxicity:
not specified

Overall reproductive toxicity

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

Applicant's summary and conclusion

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).