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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP status unknown, non-guideline study, published in peer reviewed literature, no restrictions, fully adequate for assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Two generation reproduction study of ethylbenzene by inhalation in Crl-CD rats
Author:
Faber WD, Roberts LSG, Stump DG, Tardif R, Krishnan K, Tort M, Dimond S, Dutton D, Moran E and Lawrence W
Year:
2006
Bibliographic source:
Birth Defects Res (Part B) 77:10-21

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- CAS number: 100-41-4
- Source: The Dow Chemical (Midland, MI) via Ashland Chemical (Morrisville, PA, USA)
- Storage: under refrigeration
- Analytical purity: at least 99.9%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Laboratories (Raleigh, NC, USA) on 8 April 2003
- Age on arrival: approximately 38 days
- Randomisation: based on body weight
- Housing:
prior to pairing: individual housing in wire-mesh cages suspended above cage-board
cohabitation: home cage of the male
post mating: females transferred to plastic maternity cages with nesting material where housed until weaning of the litter
- Animal facility: AAALAC accredited
- Animal room environmentally controlled (no further details)
- Diet: PMI Nutrition International Inc. certified rodent LabDiet 5002, ad libitum
- Water: municipal water reverse-osmosis treated delivered by automatic system, ad libitum except during inhalation exposure

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
Exposure concentrations were based on death, clinical signs and reduced body weight gain of weanling rats exposed to 500 or 1000 ppm ethylbenzene in a preliminary study. Clinical signs included laboured respiration, unsteady gait and prostration.
Details on mating procedure:
1 male:1 female for 14 days or until confirmation of mating observed. Sibling pairings were avoided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In exposure chambers, analysed by gas chromatography with flame ionization detection. Analysed concentrations were very close to nominal.
In dosing solutions, analysed by gas chromatography with mass spectroscopy.

Duration of treatment / exposure:
Inhalation: 6 hours daily, 7 days per week. F0 and F1 animals exposed for at least 70 consecutive days prior to mating. Males continued on the same regime whilst on study. The females were similarly exposed throughout mating and gestation, through to day GD 20; exposure recommenced on day 5 of lactation (LD5) and continued through to weaning (LD21).
Oral gavage: females only days 1-4 of lactation. Ethylbenzene administered in corn oil at dose levels of 0, 26, 90, and 342 mg/kg/day (dose levels selected to result in similar internal maternal exposure based on PBPK modelling). Each dose was divided into 3 equal amounts and administered approximately two hours apart.
Frequency of treatment:
daily
Details on study schedule:
After a pre-mating period of at least 70 days, each female was housed with a randomly selected male from the same exposure group, avoiding sibling matings. Each female was examined daily for the presence of sperm in a vaginal smear or the presence of a copulatory plug. The day of confirmation of mating was gestation day 0 (GD 0). Females failing to mate were given a second opportunity with another male. Females were allowed to litter naturally and rear their young to weaning (PND 21).
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 100 and 500 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
30 F0, 25 F1

Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily checks for mortality, clinical signs and changes in appearance and behaviour. Animals visible in inhalation chambers (50%) were observed for changes in appearance and behaviour at the mid point of exposure.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule: weekly and prior to necropsy. Females on GD 0, 4, 7, 11, 14, 20 and LD 1, 4, 5 (pre-exposure), 7, 14, and 21

FOOD CONSUMPTION:
- Time schedule: weekly except during the mating period. Females on GD 0, 4, 7, 11, 14, and 20 and LD 1, 4, 5, 7, 14, and 21

MATING:
- Pre-coital interval was calculated according to the number of 12-hr dark cycles before evidence of mating

BLOOD ANALYSIS:
- Blood samples from the tail vein were collected 1 hour after the third gavage dose from 4 F1 females per group on lactation day 4 and from the same females following the 6 hour period of inhalation exposure on day 22. The blood was analysed for ethylbenzene concentration.

Oestrous cyclicity (parental animals):
Daily vaginal smears to determine the stage of oestrus for 21 days before pairing and until evidence of mating observed or until the end of the mating period. The average cycle length was determined for complete oestrous cycles.
Sperm parameters (parental animals):
Sperm motility was assessed for all males using the Hamilton-Thorne HTM-IVOS Version 12 computer-assisted sperm analysis (CASA)
system. Sperm morphology was evaluated by light microscopy using a modified wet mount evaluation technique. Homogenization-resistant spermatid count and sperm production rate were determined using a DNA-specific fluorescent dye and the HTM-IVOS CASA system.
Litter observations:
OBSERVATIONS:
- Each litter was examined twice daily for survival and signs of toxicity.

CULLING:
- To reduce variability, 10 F1 and F2 pups per litter were randomly selected on PND 4.

BLOOD SAMPLING:
- Pups from 4 F2 litters/group were used for analysis of ethylbenzene in blood on lactation day 4. Four male plus 4 female F2 pups were used to obtain blood samples 1 hr after completion of a single 6-hr inhalation exposure to ethylbenzene on PND 22.

SEX & BODY WEIGHTS
- Pups were sexed on PND 0, 4, 7, 14 and 21. F1 pups were individually weighed on PND 1, 4, 7, 14, and 21; F2 pups were individually weighed on PND 1, 4, 7, 11, 13, 17 and 21. Pups were weighed on the day of sexual maturation.

DEVELOPMENTAL LANDMARKS
- Assessed in F1 and F2 offspring: pinna detachment, hair growth, incisor eruption, eye opening, balanopreputial separation and vaginal patency.


Postmortem examinations (parental animals):
SACRIFICE: All adult animals killed using isoflurane inhalation and exsanguination. Vaginal smears taken from all females on the day of euthanasia to determine stage of oestrus. Males were terminated after completion of parturition. All females that littered were terminated 6-10 days after weaning their litters. Females that mated but did not litter were terminated on presumed GD 25. Females that had total litter loss were terminated within 24 hr.

GROSS NECROPSY: yes, all animals

ORGAN WEIGHTS: brain, liver, kidneys, spleen, lungs, thyroids, prostate

HISTOPATHOLOGY: Selective histopathological examination was undertaken (no further details). Ovarian primordial follicle counts were recorded for all F1 females in the control and high dose group.
Postmortem examinations (offspring):
SACRIFICE: Intact offspring dying PND 0-4 were necropsied. A detailed necropsy was carried out on any pup dying PND 4-21 and for all F1 pups dying PND 22-42. All F1 and F2 pups not selected were killed using CO2 inhalation on PND 21. All F2 pups not selected for neuropathological assessment were killed on PND 21. F2 pups selected for toxicokinetic evaluation were killed on PND 22.

GROSS NECROPSY: Yes all pups

ORGAN WEIGHTS: selected organs were weighed (no further details)

HISTOPATRHOLOGY: selective histopathological examination was undertaken (no further details)
Statistics:
Test and control groups were compared by sex and the analyses used two-tailed tests for 5% significance. Chi squared test with Yates’ correction was used to analyse mating and fertility indices. Analysis for heterogeneity of variance and normality were applied to mean body weights, food consumption, food efficiency, organ weights, oestrous cycles, pre-coital intervals, gestation lengths, implantation sites, ovarian primordial follicle counts, pups born, live litter size, epididymal and testicular sperm numbers, sperm production rates, and days of developmental landmarks. A parametric one-way analysis of variance (ANOVA) was used to determine intergroup differences where data were homogeneous and normal. Dunnett’s test compared the control and test groups when ANOVA was significant (p<0.05). The non parametric ANOVA Kruskal-Wallis test was used where data were not homogeneous and normal to determine intergroup differences. The Mann-Whitney U-test was used where ANOVA showed statistical significance (p<0.05). Pup weights were analysed by sex using a nested analysis of covariance (ANCOVA) where the number of pups born was the covariate. ANCOVA made the following assumptions: homogeneity of regression slopes, linear relationship between pup weights and number of pups born, additive group and regression effects. A two-tailed Fisher ’s Exact test compared histopathological findings in test and control groups. Kruskal-Wallis non parametric ANOVA test to determine intergroup differences was used for mean litter proportions (%/litter) of pup survival, sex ratio at birth (% of males/ litter), percentage of motile and progressively motile sperm and percentage of sperm with normal morphology. Mann-Whitney U-test compared the test and control groups where ANOVA showed statistical significance (p<0.05).

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Increased relative (to final body weight) liver and kidney weights observed in F0 males (13.3% and 12.5%, respectively) and F1 males (8.1% and 9.8%, respectively) exposed to 500 ppm ethylbenzene were considered to be treatment-related. Increased relative liver weights were slightly increased (3- 7%) in F0 and F1 females exposed to 500 ppm.

Effect levels (P0)

Dose descriptor:
NOAEC
Remarks:
reproductive performance
Effect level:
>= 500 ppm
Sex:
male/female
Basis for effect level:
other: highest concentration tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEC
Remarks:
reproductive performance
Generation:
F1
Effect level:
>= 500 ppm
Sex:
male/female
Basis for effect level:
other: highest concentration tested
Dose descriptor:
NOAEC
Remarks:
developmental toxicity
Generation:
F1
Effect level:
>= 500 ppm
Sex:
male/female
Basis for effect level:
other: no effect on number of pups born live, survival, clinical condition, body weight, acquisition of developmental landmarks

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEC
Remarks:
developmental toxicity
Generation:
F2
Effect level:
>= 500 ppm
Sex:
male/female
Basis for effect level:
other: no effect on number of pups born live, survival, clinical condition, body weight, acquisition of developmental landmarks

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOEC and NOAECs of ethylbenzene for parental systemic toxicity were 100 and 500 ppm respectively and the NOAEC for reproductive and developmental toxicity was >500 ppm (equivalent to 2171 mg/m3).
Executive summary:

Male and female Crl:CD(SD)IGS BR rats were exposed by inhalation to 0, 25, 100 or 500 ppm ethylbenzene for 6 hr/day throughout the 2-generation study except for females between lactation days 1 and 4 when the dose was administered in corn oil by oral gavage at dose levels of 26, 90 or 342 mg/kg/day (to provide similar maternal blood (AUC) as provided by inhalation).

At 500 ppm ethylbenzene, the males of both generations showed reduced body weight gain and increased liver and kidney weights whilst only liver weights were affected in females. As the reduced body weight gain in the 500 ppm males was transient and there were no histopathological changes associated with the increased organ weights in the 500 ppm males and females, these changes were considered not to be adverse. Therefore, for parental systemic toxicity the NOEC was considered to be 100 ppm and the NOAEC 500 ppm. There were no adverse effects of ethylbenzene on reproductive performance in either generation. Neither were there any effects of treatment on spermatogenic endpoints, ovarian follicle counts, reproductive organ weights or macroscopic pathology. The NOAEC for reproductive toxicity and offspring development was 500 ppm ethylbenzene.