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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 1987 - December 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1997
Title:
Unnamed
Year:
1988

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
Reproductive Assessment by Continuous Breeding (RACB) protocol of the US National Toxicology Program (NTP) according to Lamb (1985).
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
RACB protocol consists of Task 1 (initital dose-setting study), Task 2 (continuous breaading phase) Task 3 (crossover mating trial; only in case Task 2 is positive), and Task 4 (F1 offspring dose). In this summary the performance and results of Task 2 are decsribed. Task 3 was not performed because results were negative at Task 2.
Task 2: F0 parents (n=40 in control and n=20 in the 3 test groups) are exposed to the chemical during a 7-day pre-mating period, randomly assigned to a mating pair and treated with the same chemical and dose throughout the 98-day period of continual cohabitation during which multiple litters are born. To encourage immediate remating, in early pregnancies, neonates are removed from the dam within 12 h. The principal toxicity is aberrant reproductive performance in F0 rodents as indicated by alterations in the number of litters per breeding pair or neonatal body weight and sex ratio. After 98 days, breeding pairs are separated and continuously treated until the F1 generation is delivered and weaned. Following the protocol of a negative study, at weaning the pups from the low and mid dose groups were killed and discarded, and the pups from the control and high dose groups were reared by the dams until weaning (PND 21) at which time direct dosing of F1 offspring from the control and high dose groups was initiated for assessment of fertility during a 7-day cohabitation period at the time of sexual maturity (approx. postnatal day 74), and then until necropsy.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Chloroform
EC Number:
200-663-8
EC Name:
Chloroform
Cas Number:
67-66-3
Molecular formula:
CHCl3
IUPAC Name:
trichloromethane
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: Swiss CD-1 (ICR)BR
Sex:
male/female
Details on test animals or test system 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.
All study animals were separately identified and assigned to treatment groups using a stratified randomization procedure based on body weights.
Details on mating procedure:
continuous cohabitation phase (14 weeks); see above for the ful description of the RACB protocol
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 and 3 weeks thereafter); 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 pups from the low and middle dose groups were killed and the pups from the control and high dose groups were reared and dosed throughout the mating period (at approximately postnatal day 74) until necropsy.
Doses / concentrationsopen allclose all
Dose / conc.:
6.6 mg/kg bw/day (actual dose received)
Remarks:
by gavage
Dose / conc.:
15.9 mg/kg bw/day (actual dose received)
Remarks:
by gavage
Dose / conc.:
41.2 mg/kg bw/day (actual dose received)
Remarks:
by gavage
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 (Task 1).
Positive control:
Not used

Examinations

Parental animals: Observations and examinations:
Clinical signs, mortality, body weight, food intake and average consumption of drinking water during representative weeks in both F0 and F1 groups.
Oestrous cyclicity (parental animals):
At the end of the study estrous cyclicity in F1 animals.
Sperm parameters (parental animals):
Epididymal sperm motility, sperm morphology, sperm count In F1 groups
Litter observations:
Number of litters per pair, number of live pups per litter, pup weight per litter, cumulative days to litter (from F0 generation)
Fertility index, number of live pups per litter, pup weight per litter (from F1 generation), pup sex ratio, pup survival.
Postmortem examinations (parental animals):
At weaning the F0 mice were killed and discarded without necropsy
Weight of selected organs: testes, epididymides, prostate, seminal vesiicles, kidneys and liver in F1 groups
Histopathology of liver, lung, thyroid, spleen, esophagus, and accessory sex organs in F1 groups
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.
Reproductive indices:
Fertility index (F1 groups)

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
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 F0 animals died during the study; none of these deaths was considered treatment-related. There were no treatment-related changes in F0 body weights (difference by no more than 2%) or average weekly water consumption per cage. There were no treatment-related effects on F0 fertility and no treatment-related effects were observed on the number of litters per breeding pair, days between litters, live pups per litter, pup sex ratio, or birth weights. Body weights of lactating dams did not differ among control and treated groups with the exception of the high dose dams weighing significantly less than controls on PND 14 (final litter). Postnatal survival, number of still births, and pup weights also were not significantly different between control and treated groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 41.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on BW gain and reproduction parameters

Results: P1 (second parental generation)

Details on results (P1)

In the assessment of fertility in the control and high dose F1 offspring from the last litters, five control and two treated F1 animals died or were sacrificed in extremis during the period between weaning and mating; none of these deaths were considered treatment-related. Female body weight-adjusted liver weight was elevated by 14% compared to the control group in the F1 high dose group ( 41.2 mg/kg body weight/day) by post-natal week 31; this findin was considered non-adverse. Average weekly water consumption was similar between the control and high dose groups.
At necropsy, most F1 organ weights were similar between control and treated animals; however, female relative liver weights and male right epididymis absolute weights were significantly increased in the treated animals compared to controls. However, when the relative (to body weights) epididymal weights were calculated , there was no difference between these two groups. There were no differences between the groups in epididymal sperm parameters (number, motility and morphology); mild to minimal ductal epithelial degeneration was noted in the high dose males; the study investigators, however, considered that these findings may not be treatment-related. All treated females of the high dose group 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.
Fertility was significantly increased in the high dose animals compared to controls although the mating index was unaffected by treatment; of the 20 cohabitated control pairs only 14 delivered live pups while 19 of 20 high dose pairs delivered live pups, the treated pairs also delivered 12 pups per litter versus 10 pups in the control animals; the higher numbers in treated animals are not considered to be treatment-related as in case of reproductive toxicity lower numbers would be expected. No significant differences with treatment were evident in the number of male pups per litter, proportion of male pups, live pups per litter, or birth weight; the number of female pups per litter and litter size were significantly increased in the treated group compared to controls.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Effect level:
15.9 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increase in relative liver weight (females) at the next higher dose level
Dose descriptor:
NOAEL
Effect level:
>= 41.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on reproduction

Results: F1 generation

General toxicity (F1)

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)

The last litter from all dose groups was reared by the dam until weaning. There were no treatment-related alterations in pup-viability (post-natal survival) or in body weight.
At weaning the pups from the low and mid dose groups were killed and discarded, the pups from the control and high dose groups were reared and dosed through the mating period until necropsy

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 41.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on pup viability and fetal weight

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 41.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted on pups

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Reproductive toxicity of chloroform in Swiss mice

Reproductive toxicity in F0 generation

6.6 mg/kg

Male, female

15.9 mg/kg

Male, female

41.2 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

6.6 mg/kg

Male, female

15.9 mg/kg

Male, female

41.2 mg/kg

Male, female

Fertility index

-

-

Increase b)

# live pubs/litter; pub weight/litter

-

-

Increase b), No change

Absolute testis, epididymis weight a)

-

-

No change, Increase (b)

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

-

-

No change

Epidid. Sperm parameters (#, motility, morphology)

-

-

No change

Estrous cycle length

-

-

No observation

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

-) An F2 generation was only generated using pups from the control and high dose groups only

Table 2: General toxicity of chloroform in Swiss mice

General toxicity in the F0 generation

6.6 mg/kg

Male, female

15.9 mg/kg

Male, female

41.2 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

6.6 mg/kg

Male, female

15.9 mg/kg

Male, female

41.2 mg/kg

Male, female

Pub growth to weaning

-

-

No change

Mortality

-

-

No change

Adult body weight

-

-

No change

Kidney weight a)

-

-

No change

Liver weight a)

-

-

No change, Increase b)

Feed consumption

-

-

No observation

Water consumption

-

-

No change

Clinical signs

-

-

No change

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

-) An F2 generation was only generated using pups from the control and high dose groups only

Applicant's summary and conclusion

Conclusions:
Chlorofom had no adverse effect on mouse reproductive endpoints; the NOAEL for reproduction was at least 41.2 mg/kg bw/day. The NOAEL for parental toxicity was 15.9 mg/kg bw, based on increased relative liver weight and hepatocellular degeneration in P1 females at 41.2 mg/kg bw/day. No treatment-related changes were observed in epididymal sperm parameters (number, motility and morphology) and relative weight of epididymides and testis.
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 bw/day. The second generation reproductive assessments were conducted using pups from the control and high dose groups only due to absence of effects on reproduction producing the first generation. 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 P1 parent generation showed an increased relative liver weight (14%) and some degree of hepatocellular degeneration in females treated at 41.2 mg/kg bw/day. In males, the only difference between the control and the high dose groups was an increase in absolute (7%) but not relative epididymis weight in treated animals; there were no differences in epididymal sperm parameters (number, motility and morphology) and no change in weight of testis. Based on these findings, the NOAEL for reproduction was at least 41.2 mg/kg bw/day whereas the NOAEL for parental toxicity (females) was 15.9 mg/kg bw.

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