Dibromomethane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and appropriate guidelines

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines: OPPTS 870.3550 Reproduction/ Developmental Toxicity Screening test, July 2000

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: TSCA test guidleine 40 CFR 799.9355 (comparable to OECD TG No. 421)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dibromomethane

EC Number:

200-824-2

EC Name:

Dibromomethane

Cas Number:

74-95-3

Molecular formula:

CH2Br2

IUPAC Name:

dibromomethane

Details on test material:

99.4% purity, DBM clear liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment, the males weighed 268 to 336g;
the females weighed 176 to 226g and were approximately eight weeks old.
Environmental conditions were continuously monitored by a computerised system and print-outs of hourly mean temperatures and humidities are included in the study records. The temperature and relative humidity controls were set to achieve target values of 21±2ºC and 55 ±15% respectively.

Administration / exposure

Route of administration:

oral: gavage

Details on exposure:

The test material was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 polyethylene glycol 400.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A
vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation

Details on analytical verification of doses or concentrations:

Samples were taken of each test material formulation and were analysed for concentration of Dibromomethane at Safepharm Analytical Laboratory. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.

Duration of treatment / exposure:

40 days (14 days premating, throughout mating, gestation, parturition and early lactation)

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 150, 500 mg/kg
Basis:

No. of animals per sex per dose:

Three groups each of 10 male and 10 female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats

Control animals:

yes

Details on study design:

The test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for approximately forty days (to include a two week maturation phase, pairing, gestation and early lactation) at dose levels of 500, 150 and 50 mg/kg/day. A control group of ten males and ten females was similarly dosed with vehicle alone (Polyethylene glycol 400).
Clinical signs, bodyweight development, food and water consumption were monitored during the study.
Pairing of animals within each dose group was undertaken on a one male: one female basis on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.
During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size, offspring weights and assessment of righting reflex.
Surviving males were terminated on Day 43 of the study, with all the surviving females and offspring being killed on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Examinations

Parental animals: Observations and examinations:

All animals were examined for overt signs of toxicity, ill-health and behavioural change
immediately before and after dosing, and one and five hours after dosing, during the working
week. Animals were observed immediately before and after dosing, and one hour after dosing at
weekends (except for females during parturition where applicable).

Litter observations:

On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded.
For each litter the following was recorded:
i) Number of offspring born
ii) Number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii) Clinical condition of offspring from birth to Day 4 post partum
iv) Individual offspring and litter weights on Day 1 and 4 post partum

Postmortem examinations (parental animals):

Adult males and females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. Surviving males and a single female that failed to mate were killed on Day 43 of the study. One female that failed to litter was killed on Day 27 post coitum. Females that showed total litter loss were killed on the day the last offspring was found dead. Surviving females were killed on Day 5 of lactation. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone at Day 5 of age.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. The procedure was enhanced (where necessary) by staining the uteri with a 1% ammonium polysulphide solution.

Statistics:

Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance.
Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using nonparametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant treatment-related clinical signs of toxicity observed at 50, 150 or 500 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day,lower bodyweight gain was apparent for females during gestation, compared to control, but this was considered to be a consequence of the lower litter size at this dosage. No similar effects were apparent for animals at 50 or 150 mg/kg/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day,lower bodyweight gain was apparent for females during gestation, compared to control, but this was considered to be a consequence of the lower litter size at this dosage. No similar effects were apparent for animals at 50 or 150 mg/kg/day

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination of adult reproductive tissues did not reveal any treatment-related effects at dosages of 50, 150 or 500 mg/kg/day.

Details on results (P0)

Mating performance, fertility and gestation length - At 500 mg/kg bw/day, there was an increase in pre-coital interval compared with control, with only four females mating during the first four days of pairing. Copulation plug count was generally lower than control, but fertility was not adversely affected with eight females achieving pregnancy. Gestation length of these females tended to be longer than control, possibly influenced by lower litter size.
Litter responses At 500 mg/kg bw/day, litter size was lower than control on Day 1 due to increased post-implantation loss; this loss probably representing death of the offspring in utero. In addition, one female at this dosage was considered to have lost her litter in-utero. No similar treatment
related effects were apparent at 50 or 150 mg/kg bw/day.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

150 other: mg/kg

Sex:

male/female

Results: F1 generation

General toxicity (F1)

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy findings among adult animals and their offspring did not indicate any effect of maternal treatment at dosages of 50, 150 or 500 mg/kg/day.

Histopathological findings:

no effects observed

Description (incidence and severity):

Microscopic examination of adult reproductive tissues did not reveal any treatment-related effects at dosages of 50, 150 or 500 mg/kg/day.

Details on results (F1)

At 50, 150 or 500 mg/kg bw/day, offspring bodyweight on Day 1 and subsequent survival, growth and development to Day 4 were unaffected by maternal treatment; lower litter weights at 500 mg/kgw/day reflected the lower litter size. One control female and one female receiving 500 mg/kg bw/day showed total litter loss on Day 1 post partum.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

150 other: mg/kg

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Treatment with dibromomethane at 500 mg/kg bw/day was associated with effect on mating performance and a reduction in litter size at birth, most probably due to increase in in-utero mortality. The ‘No Observed Adverse Effect Level’ (NOAEL) for reproduction observed in this study was considered to be 150 mg/kg/day.

Executive summary:

Oral (Gavage) Reproduction/Development Toxicity Screening Test in the Rat (Dhinsa and Fulcher 2007) was conducted under GLP standards and in accordance with OECD TG for Testing of Chemicals No.421. (adopted 27 July 1996) and EPA Health Effects Test Guidelines: OPPTS 870.3550 (July 2000). The study was identified as a key study (klimisch rating 1).

The test material (99.4% purity, dibromomethane) was administered by gavage to three groups each of 10 male and 10 female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, at dose levels of 50, 150 and 500 mg/kg/day, for approximately forty days (two week maturation phase, pairing of maximum 14 days, gestation and early lactation). 

A control group was similarly dosed with vehicle only (Polyethylene Glycol 400).

Clinical signs, bodyweight development, food and water consumption were monitored during the study. Animals were paired one male:one female on day 15 of the study, with females subsequently being allowed to litter and rear their offspring to day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size, offspring weights and assessment of righting reflex.

Surviving males were terminated on Day 43 of the study, with all surviving females and offspring being killed on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

 

Effects on Fertility

Mortality: One male, receiving 500 mg/kg bw/day, was killedin extremison Day 2 of the study and one control female was found dead on Day 4 of the study; neither of these deaths were considered to be a consequence of treatment. 

Clinical Observation: There were no significant treatment related-clinical signs of toxicity observed at 50, 150 or 500 mg/kg bw/day.

Bodyweights:At 500 mg/kg bw/day, lower bodyweight gain was apparent for females during gestation, compared to control, but this was considered to be a consequence of the lower litter size at this dosage. No similar effects were apparent for animals at 50 or 150 mg/kg bw/day.

Food consumption and food conversion efficiency:At 500 mg/kg bw/day, lower food conversion efficiency was apparent for females during gestation, compared to control, reflecting the lower weight gain observed during this phase of the study. Food intake was lower during lactation and was attributed to the lower physiological demand on parent females due to the smaller litter size. No similar effects were apparent for animals at 50 or 150 mg/kg bw/day.

Mating performance, fertility and gestation length:At 500 mg/kg bw/day, there was a clear increase in pre-coital interval, compared with control, with only four females mating during the first four days of pairing. Copulation plug count was generally lower than control, but fertility was not adversely affected with 8/ 10 females achieving pregnancy. Gestation length of these females tended to be longer than control, possibly influenced by lower litter size. At 50 or 150 mg/kg bw/day, mating performance, fertility and gestation length were considered to be unaffected by treatment.

Litter responses:at 500 mg/kg bw/day, litter size was lower than control on Day 1 due to increased post-implantation loss; this loss probably representing death of the offspringin-utero.In addition, one female at this dosage was considered to have lost her litterin-utero. No similar treatment related effects were apparent for animals at 50 or 150 mg/kg bw/day.

At 50 or 150 mg/kg bw/day, offspring bodyweight on day 1 and subsequent survival, growth and development to Day 4 were unaffected by maternal treatment; lower litter weights at 500 mg/kg bw/day reflected the lower litter size. One control female and one female receiving 500 mg/kg bw/day showed total litter loss on Day 1post partum.

Necropsy:Necropsy findings among adult animals and their offspring did not indicate any effect of maternal treatment at dosages of 50, 150 or 500 mg/kg bw/day.

Organ weight:At 500 mg/kg bw/day, males showed lower absolute and bodyweight-relative epididymal testes weights compared to control. At 150 mg/kg bw/day, absolute and bodyweight-relative testes weights were also lower than control. In the absence of any treatment-related histopathological effects at 500 mg/kg bw/day, these differences in male reproductive organ weights were considered to be of equivocal toxicological significance.

Histopathology: Microscopic examination of adult reproductive tissues did not reveal any treatment related effects at dosages of 50, 150 or 500 mg/kg bw/day.

Conclusion: Treatment with dibromomethane at 500 mg/kg bw/day was associated with effect on mating performance and a reduction in litter size at birth, most probably due to increase inin-uteromortality. The ‘No Observed Adverse Effect Level’ (NOAEL) for reproduction observed in this study was considered to be 150 mg/kg/day.