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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:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The principles of the study are comparable to the relevant guidelines with acceptable restrictions.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other:
Principles of method if other than guideline:
Reproductive Assessment by Continuous Breeding (RACB) protocol of the US National Toxicology Program (NTP) according to Lamb (1985), J. Amer. Coll. Toxicol. 4, 163-171 and Reel et al. (1985), J. Amer. Coll. Toxicol. 4, 147-162
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Chloroform
- Supplier: Aldrich Chemical Company, via Research Triangle Institute (Research Triangle Park, North Carolina)
- Analytical purity: >99 %
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: 0.75 % ethanol as inhibitor
- Lot/batch No.: no data
- Storage condition of test material: stable when stored for two weeks protected from light at ambient temperatures (20 to 25 °C)

Test animals

Species:
mouse
Strain:
other: CD-1 (ICR)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Kingston, New York
- Age at study initiation: 11 weeks
- Weight at study initiation: (P) Males: 31.96-38.92 g; Females: 23.18-29.66 g; (F1) Males: 32.68 +/- 0.52 g; Females: 26.56 +/- 0.55 g
- Housing: 2 per cage by sex during quarantine and the 1-week pre-mating using solid bottom polycarbonate cages with stainless steel wire lids; animals were subsequently housed as breeding pairs or individually
- Diet (e.g. ad libitum): NIH-07 diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks of quarantine, 1 week of acclimation


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 14 hours light/10 hours darkness

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Animals were treated with 8, 20 or 50 mg/kg body weight/day by gavage. The test chemical was mixed with Mazola corn oil on a weight to volume basis. Corn oil was tested for peroxide content prior to formulation and discarded if peroxide level was greater 10 meq. Each dose level was independently formulated. The concentration of chloroform was adjusted so that all doses were administered in 10 mL/kg body weight. Dose formulations were prepared at minimum every three weeks and aliquotted at the time of formulation into vials for daily use. Aliquots were stored at approximately 4 °C until use.
Details on mating procedure:
continuous cohabitation phase (14 weeks)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
An aliquot of each formulation, the control and the bulk chemical were sent to Research Triangle Institute for reference analysis during weeks 1, 6, 10 and 14 of the F1 study. Dosage formulation studies performed by Research Triangle Institute indicated no problems with the preparation of corn oil solutions at the 40 mg/mL level. Stability studies on corn oil solutions of chloroform (5 mg/mL level) indicated no significant loss of chemical after three weeks storage in sealed glass bottles in the dark at room temperature. Analysis of dosing solutions by gas chromatography showed that the actual doses administered to the animals were closer to 6.6, 15.9 and 41.2 mg/kg body weight/day.
Duration of treatment / exposure:
Continuous for 18 weeks (1 week prior to cohabitation, 14 weeks of cohabitation 3 weeks therefater) : Treatment of F0 and F1 generations
Frequency of treatment:
daily
Details on study schedule:
At the end of the dosing phase, the last litter from all dose groups was reared by the dam until weaning. At weaning, the F0 animals were killed. The pubs from the low and middle dose groups were killed and the pubs from the control and high dose groups were reared and dosed through the mating period (at approximately postnatal day 74) until necropsy.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 6.6, 15.9 and 41.2 mg/kg body weight/day
Basis:
analytical conc.
No. of animals per sex per dose:
20 breeding pairs for dose groups, 40 breeding pairs for control
Control animals:
yes, concurrent vehicle
Details on study design:
-Dose selection rationale: Data on body weights, clinical signs, and food and water consumption from a 2-week dose-range-finding study.

Examinations

Parental animals: Observations and examinations:
Clinical signs, parental body weight and average consumption of drinking water during representative weeks
Oestrous cyclicity (parental animals):
As monitored by vaginal lavage for the 12 days preceding necropsy
Sperm parameters (parental animals):
Epididymal sperm motility, sperm morphology, sperm count
Litter observations:
Litters per pair, live pups per litter, proportion of pups born alive, sex of live pups and the pup body weights
Postmortem examinations (parental animals):
Selected organs: testes, epididymides and ovaries
Statistics:
The Cochran-Armitage test (Armitage 1971, Statistical Methods in Medical Research, John Wiley & Sons, New York) was used to tes for a dose-related trend in fertility. Dose group means for the number of litters, the number of live pups per litter, the proportion of live pups (number of pups born alive divided by the total number of pups produced by each pair), and the sex ratio (total number of male pups born alive out of the total number of live pups born to each fertile pair) were tested for overall differences using the Kruskal-Wallis test and for ordered differences using Jonckheere's test. Pairwise comparisons of treatment group means were performed by applying the Wilcox-Mann-Whitney U test. To remove the potential effect of the number of pups per litter on the average pup weight, an analysis of covariance (Neter and Wasserman 1974, Applied Linear Statistical Models) was performed. Least squares estimates of dose group means, adjusted for litter size, were computed and tested for overall equality using an F-test and pairwise equality using a t-test (carried out for males, females and both sexes combined). An analysis of covariance was used to adjust organ weights for total body weight. Unadjusted body and organ weights were analysed using the Kruskal-Wallis and Wilcox-Mann-Whitney U tests. Dose-related trends were tested for by Jonckheere's test.

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:
not examined
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed

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)

Five animals died during the dosing phase; these deaths were scattered throughout the dose groups and were not judged related to treatment. Food and water consumption was not affected by treatment; group mean body weights during the dosing phase differed by no more than 2 %. There were no treatment-related changes in any endpoint related to reproductive function. All F0 mice were killed at weaning.

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:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
effects observed, treatment-related

Details on results (F1)

Number of litters and number of pub per litter were all unchanged. There were no treatment-related alterations in pub-viability or increase in body weight.
Female body weight-adjusted liver weight was elevated by 14 % compared to the control group in the F1 treated group (dose of 41.2 mg/kg body weight/day). Vaginal cytology was not evaluated in these animals. In males, the only difference between the control and treated groups was a 7 % increase in relative epididymis weight. There were no differences between the groups in epididymal sperm measures.
All treated females showed some degree of hepatocellular degeneration. Treatment-related histologic alterations in males included hepatitis and hepatocellular degeneration (1 case each).
No changes were seen in lung, thyroid, spleen, esophagus, or the accessory sex organs.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
15.9 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Based on increased fertility index, relative epididymis weight and liver weight

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Reproductive toxicity of chloroform in Swiss mice

Reproductive toxicity in F0 generation

7 mg/kg

Male, female

16 mg/kg

Male, female

41 mg/kg

Male, female

Average # litters/pair

No change

No change

No change

# live pubs/litter; pub weight/litter

No change

No change

No change

Cumulative days to litter

No change

No change

No change

Absolute testis, epididymis weight a)

No observation

No observation

No observation

Sex accessory gland weight a) (prostate, seminal vesicle)

No observation

No observation

No observation

Epidid. Sperm parameters (#, motility, morphology)

No observation

No observation

No observation

Estrous cycle length

No observation

No observation

No observation

Reproductive toxicity in F1 generation

7 mg/kg

Male, female

16 mg/kg

Male, female

41 mg/kg

Male, female

Fertility index

No observation

No observation

Increase b)

# live pubs/litter; pub weight/litter

No observation

No observation

Increase b), No change

Absolute testis, epididymis weight a)

No observation

No observation

No change, Increase (b)

Sex accessory gland weight a) (prostate, seminal vesicle)

No observation

No observation

No change

Epidid. Sperm parameters (#, motility, morphology)

No observation

No observation

No change

Estrous cycle length

No observation

No observation

No observation

a) adjusted to bodyweight; b) statistically significant change (p 0.05)

Table 2: General toxicity of chloroform in Swiss mice

General toxicity in the F0 generation

7 mg/kg

Male, female

16 mg/kg

Male, female

41 mg/kg

Male, female

Body weight

No change

No change

No change

Kidney weigh a)

No observation

No observation

No observation

Liver weight a)

No observation

No observation

No observation

Mortality

No change

No change

No change

Feed consumption

No observation

No observation

No observation

Water consumption

No change

No change

No change

Clinical signs

No change

No change

No change

General toxicity in the F1 generation

7 mg/kg

Male, female

16 mg/kg

Male, female

41 mg/kg

Male, female

Pub growth to weaning

No observation

No observation

No change

Mortality

No observation

No observation

No change

Adult body weight

No observation

No observation

No change

Kidney weight a)

No observation

No observation

No change

Liver weight a)

No observation

No observation

No change, Increase b)

Feed consumption

No observation

No observation

No observation

Water consumption

No observation

No observation

No change

Clinical signs

No observation

No observation

No change

a) adjusted to bodyweight; b) statistically significant change (p 0.05)

Applicant's summary and conclusion

Executive summary:

A two-generation reproductive toxicity test was carried out with chloroform using Swiss mice according to the Reproductive Assessment by Continuous Breeding (RACB) protocol. Chloroform was administered by oral gavage at actual doses of 0, 6.6, 15.9 and 41.2 mg/kg bodyweight/day.

The second generation reproductive assessments were conducted using pups from the control and high dose (41.2 mg) groups only. Of the 20 cohabited control pairs, 14 delivered live pups, while 19 of 20 high dose pairs delivered live pups. The controls delivered 10 pups per litter, the treated groups 12 pups. There were no other differences between the groups.

The necropsy of the F1 parent generation showed that there was no difference in female body weights, while female body weight-adjusted liver weight was elevated by 14 % in the treated group. All females showed some degree of hepatocellular degeneration. In males, the only difference between the control and the high dose groups was a 7 % increase in relative epididymis weight of the treated animals. There were no differences in epididymal sperm measures.

In summary, based on the findings, the NOAEL of the study for the reproduction functions was 15.9 mg/kg body weight/day.