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Key value for chemical safety assessment

Effects on fertility

Description of key information

In the key study (NTP, 1988; Chapin, 1997), a two-generation reproductive toxicity test was carried out; 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 (highest dose tested) whereas the NOAEL for parental toxicity (females) was 15.9 mg/kg bw.

In a supporting study (Borzelleca, 1982), the reproductive toxicity of chloroform dissolved in drinking water was tested in a modified multigeneration study in ICR mice, receiving the substance at concentration levels of 0, 100, 1000 or 5000 mg/L. Chloroform exhibited considerable hepatotoxic effects at 5000 mg/L. Exposure of mice to this concentration level significantly decreased the mating and gestation index in the P0 and P1 generations and the number of pups per litter. The 1000 mg/L concentration produced only marginal toxicity in the P0/P1 and F1/F2 generations and the reproductive or developmental toxicity parameters were were not affected at the 100 mg/L concentration. No changes were observed in the dominant lethal part of the study, and no teratogenicity was noted. Thus, the NOAEL for general toxicity and reproductive effects of chloroform in ICR mice was 100 mg/L, which would correspond to 17 mg/kg bw/day in case of a mean drinking water intake of 5 mL and a body weight of 30 g (as indicated by the authors).

In a study on developmental neurotoxicity of chloroform in mice it was concluded that the substance did not show such effects at the tested oral dose of 31.1 mg/kg bw/day administered to the dams (Balster, 1982).

Link to relevant study records
Reference
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
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
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
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.
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
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)
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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
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.
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
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
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

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
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
Reproductive effects observed:
no

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

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
15.9 mg/kg bw/day
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Information from animal testing

In a 2 -generation study in mice, chlorofom did not induce adverse effect on reproductive endpoints; the NOAEL for reproduction was at least 41.2 mg/kg bw/day (highest dose tested). 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 (NTP, 1988; Chapin, 1997).  

In the supporting study (Borzelleca, 1982), the ingestion of chloroform dissolved in drinking water at a concentration of 5000 mg/L (corresponding to a nominal dose of 855 mg/kg bw/day) by ICR mice induced a reduction in body weight gain and survival, resulted in increased liver weight and hepatotoxicity and resulted in a decreased mating and gestation index. It also resulted in a reduced number of pups per litter, pup viability and lactation index, and fetal body weight. The 1000 mg/L concentration (corresponding to a nominal dose of 171 mg/kg bw/day) produced liver toxicity in the P0 and P1 generation, but marginal fetal toxicity in the F1 and F2 generations, which was not consistently seen in all subgroups. No changes were observed in the dominant lethal part of the study, and no teratogenicity was seen. The reproductive or developmental toxicity parameters were not affected at 100 mg/Lwhich would correspond to a nominal dose of 17 mg/kg bw/day in case of an assumed (by the authors) mean drinking water intake of 5 mL and a body weight of 30 g. However, it is noted that females have a much higher water intake during lactation. Thus the existence of embryotoxicity was indicated by effects on fertility, litter size, and the gestation index at the high dose of 5000 mg/L. Postnatal survival was marginally affected by the high concentration (the viability index more than the lactation index). Athough no statitically significant effects were observed in the teratology part of the study, the number of total implants and live fetuses per litter appeared to be decreased by the high concentration of CFM. This suggested that the effects observed on reproduction may have been due to a toxic effect on the dams, thus due to the significant maternal toxicity observed, as females appeared more sensitive to the toxic effects than males. Also, there does not appear to be any multigenerational effect associated with the ingestion of CFM; the effects observed in the offspring were also seen in the parents, and effects seemed to decrease in severity from the first generation to the second generation.

In a study on neurodevelopmental toxicity of chloroform in mice it was concluded that the substance did not show such effects at the tested oral dose of 31.1 mg/kg bw/day administered to the dams (Balster, 1982).

Overall, the highest NOAEL for parental toxicity was more or less the same, viz. 15.9 and 17 mg/kg bw/day.

The highest NOAEL for reproductive toxicity was 41.2 mg/kg bw/day (key study); the lowest effect concentration for reproduction toxicity (marginal fetal toxicity) was 171 mg/kg bw/day. Effects on reproduction were seen in the presence of maternal toxicity.

Human information

In a review by Williams (2018), there is no direct evidence of effects on human fertility caused by chloroform exposure. An analysis of the effect of laboratory vs. non-laboratory work on the incidence of spontaneous abortions (loss of the embryo before gestational week 20) was performed on Swedish women born after 1945, who had worked for at least one year between 1990 and 1994 and given birth to at least one child during the period of 1990 to April 1995 (Wennborg et al. 2000). To this end, a cohort of 1052 women was identified and questionnaires were sent to these women. The final analysis was performed on 622 women (856 pregnancies) who had responded to the questionnaire. The study found a slightly increased risk for spontaneous abortions among women working in laboratories with chloroform. The study did include other exposure variables for which no increased risk was found (such as contact with radioactive material, viruses, bacteria). However, the authors of the study did not perform measurements of chloroform concentrations at the work places of women so that a link between chloroform exposure and increased rate of abortion in women is uncertain.

Effects on developmental toxicity

Description of key information

In the key study by Baeder (1991), pregnant Wistar rats were exposed to chloroform vapours at target concentrations of 3, 10 and 30 ppm for 7 hours per day during 10 days ( Day 7 up to and including Day 16 after mating; Day 1 presence of sperm noted). The actual delivered concentrations of chloroform were 0, 3.1, 10.7, or 30.2 ppm (0, 15, 52.2, or 147 mg/m3). Based on decreased body weight gain in dams, a NOAEC of 3 ppm (15 mg/m3) for maternal toxicity was identified. There were small increases in low-weight fetuses at the two lowest concentrations but this effect was not considered adverse; therefore, the NOAEC for developmental effects in this study was 10.7 ppm (52.2 mg/m3) based on the weight of evidence of the data, including comparison to historical controls and the results of a first study in which higher concentrations were investigated (Baeder, 1989). In the other inhalation studies in rats and mice (Schwetz, 1974; Murray, 1979) higher concentrations were tested resulting in LOAECs of 30 or 100 ppm for fetotoxicity. In all studies, feteotoxicity was seen at maternally toxic concentrations, but no indication of a teratogenic effect of chloroform was found in the fetuses examined.

In the oral studies in rats, mice and rabbits (Thompson, 1974; Ruddick, 1983; Balster, 1982) NOAELs for fetotoxicity were as follows: 50 and 200 mg/kg bw/day (rats), at least 50 mg/kg bw/day (rabbits) and at least 31 mg/kg bw/day in mice. Fetotoxicity was seen at maternally toxic doses, but chloroform was not teratogenic in these studies.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Exposure 7 h/day rather than 6 h/day.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: company's own breeding (Hoe:WISKf(SPF71))
- Age at study initiation: 65-70 days
- Weight at study initiation: 195 +/- 9 g
- Fasting period before study: no data
- Housing: one per cage in plastic cages on wood shavings; in pairs in a metal-grid cage, separated by a metal partition dividing (during 10 days of exposure)
- Diet (e.g. ad libitum): standard diet Altromin 1310 pellets (Altromin GmbH, Lage/Lippe, Germany) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 to 23 °C; 21 to 22.5 °C (during 10 days of exposure)
- Humidity (%): 56 to 70 %; 42 to 74 % (during 10 days of exposure)
- Air changes (per hr): 16 to 20 changes per hour; 10 changes per hour (during 10 days of exposure)
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Chloroform was continuously applied by means of a slow infusion injector onto an evaporation head and was vaporised at 80°C. A flow of 800 L/h fed the air-gas mixture into the inhalation chamber via a connecting tube.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of chloroform in the inhalation chamber was measured using a Miran 80 single-beam photometer. Concentrations were measured every 30 minutes.
Target concentrations were 0, 3, 10 and 30 ppm; the actual delivered concentrations of chloroform were 0, 3.1, 10.7, or 30.2 ppm (0, 15, 52.2, or 147 mg/m3).
Details on mating procedure:
Female rats in oestrus were mated with fertile males overnight (15.30 to 7.30 hours) before the start of the study. Day of mating (identified based on the presence of a sperm plug) was defined as gestation day 1.
Duration of treatment / exposure:
From day 7 to 16 after mating (day of mating is GD 1).
Frequency of treatment:
7 hours per day
Duration of test:
Killing of animals, Caesarian section and autopsy on Day 21 of pregnancy
Dose / conc.:
15 mg/m³ air (analytical)
Dose / conc.:
52.2 mg/m³ air (analytical)
Dose / conc.:
147 mg/m³ air (analytical)
No. of animals per sex per dose:
20 pregnant females per concentration group
Control animals:
yes, concurrent no treatment
Maternal examinations:
Daily examination of behaviour and general physical conditions; body weight and feed consumption monitored once a week. The dams were dissected and their organs examined macroscopically. The heart, liver, kidneys and spleen were weighed.
Ovaries and uterine content:
The uterus was opened on Day 21 after mating and the live and dead conceptuses, embryonic primordia under resorption, placentae and corpora lutea in the ovaries were counted and examined macroscopically. The diameter of the embryonic resorption sites and the placental weights were determined. Pregnancy status of dams without fetuses was confirmed via staining the uterus with ammonium sulphide.
Fetal examinations:
Foetuses were examined for signs of life, outward appearance and detectable anomalies. The body weight of foetuses was determined and the crown-rump length was measured. The sex was determined at autopsy.
Half of the foetuses from each litter and those found dead in utero were fixed in alcohol, dissected under a magnifying glass, eviscerated and cleared in potassium hydroxide solution. Skeletons were stained with alazarin red S and examined under a stereomicroscope with regard to developmental stage and skeletal anomalies. The remaining foetuses were fixed in Bouin's solution, cut into cross sections and examined under a stereomicroscope for organ (visceral) anomalies.
Statistics:
In the comparison with the simultaneous control group, a standard MANOVA with sequentially rejective multiple comparisons was used to evaluate the body and organ weights, while a non-parametric linear model of Puri and Sen (1985) with sequentially rejective multiple comparisons was used to evaluated the relative feed consumption.
Implants and corpora lutea were analysed using the Mantel-Haenszel chi-squared test, as were the quotas of live and dead foetuses and of embryonic primordia under resorption. Foetal weights, crown-rump lengths and placental weights were evaluated by analysis of variance which had been corrected by the random litter effect. In the procedures mentioned, a 5% probability of error per parameter group was adhered to. The findings obtained at autopsy and at body cross-section and skeletal examination of the foetuses were evaluated separately for the foetuses and for the litters by the exact Fisher test.
Historical control data:
To compare the study data with previous control data, normal ranges were calculated for groups. They are fixed in such a way that, at probability of 95 %, they contain at least 75 % of the values of a group control of animals. The parallelogram method of Abt (1982) was used to evaluate body and organ weights. The parallelopiped analysis of Wald (1943) was used to evaluate body weight development and feed consumption of the dams and the litter means of the foetal bodyweight, crown-rump length and placental weight. Corpora lutea, implants and survival rates were compared with univariate normal ranges after Wilks (1942). For the quotas of live and dead foetuses and conceptuses under resorption, the ranges were determined by triangular analysis after Rosenkranz (1988).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a slight reduction in weight gain during the exposure period in dams of the 10 and 30 ppm groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The dams in the 30 ppm group displayed a slight reduction in feed consumption during the entire exposure period, this being rather more marked in the first week than in the second.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the 30 ppm group, there was a very slight gain in kidney weights as compared to the control dams, but this observation remained within the range of the historical control data.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
All dams in the treated groups carried live fetuses to term with the exception of one dam in the 30 ppm group, which presented with a total litter resorption having 13 empty implantation sites.
Description (incidence and severity):
20 dams in the 3 and 10 ppm groups and 19 dams in the 30 ppm group carried live foetuses to term. One dam in the 30 ppm group had no fetuses; instead there were merely 13 empty implantation sites in utero. Three dams in the 3 ppm group had a fairly high number of necrotic biastocysts (35.7 to 80%). Two dams in the 10 ppm group had an extremely large number of corpora lutea (20 and 23) in the ovaries. In the dam of the 30 ppm group which had been found to have only empty implantation sites in the uterus, the corpora lutea were small and could not be determined exactly. The final autopsy revealed that one dam in the 3 ppm group, three dams in the 10 ppm group and four dams in the 30 ppm group had moderate to severe unilateral or bilateral renal pelvic dilatation.
Key result
Dose descriptor:
NOAEC
Effect level:
15 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: reduction in BW gain at the next two higher levels tested
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
It was noted that in the 10 ppm and 30 ppm groups, 24 and 26.9% of the foetuses had body weights below 3.0 g, whereas in the control and 3 ppm groups, only 3.2 and 14.2% of the foetuses had body weights below 3.0 g. The mean fetal weight, although statistically significantly lower in the 30 ppm group, was not really different from the fetal weight in the control group and was still within the normal range.
Description (incidence and severity):
The morphological examination revealed no abnormalities in the control or exposure groups, with one exception of an internal hydrocephalus in a live foetus of the 3 ppm group. Numerous foetuses in all exposure groups showed poorly or not yet ossified cranial bones and/or sternebrae, and several foetuses also exhibited fewer than two ossified caudal vertebral centres and or missing ossification of the 5th metacarpal bone. Poorly or not yet ossified vertebral arches and or centres, ribs and phalanges were observed only in a few foetuses in various exposure groups. In many foetuses there was no ossification of the 5th metacarpal bone. The number of control foetuses in which fewer than two caudal vertebral centres and sternebrae were only poorly or not yet ossified was small. On comparison of the findings observed in the compound and control groups, it can be seen that the number of foetuses in the 30 ppm group with poorly ossified cranial bones was statistically higher when compared to the number of affected control foetuses. The same was true for the foetuses in the three exposure groups which exhibited poor ossification of the caudal vertebrae and sternebrae. The number of foetuses in the 10 ppm group which displayed waved and or thickened ribs was greater when compared to the control foetuses. All findings, nevertheless, lay within the range of the historically found spontaneous rate.
Details on embryotoxic / teratogenic effects:
One dam exposed to 30 ppm chloroform via inhalation exhibited only empty implantation sites (i.e., no fetuses were present). A statistically significant increase in the incidence of fetuses with body weights <3 grams and in the incidence of fetuses with slight or no ossification of individual skull bones was observed in the 30-ppm exposed group when compared with controls. The incidence of fetuses with body weights <3 grams was increased in a concentration-related fashion (3.2%, 14.2%, 24%, and 26.9% at 0, 3, 10, and 30-ppm, respectively); this trend did not appear to be due to variations in litter size. However, when the litter was used as the statistical unit of comparison, only litters from the high-concentration group had a significant number of fetuses weighing 3 grams or less. The mean crown-rump length, although statistically significantly lower in the 30 ppm group, was not really different from the crown-rump length in the control group, and was still within the normal range.
A significant increase in the incidence of fetuses with ossification of less than two caudal vertebral centers was observed at all concentrations. A dose response was observed for the incidence of litters with this effect; however, the effect was not statistically significant. Finally, all exposure groups exhibited a significant increase in the incidence of both litters and fetuses with nonossified or weakly ossified sternebrae; however, there was no statistically significant concentration-related trend for this effect. Even though there were increases in low-weight fetuses at the two lowest concentrations, this effect was not considered adverse. No indication of a teratogenic effect of chloroform was found in the foetuses examined.
The skeletal ossification of the foetuses of the dams exposed to chloroform corresponded to the stage of pregnancy. The statistically greater number of foetuses in the 30 ppm group, as compared to the control group, with poorly or not yet ossified cranial bones, and the not yet or only poorly ossified sternebrae and less ossified caudal vertebrae in all three compound groups is not relevant for the evaluation of chloroform, as the number of affected foetuses lies within the range of the control values. As these show, the number of control foetuses in which sternebrae or caudal vertebrae were poorly or not yet ossified was extremely low in comparision with the previous control values. The lack of a relationship between dose and
effect and the fact that, in the previous study (1989), concentrations of 30 to 300 ppm had not affected skeletal ossification in foetuses also tend to contradict a causal relationship with exposure to chloroform.
Therefore, the NOAEC for developmental effects in this study was 10.7 ppm (52.2 mg/m3) based on the weight of evidence of the data, including comparison to historical controls and the higher concentration study (1989).
Key result
Dose descriptor:
NOAEC
Effect level:
52.2 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased fetal weight and ossification at the next higher level
Developmental effects observed:
yes
Lowest effective dose / conc.:
147 mg/m³ air (analytical)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Table 1: Survey of results during gestation and at Caesarian section

Observations

Type

Group 1: 0 ppm

Group 2: 3 ppm

Group 3: 10 ppm

Group 4: 30 ppm

Number of pregnancies

Total

20

20

20

20

Number of females at term with live foetuses

Total

20

20

20

19

Number of corpora lutea

Total

290 N

291 N

301 N

267 N

Mean

14.5

15.6

15.1

14.1

S.D.

1.4

1.7

2.9

1.4

Number of implantations

Total

259 N

255 N

273 N

254 N

Mean

13.0

12.8

13.7

13.4

S.D.

2.2

3.5

2.9

1.5

Pre-implantation loss %

Mean

10.87

13.68

9.41

4.83

Post-implantation loss %

Mean

4.55

3.17

5.80

6.11

Number of total intrauterine deaths

Total

11 N

8 N

15 N

16 N

Mean

0.55

0.40

0.75

0.84

S.D.

0.89

0.60

1.02

1.42

Number of live foetuses

Total

248 N

247 N

258 N

238 N

Mean

12.4

12.4

12.9

12.5

S.D.

2.4

3.5

3.0

1.9

N: within the normal range

Table 2: Survey of results in live foetuses at Caesarian section

Observations

Type

Group 1: 0 ppm

Group 2: 3 ppm

Group 3: 10 ppm

Group 4: 30 ppm

Number of foetuses

Total

248 N

247 N

258 N

238 N

% of implantations

Mean

95.45

96.83

94.20

93.89

Males

%

47.58

44.13

50.39

50.84

Body weight (in g)

Mean

3.4 N

3.2 N

3.2 N

3.2 N *

S.D.

0.3

0.3

0.3

0.3

Crown/Rump length (in mm)

Mean

35.8 N

35.5 N

34.4 N

34.8 N *

S.D.

2.0

2.1

2.6

1.9

Placental weight (in g)

Mean

0.48 N

0.45 N

0.48 N

0.45 N

S.D.

0.07

0.07

0.09

0.07

N: within the normal range; * implants and corpora lutea were analysed using the

Mantel-Haenszel chi-squared test, as were the quotas of live and dead foetuses

and of embryonic primordia under resorption. Foetal weights, crown-rump lengths

and placental weights were evaluated by analysis of variance which had been corrected

by the random litter effect. In the procedures, a 5 % probability of error per parameter

group was adhered to.

Table 3: Observations for individual foetuses - external or visceral defects found at autopsy

Observations

Type

Group 1: 0 ppm

Group 2: 3 ppm

Group 3: 10 ppm

Group 4: 30 ppm

Number of foetuses examined

Total

130

129

135

123

External: retarded foetus

Number

1

2

4

1

%

0.8

1.6

3.0

0.8

External/visceral: no abnormalities detected

Number

121

120

129

112

%

93.1

93.0

95.6

91.1

Blood in thoracic cavity

Min Number

0

2

0

0

%

0

1.6

0

0

Blood in abdominal cavity

Min Number

0

0

0

1

%

0

0

0

0.8

Kidney: pelvis distended – uni- or bilateral

Min Number

8

5

2

9

%

6.2

3.9

1.5

7.3

Skeleton: no abnormalities detected

Number

35

42

36

31

%

26.9

32.6

26.7

25.2

Skull: splitting of bone on parietal bone – left

Min Number

1

0

0

0

%

0.8

0

0

0

Skull: individual skull bones – slight or non-ossification

Ret Number

42

47

48

60 *

%

32.3

36.4

35.6

48.8

Thoracic vert. arch: weakly ossified – amidst normally ossified thoracic vertebrae – right

Ret Number

0

0

0

1

%

0

0

0

0.8

Fragmented thoracic vert. centra

Min Number

1

0

0

0

%

0.8

0

0

0

Lumbar vert. arch: weakly ossified – uni- or bilateral

Ret Number

1

0

3

2

%

0.8

0

2.2

1.6

Sacral vert. arch centra: non-ossified

Ret Number

0

1

1

0

%

0

0.8

0.7

0

Caudal vert. centra: ossification of less than 2 vertebral centres

Ret Number

4

14 *

16 *

14 *

%

3.1

10.9

11.9

11.4

Extra vertebra/extra rib: anlage of a 14 thoracic vertebra with an analogous 14th rib – short and/or normally long – uni- or bilateral

Var Number

1

0

1

2

%

0.8

0

0.7

1.6

Sternebrae: fragmented, longitudinally displaced or dysplasia

Min Number

5

1

4

2

%

3.8

0.8

3.0

1.6

Sternebrae: non-ossified or weakly ossified

Ret Number

7

32 *

35 *

18 *

%

5.4

24.8

25.9

14.6

Rib: weakly ossified – distal part – left or bilateral

Ret Number

0

0

1

0

%

0

0

0.7

0

Rib: shortened 13th – left

Min Number

1

0

0

0

%

0.8

0

0

0

Rib: wavy and/or thickened

Min Number

10

11

22 *

15

%

7.7

8.5

16.3

12.2

Extra rib: at 7th cervical vertebra – short – unilateral

Var Number

0

1

3

0

%

0

0.8

2.2

0

Extra rib: at 1st lumbar vertebra – short – uni- or bilateral

Var Number

52

41

39

30

%

40.0

31.8

28.9

24.4

Pectoral girdle: scapula bent costad and/or shortened – right or bilateral

Min Number

0

1

3

1

%

0

0.8

2.2

0.8

Forepaw: non-ossified metacarpal 5

Ret Number

13

13

21

11

%

10.0

10.1

15.6

8.9

Forepaw – phalanx: phalanx III of 1st to 5th toes non-ossified

Ret Number

0

0

1

0

%

0

0

0.7

0

Hindpaw  - phalanx: phalanx III of 1st to 5th toes non-ossified

Ret Number

0

1

2

0

%

0

0.8

1.5

0

* p 0.05 one sided obtained with Fisher's exact test: group 1 compared with groups 2, 3, 4

Table 4: Observations for individual foetuses - external or visceral defects obtained at body cross-section examination

Observations

Group 1: 0 ppm

Group 2: 3 ppm

Group 3: 10 ppm

Group 4: 30 ppm

# of foetuses examined

Total

118

118

123

115

External/visceral: no abnormalities detected

Number

109

108

111

110

%

92.4

91.5

90.2

95.7

Brain: Hydrocephalus internus

Maj Number

0

1

0

0

%

0

0.8

0

0

Blood in abdominal cavity

Min Number

2

3

1

1

%

1.7

2.5

0.8

0.9

Liver: Lobus dexter or lobus sinister - haematoma

Min Number

1

2

1

0

%

0.8

1.7

0.8

0

Kidney: blood in kidney and vicinity – left – or haematoma in kidney – right

Min Number

1

0

0

1

%

0.8

0

0

0.8

Kidney: pelvis distended – uni- or bilateral

Min Number

5

5

8

3

%

4.2

4.2

6.5

2.6

Kidney/ureter: pelvis and ureter distended – left of bilateral

Min Number

0

1

2

0

%

0

0.8

1.6

0

Conclusions:
Based on decreased body weight gain in dams a NOAEC of 3 ppm could be identified. However, even though there were increases in low-weight fetuses at the two lowest concentrations, this effect was not considered adverse. No indication of a teratogenic effect of chloroform was found in the foetuses examined. Therefore, the NOAEC for developmental effects in this study was 10.7 ppm (52.2 mg/m3) based on the weight of evidence of the data, including comparison to historical controls and the higher concentration study (1989).
Executive summary:

In this supplementary study, groups consisting of 20 female Wistar rats were exposed to chloroform in concentrations of 3, 10 or 30 ppm for 7 hours daily between day 7 and day 16 of pregnancy. A simultaneous control group of the same size inhaled air without the addition of the test compound. On day 21 of pregnancy, the dams were killed and delivered by caesarian section. The foetuses were then examined morphologically for developmental anomalies.

The examinations revealed that repeated inhalation of chloroform in a concentration of 3 ppm during the sensitive phase of embryofoetal organogenesis did not lead to an impairment of the general state of health, nor were there adverse effects upon the feed consumption and body-weight development of the dams or the intrauterine development of the conceptuses. After concentrations of 10 ppm and 30 ppm the dams displayed a slight reduction in feed consumption and body weight development. The foetuses were slightly stunted. On exposure to 30 ppm all embryonic primordia of one dam died in utero. The findings observed in the dams and conceptuses after exposure to 30 ppm correspond with those determined in the previous study (1989) at this concentration.

It can be concluded that, under the study conditions selected, the inhalation of chloroform in a concentration of 3 ppm is tolerated by both pregnant rats and conceptuses. Concentrations of 10 ppm

and above led to slight adverse effects on feed consumption and bodyweight development in the dams. Intrauterine embryonic death seemed to occur at concentrations of

30 ppm and above. The intensity of the maternal and embryonal findings was dose-dependent. No indication of a teratogenic effect of chloroform was found in the foetuses examined. The toxic effect of chloroform on dams and conceptuses which was described in the literature was confirmed, although some findings were different, but no teratogenic potential was observed.

On the basis of the results of the embryotoxicity studies, it can therefore be concluded that the NOAEC for chloroform in the rat, in the case of inhalative exposure, lies at 3 ppm (15 mg/m3) for maternal toxicity and 10 ppm (52.2 mg/m3) for fetotoxicity.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
no guideline available
Principles of method if other than guideline:
The test substance was administered by oral intubation to pregnant rabbits (15/group) on days 6-18 of gestation.
Range-finding test: Total daily doses of 0, 63, 100, 159, 25] or 398 mg/kg/day of chloroform (100 mg/ml in USP corn Oil) were administered twice daily by stomach tube to groups of 5 females each on days 6—18 of gestation. During the course of the study an additional group of 5 does was placed on test and similarly given a total daily dose of 25 mg/kg/day. Range-finding study: During the course of the study, moribund condition or death occurred in 3 of 5 dams given 100 mg/kg/day and in all dams at the 3 higher doses. Necropsy of these females revealed severe acute toxic hepatitis and nephrosis. Doses of 63 mg/kg/day produced anorexia, weight loss, diarrhea, abortion and 1 death. No overt signs of toxicity other than mild diarrhea and intermittent anorexia were observed in 5 dams given 25 mg/kg/day. Each of these dams was pregnant with an average of 7 fetuses per litter. Two of the 4 surviving dams given 63 mg/kg/day were pregnant and each had 6 fetuses. No resorptions were observed in dams from either of these 2 groups. One of the 2 surviving dams given 100 mg/kg/day had 4 resorption sites but no viable conceptuses. The other survivor was not pregnant. Gross examination of fetuses revealed arthrogryposis and cleft palate in a single control fetus; all other fetuses were grossly normal. Of the survivors in the groups given 0, 25, 63 and 100 mg/kg/day, treatment-related histopathologic changes were restricted to mild fatty change of the liver and kidneys in 1 of 2 females given 100 mg/kg/day.
GLP compliance:
no
Remarks:
did not exist at the time
Limit test:
no
Species:
rabbit
Strain:
other: Dutch-Belted
Details on test animals or test system and environmental conditions:
Sexually mature, virgin female Dutch-Belted rabbits,5 1.7—2.2 kg, were housed individually in wire-bottom cages in a controlled environment with a 12-hr daily light period and were conditioned a minimum of 3 weeks. Food‘ and water were available
ad libitum.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Groups of 15 dams each were given oral doses of 0, 20, 35 or 50 mg/kg/day of chloroform (20 mg/mL) on days 6—18 of gestation. Because treatment twice daily increased the incidence of injury to the gingivae by the mouth gag and to the esophagus by the stomach tube, dams were treated once daily. Controls were given an equivalent daily dose of vehicle. Dosing volume was not reported but differed depending on dose.
Details on mating procedure:
The females were given a single 50 IU iv injection of human chorionic gonadotropin 3% min prior to artificial insemination (Gibson et al., 1966). The day of insemination was considered day 0 of gestation.
Duration of treatment / exposure:
from days 6 through 18 of gestation
Frequency of treatment:
daily
Duration of test:
until day 29 of gestation
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
35 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15
Control animals:
yes
Maternal examinations:
Dams were observed daily, and body weights were obtained on days 0, 6, 19 and 29 of gestation. Surviving dams were killed on day 29 by atlanto—occipital dislocation, and the fetuses were removed, weighed, and examined. Representative samples of maternal heart, liver and kidneys were processed for microscopic examination to confirm gross findings.
Ovaries and uterine content:
The number of viable fetuses, implantations, resorptions, corpora lutea and litter size were recorded for each female.
Fetal examinations:
Following cesarean section of does on day 29, fetuses were incubated for 24 hr to determine viability. All fetuses were then sacrificed, weighed, examined for sex determination, skinned, injected intrathoracically and ip with 95% ethanol, and stored in ethanol. Each fetus was dissected and examined for external and visceral abnormalities, then cleared and stained with alizarin red-S for skeletal examinations.
Statistics:
Statistical evaluation of maternal body weight gains, food consumption, implantations, corpora lutea, resorptions, litter size, and fetal weights was made by an analysis of variance and Dunnett’s Two-Tailed Multiple Range test (Steel and Torrie, 1960). Sex ratios and frequency ofanomalies among the fetal population and among litters were analyzed by the chi-square test (Siegel, 1956). In all analyses, the level of significance chosen was p < 0.05.
Details on maternal toxic effects:
During the treatment period, some rabbits from all groups (0, 20, 35 and 50 mg/kg/day) became anorectic and had mild to severe diarrhea. Body weight gains were significantly depressed in dams given 50 mg/kg/day. Abortion, which occurred in dams from both control and treated groups, did not show a dose-response relationship (Table 3, attached). Seven spontaneous deaths (two controls, one in the low dose group and four in the high dose group) occurred among the dams. Death was attributed to acute fibrinous peritonitis in a dam given 20 mg/kg/day and to hepatotoxicity in the 4 dams given 50 mg/kg/day. Examination of dams on day 29 did not reveal treatment-related changes of the liver, kidneys, or other tissues. See tables attached. The mean numbers of corpora lutea, implantations, resorptions, live fetuses and the fetal sex ratios were not significantly different from controls in any dose group.
Key result
Dose descriptor:
NOAEL
Effect level:
35 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: decreased BW gain, and hepatotoxicity at 50 mg/kg/day
Details on embryotoxic / teratogenic effects:
Of the reproductive parameters examined, only the mean body weights of fetuses from dams given 20 or 50 mg kg/day were significantly different from controls, thus no dose-response relationship was obtained (Table 3). The 24-hr fetal survival rate was not adversely affected. Fetal examination did not reveal any major anomalies attributable to maternal chloroform treatment (Table 4). Major cardiovascular anomalies in 2 fetuses from the low dosage group were considered spontaneous as were multiple soft tissue and skeletal defects observed in a control fetus. The incidence of incompletely ossified skull bones (usually parietals) was significantly increased among fetuses, but not litters, of dams given 20 or 35 mg/kg/day, and not at 50 mg/kg/day, thus no dose-response relationship was obtained.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slight effects noted did not show a dose-response relationship
Developmental effects observed:
no
Conclusions:
A NOAEL of at least 50 mg/kg/day was identified for developmental effects; the maternal NOAEL was set at 35 mg/kg/day.
Executive summary:

In a preliminary study, pregnant Dutch-belted rabbits were administered 0, 25, 63, 100, 159, 251, or 398 mg chloroform/kg/day in corn oil on days 6–18 of gestation. Results showed decreased maternal survival in dams administered 100 mg/kg/day or greater. Anorexia, weight loss, diarrhea, abortion, and one maternal death were observed in females administered 63 mg/kg/day. Dams administered 25 mg/kg/day showed signs of mild diarrhea and intermittent anorexia. Oral doses of 100 mg/kg/day or higher were toxic to both the dam and the fetus.

A main study was conducted in which 0, 20, 35, or 50 mg chloroform/kg/day via oral intubation was administered to pregnant rabbits (15/group) on days 6–18 of gestation. Decreased weight gain was reported in dams in the high-dose group. Hepatotoxicity was the cause of four maternal deaths in the high-dose group. No microscopic treatment-related effects were reported in the liver, kidney, or other tissues of the high-dose dams. A statistically significant, non-dose related, decrease in body weight was observed in fetuses of the 20 and 50 mg/kg/day groups when compared to controls. Fetuses from the 20 and 35 mg/kg/day groups, but not of the 50 mg/kg/day group, had a statistically significant increase in the frequency of incompletely ossified skull bones when compared with controls; this effect was not statistically significantly increased when the litter was used as the statistical unit of comparison and was observed in the absence of a dose-response. These findings were not considered evidence of teratogenicity or fetotoxicity by the study authors. Therefore, a NOAEL of at least 50 mg/kg/day was identified for developmental effects; the maternal NOAEL was set at 35 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
In total 3 oral developmental toxicity studies available in rats and rabbits, all showing comparable results, viz. absence of teratogenic effects.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
52.2 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
In total 4 inhalation developmental toxicity studies available in rats and mice, all showing comparable results, viz. absence of teratogenic effects.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Information from animal testing

Pregnant Wistar rats were exposed to chloroform vapours for 7 hours per day for 10 days (Day 7 up to and including Day 16 after mating; Day 1 is presence of sperm) and all animals were killed on Day 21 of gestation in order to examine dams and foetuses (Baeder, 1991). Concentrations of 10 ppm (52 mg/m3) resulted in a decrease in body weight gain of the dams, and 30 ppm (147 mg/m3) led to slight retardation in the conceptuses. Intrauterine embryonic death seemed to occur at concentrations of 147 mg/m3 and greater. This was confirmed in a previous test using higher vapour concentrations (Baeder, 1989). All observed effects were judged as fetotoxic. No indication of a teratogenic effect of chloroform was found in the fetuses examined. It was concluded that a concentration of 3 ppm (15 mg/m3) is tolerated by pregnant rats; the NOAEC for fetotoxicity was 10 ppm (52 mg/m3). In two other studies, higher vapour concentrations were tested in rats or mice; the LOAEC for fetotoxicity was 30 ppm in rats (Schwetz, 1974) and 100 ppm in mice (Murray, 1979). Also in these studies no teratogenicity was observed, and fetotoxicity was seen in the presence of maternal toxicity.

Oral developmental toxicity studies are available in rats and rabbits (Thompson, 1974; Ruddick, 1983). These studies in rats provided information that chloroform administration during gestation may cause adverse effects in the fetuses including decreases in foetal weight and crown-rump length and delayed ossification of the sternebrae and skull bones but no teratogenicity. These fetotoxic effects were seen in the presence of maternal toxicity. In the rabbit study, no developmental toxicity was seen at the highest level tested (50 mg/kg bw/day), whereas the materna NOAEL was set at 35 mg/kg bw/day.

In summary, the available data indicate that chloroform acts in a fetotoxic manner causing decreases in fetal weight and crown-rump length, and delayed ossification. A teratogenic mode of action of chloroform was not observed.

Human information

A population-based case-control analysis of the association of chloroform and other trihalomethanes in drinking water with low birth weight, prematurity and intrauterine growth retardation was performed. The analysis used Iowa birth certificates data from January 1, 1989, to June 30, 1990. The study was restricted to singleton live infants born to non-Hispanic white women 19 years of age or older who were residents of Iowa towns with 1000-5000 inhabitants that derived 100% of their public drinking water from a single source. Exposure was classified using data from a 1987 municipal drinking water survey measuring chloroform in a unified manner. Three exposure categories were used: undetectable chloroform, 1-9 microgram/litre of chloroform, > 10 microgram/litre of chloroform. 159 infants exhibiting birth weights below 2500 g were compared to 795 randomly selected control infants, 342 infants exhibiting prematurity were compared to 1710 randomly selected control infants, and 187 infants with growth retardation were compared to 935 randomly selected control infants. Comparisons showed that there was an increased risk of intrauterine growth retardation associated with higher concentrations of waterborne chloroform and dichlorobromomethane. The elevated risk associated with chloroform remained even when the analysis was restricted to chlorinated water sources, indicating that chloroform has an association over and above that attributable to chlorination alone.

Toxicity to reproduction: other studies

Additional information

Review

Williams (2018) conducted a systemic assessment of the animal and epidemiological data for chloroform to determine if the available evidence indicates that chloroform causes developmental and/or reproductive toxicity. As a first step in this effort, a scoping exercise was undertaken to identify the primary developmental/reproductive concern(s) for chloroform (i.e., female reproductive toxicity, male reproductive toxicity, or developmental toxicity). Based on the results of this scoping effort, a more focused analysis was conducted on the developmental toxicity potential of chloroform. This evaluation involved separate analyses of the animal and epidemiological studies, followed by an integrated assessment of the data.

Initial scoping identified developmental toxicity was the primary area of concern. At levels producing maternal toxicity in rats and mice, chloroform caused decrements in fetal weights and associated delays in ossification. In a single mouse inhalation study, exposure to a high concentration of chloroform was associated with small fetuses and increased cleft palate. However, oral exposure of mice to chloroformat a dose 4 times higher was negative for cleft palate; multiple inhalation studies in rats were also negative. Epidemiologic data on low birth weight and small for gestational age were generally equivocal, preventing conclusions from being drawn for humans. The animal data also show evidence of very early (peri-implantation) total litter losses at very high exposure levels. This effect is likely maternally mediated rather than a direct effect on the offspring. Finally, the epidemiologic data indicate a possible association of higher chloroform exposure with lower risk of preterm birth (<37 weeks gestation).

The available animal data suggest that exposures lower than those causing maternal toxicity should be without developmental effects in the offspring. Also, most studies in humans rely on group-level geographic exposure data, providing only weak epidemiologic evidence for an association with development outcomes and fail to establish a causal role for chloroform in the induction of adverse developmental outcomes at environmentally relevant concentrations.

Justification for classification or non-classification

Based on the animal data available for fertility, the effects of chloroform do not justify a classification; the available data show parental toxicity first, then embryotoxicity at a higher dose, and only at a high dose of 855 mg/kg/bw/day a reduced mating and gestation index (viability index was more affected than gestation index); however at 855 mg/kg bw/day severe parental toxicity was seen consisting of reduced survival and body weight gain and liver toxicity. The available data on developmental toxicity show presence of fetotoxicity consisting of early resorptions at high concentrations or doses, and reduced fetal weight and delayed ossification at lower concentrations or doses. This suggests that the effects observed on reproduction and fetal development most probably are due to a toxic effect on the dams, thus due to the significant maternal toxicity observed, as females appeared more sensitive to the toxic effects than males in these repro studies. No teratogenicity was observed.

According to Commission Regulation (EU) No 944/2013 of October 2013, amending Annex VI to the Regulation (EC) No 1272/2008 (CLP), chloroform is classified for developmental toxicity Category 2, "suspected human reproductive toxicant", with the hazard statement H361d: suspected of damaging the unborn child.

Additional information