Bromoform

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 30 December 1986 to 21 December 1987.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Reproductive Assessment by Continuous Breeding

Version / remarks:

The test protocol was devised by United States National Toxicology Program

Principles of method if other than guideline:

The test protocol was devised by the United States National Toxicology Program (NTP) for use in reproductive toxicity testing.

The design is called "Reproductive Assessment by Continuous. Breeding" (RACB). Swiss CD-1 (ICR)BR outbred albino mice are used for the RACB protocol. It consists of four related tasks, not all of which are necessarily performed for a given compound. These tasks include Task 1, dose finding; Task 2, continuous breeding phase; Task 3, identification of the affected sex; and Task 4, offspring assessment. This test protocol is designed to provide both time and cost-effective alternatives to multigeneration studies which produce similar reproductive data.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bromoform

EC Number:

200-854-6

EC Name:

Bromoform

Cas Number:

75-25-2

Molecular formula:

CHBr3

IUPAC Name:

tribromomethane

Test material form:

liquid

Details on test material:

- Name: Bromoform
- Source: Aldrich Chemical Company
- Apperance: Pale yellow liquid
- Batch No: Lot 50304PM
- Received: 28 April 1986
- Storage conditions: Refrigeration

Specific details on test material used for the study:

Test substance samples were analysed for low level impurities by packed and capillary column gas chromatography. Chromatographic purity was determined to be greater than 99%. Bulk stability studies indicated that neat test substance is stable when stored under nitrogen at temperatures up to 60°C for 14 days. Dosage formulation studies indicated no stability problems with the preparation of corn oil solutions at the 80 mg/mL level. Stability studies conducted on corn oil solutions of the test substance (10 mg/mL level) indicated no significant loss of chemical after three weeks storage in sealed glass bottles in the dark at room temperature, when the formulation was overlaid with nitrogen or argon.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY)
- Females (if applicable) nulliparous and non-pregnant: Not stated
- Age at study initiation: 11 weeks (task 2 reproductive assessment)
- Weight at study initiation: Not stated
- Fasting period before study: Not stated
- Housing: The mice were housed 2 per cage by sex during quarantine and the 1-week pre-mating period in solid bottom polycarbonate cages with stainless steel wire lids. The animals were subsequently housed as breeding pairs or individually.
- Diet (e.g. ad libitum): pelleted rodent chow (NIH-07 diet) were provided ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days quarantine period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): Not stated
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 14 hours light / 10 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

An aliquot each from all dosing stocks was analysed for test substance content using gas chromatography. The dosing formulations for the high dose were consistently within 90% of theoretical concentration. On one occasion the mid-dose concentration was 79% of theoretical value, while the remaining values were greater than 90%. The low dose concentrations (5 mg/mL, equivalent to 50 mg/kg) averaged 90% of nominal (range 83-98%).

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Task 2

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Task 2

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Task 2

No. of animals per sex per dose:

20 breeding pairs pairs per treatment group
40 breeding pairs for control group.

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weeks 1, 2, 3, 6, 10 and 14. (week 1 represents the precohabitation phase; week 2 represents the first week of the contiuous breeding phase)


WATER CONSUMPTION: Yes
- Time schedule for examinations: Weeks 1, 2, 3, 6, 10 and 14. (week 1 represents the precohabitation phase; week 2 represents the first week of the contiuous breeding phase)

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: fertility (number producing a litter/number of breeding pairs), litters per pair, live pups per litter, proportion of pups born alive, sex of live pups and the pup body weights immediately after birth.

GROSS EXAMINATION OF DEAD PUPS: Yes for external and internal abnormalities; possible cause of death.

Statistics:

The Cochran-Armitage test was used to test for a dose-related trend in fertility (Task 2).

The number of litters and the number of live pups per litter was computed on a per fertile pair basis and then treatment group means determined. The proportion of live pups is defined as the number of pups born alive divided by the total number of pups produced by each pair. The sex ratio is expressed as the proportion of male pups born alive out of the total number of live pups born to each fertile pair. Dose group means for these parameters 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 WilcoxMann- Whitney U test.

To remove the potential effect of the number of pups per litter on the average pup weight, an analysis of covariance was performed. The covariate used is average litter size including live and dead pups. 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. To control for possible sex differences, these analyses were performed on males, females, and both sexes combined. An analysis of covariance is also 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 are tested for by Jonckheere's test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Six mice died or were sacrificed during this task. For two mice the cause of death could not be determined. The deaths of the other four mice were not considered related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights at delivery of dams receiving 200 mg/kg of test substance were consistently less than control values; these reduced body weights were statistically significant after delivering the first, second, fourth and fifth litters. During the Task 2 holding period, body weights of lactating dams did not differ among control and treatment groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

The average daily consumption of drinking water was significantly increased in males and females treated with 50 mg/kg test substance during weeks 2 and 10. In the 100 mg/kg dose group, male and female consumption was significantly increased during week 10. Consumption was also significantly increased in the 200 mg/kg dose group for males and females during weeks 2, 10, and 14.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Fertility index was 100% for control and all test substance groups. The control and test substance groups did not significantly differ in number of litters per pair, litter size, proportion of live pups and proportion of male pups, female pup weight or combined pup weight at birth. Male absolute pup weights in the 200 mg/kg test group were significantly less than control values but this difference was not significant after pup weights were adjusted for litter size. The cumulative days to litter were essentially identical across groups.

Effect levels (P0)

1

Key result

Dose descriptor:

LOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weights at delivery of dams receiving 200 mg/k bw/day of test substance

2

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

other: Body weights at delivery of dams receiving 200 mg/kg test substance were consistently less than control values.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Eleven F1 mice died during this task. The cause of death for four mice was not determined. The remaining deaths were not considered treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased male terminal body weights were observed at 200 mg/kg bw/day which were considered to be a symptom of general toxicity.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day an increase in adjusted liver weights in both sexes with accompanying hepatocellular cell degeneration, along with a decrease in adjusted kidney weights in both sexes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The livers of 5/20 test susbtance treated mice exhibited a slight reticular pattern.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dose-related histopathologic changes consistong of degeneration of hepatocytes and were observed in the liver of 19/20 test substance treated F1 males (200 mg/kg bw/day). The degeneration was limited to centrolobular regions of the hepatic lobules. The hepatocytes were pale, swollen and the cytoplasm had micro-vacuolisation and granularity. Slight to mild nuclear pleomorphism was present. Occasional individual liver cell necrosis was also present. Two males in the control group and two in the dosed group exhibited minimal to mild hepatitis. Minimal to mild fatty change was observed in the livers of four bromoformtreated male mice. This hepatocellular cell degeneration was observed in 200 mg/k bw/day treated F1 mice was considered to be related to general toxictiy.


Epididymal ductal epithelium degeneration was noted in both control and 200 mg/kg bw/day test substance treated F1 males, and was not considered treatment-related.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The postnatal survival among F1 pups in the 200 mg/kg bw/day test substance group was significantly lower compared to the control group. This decline was primarily attributed to three dams, which lost all their pups by postnatal day 4. One dam in the control group also lost her litter by postnatal day 4. Although these incidences are too low to evaluate statistically, they are consistent with a treatment effect on early maternal behaviour, early lactational failure, or postnatal developmental processes. A proper evaluation of such a connection would require additional studies and could not be assessed further.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Some symptoms of general toxicity were noted in treated F1 mice. These included decreased male terminal body weights, increased adjusted liver weights in both sexes with accompanying hepatocellular cell degeneration, and decreased adjusted kidney weights in both sexes. The teast substance had no effect on epididymal sperm density, motility or morphology in the F1 generation.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Target system / organ toxicity (F1)

1

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

2

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

testes

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Applicant's summary and conclusion

Conclusions:

Treatment with the test substance at up to 200 mg/kg bw/day had no effect reproduction or fertility in either the parental or F1 generation.  Treatment with test substance at 200 mg/kg treatment caused minimal to moderate histopathologic changes in the liver of second generation CD-1 mice.

The No Observed Effect Level (NOEL) for the study is considered to be 100 mg/kg bw/day.
 

Executive summary:

Introduction

The effect of the test substance on reproduction and fertility in Swiss CD-1 mice was assessed using a United States National Toxicology Program Reproductive Assessment by Continuous Breeding Protocol (RACB).

Method

Male and female Swiss CD-1 mice were used for the study.

The RACB protocol is divided into four tasks. Task 1 is 14-day toxicity range-finder, Task 2 is a continuous breading phase, Task 3 is a 1-week mating trial and task 4 is an assessment of offspring. As the test substance had no effect on fertility Task 3 was not conducted as part of this study.

Following dose range-finder study (Task 1) the dose levels selected for Task 2 were 50, 100 and 200 mg/kg bw/day. For task 2 there were 20 male and female breeding pairs per treatment group and 40 male and female breeding pairs for control animals). Animals were dosed daily by gavage using corn oil as the vehicle for a 7-day pre-cohabitation and a 98-day cohabitation period. The last litter born during the holding period following the continuous breeding phase is reared by the dam until weaning, after which treatment is initiated by the same route and at the same concentration as during Task 2. These animals are used for assessment of second generation fertility (Task 4).

Endpoints assessed were clinical signs, parental body weight and average consumption of drinking water during representative weeks, fertility (number producing a litter/number of breeding pairs), litters per pair, live pups per litter, proportion of pups born alive, sex of live pups and the pup body weights immediately after birth. For Task 4 the last and generally the fifth litter was reared and weaned from the control and 200 mg/kg and kept to sexual maturity (74 ± 10 days) while housed by sex two per cage (maximum) and receiving the same treatment as their parents. At sexual maturity a male and female from different litters within the same treatment group are cohabited for 7 days and then housed singly until delivery. The endpoints assessed for this mating trial are the same as Task 2 with the addition of checking for the presence of a copulatory plug.

Results

Task 2: 

 

Six mice died or were sacrificed during this task. For two mice the cause of death could not be determined. The deaths of the other four mice were not considered related to treatment.

 

Fertility index was 100% for control and test substance groups (breeding pair was designated fertile if they produced at least one live or dead pup. The control and bromoform groups did not significantly differ in number of litters per pair, litter size, proportion of live pups and proportion of male pups, female pup weight or combined pup weight at birth. Male absolute pup weights in the 200 mg/kg test substance group were significantly less than control values but this difference was not significant after pup weights were adjusted for litter size. The cumulative days to litter were essentially identical across groups.

 

Body weights at delivery of dams receiving 200 mg/kg of test substance were consistently less than control values; these reduced body weights were statistically significant after delivering the first, second, fourth and fifth litters. During the Task 2 holding period, body weights of lactating dams did not differ among control and treatment groups.

 

Male and female adults were weighed on representative weeks during Task 2 and simultaneously, the water consumption per cage, or per pair, was monitored. Test substance treatment had no significant effect on body weight of males or females. The average daily consumption of drinking water was significantly increased in males and females treated with 50 mg/kg of test substance during weeks 2 and 10. In the 100 mg/kg dose group, male and female consumption was significantly increased during week 10. Consumption was also significantly increased in the 200 mg/kg dose group for males and females during weeks 2, 10, and 14.

Task 4:

Eleven F1 mice died during this task. The cause of death for four mice was not determined. The remaining deaths were not considered treatment related. 

The postnatal survival among F1 pups in the 200 mg/kg bw/day test substance group was significantly lower compared to the control group. This decline was primarily attributed to three dams, which lost all their pups by postnatal day 4. One dam in the control group also lost her litter by postnatal day 4. Although these incidences are too low to evaluate statistically, they are consistent with a treatment effect on early maternal behaviour, early lactational failure, or postnatal developmental processes.

Test substance treatment had no apparent effect on fertility and reproduction in F1 mice. Some symptoms of general toxicity were noted in treated F1 mice. This included decreased male terminal body weights, increased adjusted liver weights in both sexes with accompanying hepatocellular cell degeneration and decreased adjusted kidney weights in both sexes. Epididymal ductal epithelium degeneration was noted in both control and bromoform-treated F1 males, and was not considered treatment-related.

 

Conclusion

Treatment with the test substance at up to 200 mg/kg bw/day had no effect reproduction or fertility in either the parental or F₁generation.  Treatment with test substance at 200 mg/kg treatment caused minimal to moderate histopathologic changes in the liver of second generation CD-1 mice.

The No Observed Effect Level (NOEL) for the study is considered to be 100 mg/kg bw/day.