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Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-status unknown, non-guideline, animal experimental study, published in peer-reviewed literature, adequate for assessment of effect on female fertility and in utero and early post-natal development

Data source

Reference
Reference Type:
publication
Title:
A female rat fertility study with inhaled benzene
Author:
Kuna RA, Nicolich MJ, Schroeder RE and Rusch GM
Year:
1992
Bibliographic source:
J. Am. College Toxicology 11, 275-282

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzene
- Supplied by: American Petroleum institute/Chemical Manufacturers' Association
- Analytical purity: 99.96%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Detail: Five groups of 26 female and 13 proven fertile males were used
- Housing: In the pre-mating treatment period they were individually caged during exposure and non-exposure periods. For mating, one male and two females were housed per cage. Females were housed individually during gestation, parturition, and with litter during lactation. All cages were stainless-steel wire mesh
- Diet: Purina Laboratory Chow ad libitum except during exposure
- Water: ad libitum except during exposure

ENVIRONMENTAL CONDITIONS
- Temperature and humidity were monitored but results not reported
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m3 stainless steel and glass chambers
- System of vapour generation: flash-evaporation. Benzene was pumped into a heated flask, flash-evaporated, and the vapour mixed with the chamber air supply. Benzene vapour was delivered to exposure chambers using a syringe pump and different concentrations were achieved by adjusting pump flow rates or chamber air flow.
- Air flow rate: between 150 and 250 L/min for the duration of the exposures

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber atmospheric concentrations were monitored using a Miran Long Pathlength Infrared Model 1A, or by Gas Chromatography

Details on mating procedure:
- M/F ratio per cage: 1/2
- Length of cohabitation: maximum of 8 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After 8 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: yes
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber atmospheric concentrations were monitored using a Miran Long Pathlength Infrared Model 1A, or by Gas Chromatography. The exposure levels were determined by comparing the observed absorbance of these samples to a previously determined standard curve generated using the test material under the same instrumental settings. The gas chromatograph was used because of interference in the infrared monitoring procedure (pathlength 21.75 in) for the 10 and 1 ppm exposure groups.
Analysed exposure concentrations were close to target concentrations (see table 1).
Duration of treatment / exposure:
6 h/day
Frequency of treatment:
5 days/week during pre-mating and mating, daily days 0-20 of gestation and 5-20 of lactation
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1.0, 10, 30, 300 ppm (0, 3.2, 32, 320, 960 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
22, 22, 24, 19 and 23 pregnant rats for the 0, 1, 10, 30 and 300 ppm groups respectively
Control animals:
yes, sham-exposed

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice/day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly until completion of the mating period. Mated females were weighed on Days 0, 7, 14, and 21 of gestation and on Days 0, 4, 14, and 21 of lactation.

FOOD CONSUMPTION: No
Oestrous cyclicity (parental animals):
Daily vaginal smears were made and evaluated for each female beginning 2 weeks prior to initiation of mating.
Sperm parameters (parental animals):
No
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 post partum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
- The following parameters were examined in offspring: number, sex and weight of pups.

GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
All dams were given a gross post mortem examination. Method of sacrifice was by overdose of ether. Abnormal tissues noted during these evaluations were saved in 10% neutral buffered formalin. Uteri were examined for the presence and number of implantation sites, and along with ovaries, were saved in 10% neutral buffered formalin solution.
Postmortem examinations (offspring):
Gross post mortem examinations, including internal gender determinations were performed on all pups sacrificed by ether overdose on Day 21 of lactation and pups found dead during lactation. The latter were also checked for the presence or absence of milk in the stomach. Liver, kidney, and in males, testes weights were recorded for each pup.
Statistics:
Dam body weights, body weight changes, gestation length and the number of pups per litter were by a standard one-way analysis of variance. Data were first tested with Bartlett's test for equality of variance. The pregnancy percentage, viability index, and lactation index were tested for equality using a Chi-square test. If this test indicated statistical differences, each treated group was compared with the control group using Fisher's Exact test. Pup body weights at Days 0, 4, 14, and 21, organ weights and organ-body weight ratios at Day 21 were tested using a nested design. Pups are taken as nested within dams, and dams nested within treatment groups. The significance of the difference between groups is determined by the ratio of dose group mean square error divided by the dam within group mean square error. If the ratio indicated a statistically significant difference in means, individual group mean differences were determined by the least significant difference (two sample t-test with the pooled variance) technique.
Reproductive indices:
Pregnancy percentage, viability index, lactation index

Results and discussion

Results: P0 (first parental generation)

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:
not examined
Histopathological findings: non-neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Effect level:
300 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: there was no evidence of toxicity, effects on body weight and/or altered reproductive performance at the highest exposure of 300 ppm
Dose descriptor:
NOAEC
Effect level:
960 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: no evidence of toxicity, effects on bodyweight and/or altered reproductive performance at the highest exposure of 960 mg/m3

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

The only statistically significant differences were lower female pup body weight (day 21) and liver weights at the 300 ppm exposure level. Female day 21 body weights were 36.3±5.20, 35.45±5.89, 37.71±7.14, 33.68±5.40 and 32.59±5.05 g for 0, 1, 10, 30 and 300 ppm respectively. Liver weights were 1.69±0.36, 1.59±0.41, 1.78±0.43, 1.56±0.33 and 1.46±0.34 g for 0, 10, 10, 30 and 300 ppm respectively.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
300 ppm
Sex:
male/female
Basis for effect level:
other: no significant adverse effects on pup survival during lactation, body weight, organ weights or in the gross post mortem evaluation
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
960 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: no significant adverse effects on pup survival during lactation, body weight, organ weights or in the gross post mortem evaluation

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Exposure of adult female Sprague-Dawley rats to benzene via inhalation at concentrations up to 300 ppm (960 mg/m3) in a one-generation reproduction study produced no evidence of toxicity, body weight, and/or altered reproductive performance. There were no treatment-related effects on pup survival, or gross pathology and no significant adverse effects on body weights or organ weights. An exposure concentration of 300ppm (960 mg/m3) is a NOAEC for both adult and offspring toxicity and female fertility.
Executive summary:

Female Sprague-Dawley rats were exposed to dose levels of 1, 10, 30 or 300 ppm benzene (6 h/day, 5 d/week) during 10 weeks pre-mating, mating, gestation and lactation periods up to p.n. day 21. Five groups of 26 females and 13 proven fertile males were used. To determine if oestrous was affected by treatment, daily vaginal smears were made and evaluated for each female beginning 2 weeks prior to initiation of mating. Observations of females for mortality and clinical signs were made twice daily. Detailed physical examinations were performed weekly throughout the study. Body weights were recorded once weekly through completion of the mating period. Mated females were weighed on days 0, 7, 14, and 21 of gestation and on days 0, 4, 14, and 21 of lactation. Pups were counted, weighed, and sexed on days 0, 4, 14, and 21 of lactation. Litters were observed twice daily. On day 4 of lactation, litters of more than 10 pups were randomly culled to 10 with equal number per gender where possible. Pups that died were weighed and sexed by internal examination. All dams were given a gross post mortem examination. Uteri were examined for the presence and number of implantation sites, and along with ovaries, were fixed and saved. Gross post mortem examinations, including internal gender determinations, were performed on all pups sacrificed on day 21 of lactation and on pups found dead during lactation. The latter were also checked for the presence or absence of milk in the stomach. Liver, kidney, and in males, testes weights were recorded for each pup. Thirty-three organs and tissues along with any abnormal lesions were fixed in 10% neutral buffered formalin from two pups per sex per litter and saved for future histopathological examination.

There were no effects on maternal body weight and body weight gain nor were there adverse effects on fertility as measured by percentage pregnant animals, mean gestational length, number of litters, litter size, and viability of the pups and the weanlings at any dose tested. The only statistically significant differences for offspring were lower female pup body weight on day 21 and lower liver weight at the 300 ppm exposure level. These differences were small and considered not to be adverse. No treatment related effects were seen in pup survival or at gross post mortem on post natal day 21.

An exposure concentration of 300ppm (960 mg/m3) is a NOAEC for both adult and offspring toxicity and female fertility.