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REACH

Sodium bromide

EC number: 231-599-9 | CAS number: 7647-15-6

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The purpose of this study was to provide information concerning the effects of sodium bromide on the male and female reproductive systems in Crl:CD(SD) rats, including gonadal function, estrous cycles, mating behavior, conception, gestation, parturition, lactation, weaning and the growth development and fertility of the offspring. The study was also designed to provide information about effects on neonatal morbidity, mortality and supplementary data on potential prenatal and postnatal development. Male and female P generation rats in Groups 1 through 3 were paired twice, owing to reduced pregnancy rate in Group 3. The first litter formed the F1a generation, dosed from Day 21 postpartum and selected for post-weaning assessments (including reproductive assessments and production of the F2a litters). The (F1b) litter from the second cohabitation of the P generation rats was terminated at day 40 postpartum. Groups 4(Male) and 5(Female; 350 or 500 mg/kg/day, respectively) were terminated at the end of the P generation owing to poor condition in parental animals and low viability in the F1a pups.

Significant adverse effectswere observed on clinical condition, body weight gain and food intake in the P generation, in males treated at 350 mg/kg/day and, to a lesser extent, in females treated at 500 mg/kg/day. Adverse effects on reproductive capacity were observed at these dose levels, with reduced male and female fertility, adverse effects on sperm count and morphology. All males also showed retained spermatid heads of minimal to moderate severity, and 10 females had depleted corpora lutea (although there was no effect on ovarian follicle counts and six of these females became pregnant). Litter size at birth was lower and pup viability, impaired by poor maternal care, was so poor as to preclude a second generation.

Similar but less marked effects on clinical condition, body weight and food intake were observed in males and females treated at 175 mg/kg/day. Mating performance was unaffected by treatment, and although fertility was reduced in both cohabitation periods, overall male fertility was within thehistorical controlrange, which may suggest transient and/or recoverable effects. Fewer males (11/23) showed retention of spermatids, the majority were described as minimal and there was no direct correlation with other effects. Five females were not pregnant at either pairing, but mating and fertility indices were within thehistorical controlrange and only 2 of these had no corpora lutea. There was no adverse effect on gestation, littering, litter size or pup survival growth and development of the F1a or F1b litters and effects in the F1 generation were limited to minimal/mild spermatid head retention in 3 males (compared to one control male) or irregularity of estrous cycle, neither of which adversely affected mating or fertility. There were no adverse effects on the F2 litters.

There were no indicators of toxicity or adverse effectson reproductive parametersin either generationevaluated at50mg/kg/dayofsodium bromide.

The NOAEL for parental toxicity, reproductive performance and pre-and postnatal development was therefore established as 50mg/kg/day

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014- 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

August, 1998.

Deviations:

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

January 22, 2001

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Justification for study design:

The following parameters and end points were evaluated in this study for the P generation: viability, clinical signs, maternal behavior, body weights, body weight changes, food consumption, estrous evaluation, reproductive capacity, gross necropsy findings, ovarian and uterine examinations, male reproductive assessments, organ weights, and histopathological examinations.
The following parameters and end points were evaluated in this study for the F1a generation: viability, clinical signs, developmental evaluations, sexual maturation, body weights, body weight changes, food consumption, estrous evaluation, reproductive capacity, maternal behavior, gross necropsy findings, ovarian and uterine examinations, male reproductive assessments, organ weights, and histopathological examinations.
The following parameters and end points were evaluated in this study for the F1b and F2 generation: viability, clinical signs, sexual maturation, developmental evaluations, body weights, body weight changes, gross necropsy findings, and organ weights.

Specific details on test material used for the study:

Identification: Sodium Bromide
CAS Number: 7647-15-6
Batch (Lot) Number: 710130023, 710120333
Expiration Date: 20 Jan 2015 or 31 May 2016, respectively
Physical Description: White crystalline powder
Storage Conditions: Kept at controlled room temperature

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study because: 1) it is an accepted rodent species for preclinical toxicity testing by regulatory agencies; 2) this species and strain has been demonstrated to be sensitive to developmental toxicants; and 3) historical data and experience exist at the Testing Facility. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test substance. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Husbandry: All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals.

Housing: The rats were individually housed in solid-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of rats was housed in the male rat’s nesting box (solid bottom cage). During the postpartum period, each dam and delivered litter was housed in individual nesting boxes until weaning.

Environmental Conditions: The study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of 10 changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters. Room temperature and humidity were monitored constantly throughout the study. Room temperature was targeted at 66°F to 77°F (19°C to 25°C); relative humidity was targeted at 30% to 70%. An automatically controlled 12-hour light:12-hour dark fluorescent light cycle was maintained. Each dark period began at 1900 hours (± 30 minutes). On five occasions, the lights were turned on during the dark cycle (up to 63 minutes) to facilitate study room activities.

Nesting Material: Bed-O'Cobs® was provided to the animals. Nesting material was changed as often as necessary to keep the animals dry and clean. Analyses for possible contamination were conducted on each lot of nesting material, and results of these analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the nesting material that could interfere with the outcome of the study.

Food: Rats were given Certified Rodent Diet® #5002 (PMI® Nutrition International, lot number SEP 26 14 3A, FEB 23 15 3A) available ad libitum from individual feeders. Chloride content was analyzed by the supplier to ensure it was not less than 0.5% (0.620% [lot SEP 26 14 3A], 0.560% [lot FEB 23 15 3A]). Only the lot of the food analyzed for chlorine content was used. Results of this analysis were provided and maintained in the raw data.
The food was analyzed for nutritional components and environmental contaminants, and results of the analysis are on file at the Testing Facility.
It was considered that there were no known contaminants in the food that would interfere with the objectives of the study.

Water: Water was available ad libitum from individual bottles attached to the cages. All water was from a local source and passed through a reverse osmosis membrane before use. Chlorine was added to the processed water as a bacteriostat; processed water was expected to contain no more than 1.2 ppm chlorine at the time of analysis. Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility.
It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment: For psychological enrichment, male and female rats were provided with a chewing object. Analyses for possible contamination were conducted on each lot of enrichment devices, and results of these analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the enrichment devices that could interfere with the outcome of the study.

Veterinary Care: was available throughout the course of the study and rats were examined by the veterinary staff as needed. Records of veterinary examinations, food supplementations and therapeutic treatments are maintained with the raw data. Food supplementation included the use of moistened meal, fruity bites, and/or pelleted food. Therapeutic treatments included the use of a warming pad, tilted food jar, cold compress, lubricating jelly, Crink L Nest, a resting platform and/or privacy liners. None of the medical examinations, food supplementation, therapeutic treatments or additional husbandry procedures had an adverse impact on the integrity of the study data or on the interpretation of the study results.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Male and female P generation rats in Groups 1 through 3 were paired twice, owing to reduced pregnancy rate in Group 3. The first litter formed the F1a generation, dosed from Day 21 postpartum and selected for post-weaning assessments (including reproductive assessments and production of the F2a litters). The (F1b) litter from the second cohabitation of the P generation rats was terminated at day 40 postpartum. Groups 4(Male) and 5(Female; 350 or 500 mg/kg/day, respectively) were terminated at the end of the P generation owing to poor condition in parental animals and low viability in the F1a pups.

Dose volumes were adjusted based on the most recently recorded body weight. The formulations were stirred continuously during dose administration. Dose administration was completed during the morning of each day. The test or control substance formulations were given using a syringe with attached gavage needle. Termination of dosing in each generation of animals was scheduled after the data were examined by the Sponsor and the possibility of further investigations had been discounted.

P generation males were given the test or control substance formulations once daily by oral gavage beginning 10 weeks before the first cohabitation period, during cohabitation(s) and, gestation, littering and post-partum periods until all F1a and for Groups 1 to 3, F1b generation pups were weaned and continuing through to the day before euthanasia (183-186 dosing days).

P generation females were given the test or control substance formulations once daily beginning 10 weeks before the first cohabitation, during the cohabitation, gestation, littering and post-partum periods until all F1a and (for Groups 1 to 3) F1b generation pups were weaned and continuing through to the day before euthanasia (>181 days). Any dam in the process of parturition was not given the test or control substance formulations until the following work day. Such events were noted in the raw data and tabulated.

The F1a and F1b generation pups were not directly given the test or control substance formulations prior to weaning, but may have been exposed to the test or control substance formulations during maternal gestation (in utero exposure) or via maternal milk or excreta during the lactation period.

One weaned pup per sex from each available litter in Groups 1 to 3 were selected for the F1a generation (Subset A, rearing and mating) were administered the test or control substance formulations once daily beginning on Day 21 postpartum, for at least 10 weeks before cohabitation, during the cohabitation, gestation, littering and post-partum periods until all F2 generation pups were weaned, and continuing through to the day before euthanasia.

The F2a generation pups were not directly given the test or control substance formulations, but may have been exposed to the test or control substance formulations during maternal gestation (in utero exposure) or via maternal milk or excreta during the lactation period.

Details on mating procedure:

Male and female P generation rats in Groups 1 through 3 were paired twice, owing to reduced pregnancy rate in Group 3. The first litter formed the F1a generation, dosed from Day 21 postpartum and selected for post-weaning assessments (including reproductive assessments and production of the F2a litters). The (F1b) litter from the second cohabitation of the P generation rats was terminated at day 40 postpartum. Groups 4(Male) and 5(Female; 350 or 500 mg/kg/day, respectively) were terminated at the end of the P generation owing to poor condition in parental animals and low viability in the F1a pups.

P generation males were given the test or control substance formulations once daily by oral gavage beginning 10 weeks before the first cohabitation period, during cohabitation(s) and, gestation, littering and post-partum periods until all F1a and for Groups 1 to 3, F1b generation pups were weaned and continuing through to the day before euthanasia (183-186 dosing days).
P generation females were given the test or control substance formulations once daily beginning 10 weeks before the first cohabitation, during the cohabitation, gestation, littering and post-partum periods until all F1a and (for Groups 1 to 3) F1b generation pups were weaned and continuing through to the day before euthanasia (>181 days). Any dam in the process of parturition was not given the test or control substance formulations until the following work day. Such events were noted in the raw data and tabulated.

The F1a and F1b generation pups were not directly given the test or control substance formulations prior to weaning, but may have been exposed to the test or control substance formulations during maternal gestation (in utero exposure) or via maternal milk or excreta during the lactation period.

One weaned pup per sex from each available litter in Groups 1 to 3 were selected for the F1a generation (Subset A, rearing and mating) were administered the test or control substance formulations once daily beginning on Day 21 postpartum, for at least 10 weeks before cohabitation, during the cohabitation, gestation, littering and post-partum periods until all F2 generation pups were weaned, and continuing through to the day before euthanasia.

The F2a generation pups were not directly given the test or control substance formulations, but may have been exposed to the test or control substance formulations during maternal gestation (in utero exposure) or via maternal milk or excreta during the lactation period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
Analyses were performed by ion selective electrode measurement using a validated analytical procedure (BRAA00).

Concentration Analysis
Duplicate (5 mL) sets of middle samples for each sampling time point were sent to the analytical laboratory as noted in Section 3.5.3. (Sample Collection and Analysis); triplicate sets of middle samples were taken in the same manner and stored in a refrigerator set to maintain 4°C at the Testing Facility as backup samples until transferred to the analytical laboratory for analysis, as required. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%.

Stability Analysis
Stability analyses performed previously in conjunction with Charles River Laboratories Study Number 20038591, demonstrated that the test substance is stable in the control substance when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Charles River Laboratories Study Number 20038591.
During the course of this study, additional stability of the prepared formulation was determined for 91 days for formulations stored in a refrigerator set to maintain a temperature of 2°C to 8°C over the concentration range 12 to 100 mg/mL. The results of this analysis are described in Appendix 4 (Dose Formulation Analysis Report).

Duration of treatment / exposure:

As described above in sections "Exposure" and "Mating procedure".

Frequency of treatment:

Once daily by oral gavage- as described above in sections "Exposure" and "Mating procedure".

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

175 mg/kg bw/day

Dose / conc.:

350 mg/kg bw/day

Remarks:

males highest dose

Dose / conc.:

500 mg/kg bw/day

Remarks:

Females highest dose

No. of animals per sex per dose:

Control animals:

yes

Details on study design:

Experimental Design - P Generation Male Rats
Group No. Test Material Dose Level (mg/kg/day) Dose Volume (mL/kg) Dose Concentration (mg/mL) No. of Animals
1 R.O. deionized water 0 5 0 24
2 Sodium Bromide 50 5 10 24
3 Sodium Bromide 175 5 35 24
4 Sodium Bromide 350 5 70 24

Experimental Design - P Generation Female Rats
Group No. Test Material Dose Level (mg/kg/day) Dose Volume (mL/kg) Dose Concentration (mg/mL) No. of Animals
1 R.O. deionized water 0 5 0 24
2 Sodium Bromide 50 5 10 24
3 Sodium Bromide 175 5 35 24
5 Sodium Bromide 500 5 100 24

Experimental Design - F1 Generation Male Rats
Group No. Test Material Dose Level (mg/kg/day) Dose Volume (mL/kg) Dose Concentration (mg/mL) No. of Animals
F1a F1b
1 R.O. deionized water 0 5 0 23 21
2 Sodium Bromide 50 5 10 22 22
3 Sodium Bromide 175 5 35 15 14
4 Sodium Bromide 350 5 70 0 -
- = Not applicable

Experimental Design - F1 Generation Female Rats
Group No. Test Material Dose Level (mg/kg/day) Dose Volume (mL/kg) Dose Concentration (mg/mL) No. of Animals
F1a F1b
1 R.O. deionized water 0 5 0 23 21
2 Sodium Bromide 50 5 10 22 22
3 Sodium Bromide 175 5 35 15 14
5 Sodium Bromide 500 5 100 0 -
- = Not applicable

Also see attachment of study schematic dosing period

Positive control:

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The rats were assessed for viability at least twice daily during the study Clinical Observations
General Appearance- once during acclimation, on the day of randomization, daily during the dose period, and on the day of scheduled euthanasia.
Postdose Observations- were recorded at approximately hourly intervals for the first 4 hours and at the end of the normal working day for the first week of administration for each sex. Beginning with the 8th dose, postdose observations were recorded between 2 and 4 hours after dose administration and at the end of the normal working day. Starting with the 58th dose, postdose observations were recorded between 2 and 4 hours after dose administration
Maternal observations- were recorded once daily during the postpartum period
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage for 14 consecutive days before initiation of the cohabitation period and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. Samples were also collected on the day of scheduled euthanasia.

BODY WEIGHT: Yes
- Time schedule for examinations:
For the males - on the day after arrival, once during the acclimation period, once weekly during the dose period and on the day of scheduled euthanasia.
For the females- on the day after arrival, once during the acclimation period, once weekly during the dose period (including but not limited to: DGs 0, 7, 10, 14, and 20, DLs 0, 4, 7, 14, and 21), and the day of scheduled euthanasia .

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER: In addition, the following parameters and end points were evaluated in this study for the P generation: maternal behavior, estrous evaluation, reproductive capacity, gross necropsy findings, ovarian and uterine examinations, male reproductive assessments, organ weights, and histopathological examinations.

Oestrous cyclicity (parental animals):

Yes. Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage for 14 consecutive days before initiation of the cohabitation period and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. Samples were also collected on the day of scheduled euthanasia.

Sperm parameters (parental animals):

Sperm motility
Sperm concentration
Sperm morphology

Litter observations:

Yes

Postmortem examinations (parental animals):

Yes

Postmortem examinations (offspring):

Yes

Statistics:

Yes

Offspring viability indices:

Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Administration of 350 mg/kg/day to males and 500 mg/kg/day of sodium bromide to females produced severe toxicity, adverse clinical observations, including dehydration, ungroomed coat, chromodacryorrhea, hunched posture, ptosis, urine-stained abdominal fur, decreased motor activity, chromorhinorrhea, ataxia, piloerection, low carriage, thin body condition, and bradypnea, with effects generally more severe in males.

In the 175 mg/kg/day dose group, similar clinical signs occurred but they were less marked and at a lower incidence, especially in females.

Administration of 50 mg/kg/day sodium bromide had no adverse effect on body weight gain or food intake in males or females of the P or F1 generation and any other findings were regarded as not adverse.

Mortality:

mortality observed, treatment-related

Description (incidence):

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Administration of 350 mg/kg/day to males and 500 mg/kg/day of sodium bromide to females produced reduced body weight gain was observed in males from week 3 onwards and body weight at the end of the dosing period was 75% of control values: food intake was also lower from week 4 onwards. In females, reduced body weight gain and food intake was observed only during late gestation and lactation.

In the 175 mg/kg/day dose group, effects on body weight were only observed in males and were more moderate, with a terminal mean body weight of 86.8% of the control value, and significantly reduced food intake from week 6 onwards. Female food intake was reduced only in early lactation.

Administration of 50 mg/kg/day sodium bromide had no adverse effect on body weight gain or food intake in males or females of the P or F1 generation and any other findings were regarded as not adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see above

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology
Test substance-related microscopic findings were noted in the reproductive tract of animals that had received 175 or 350 mg/kg/day sodium bromide, with a dose-related trend in incidence and severity.
In males, a subtle increase in spermatid head retention was identified, particularly in stage XI tubules; less frequently in stage IX, X, or XII tubules. An increase in sperm retained at the surface of the tubular lumen was also noted. Increased debris (nucleated cells and amorphous eosinophilic material, which often surrounded sperm with curled tails) in the epididymis, also showed a test substance-related trend in males All males (20) at 350 mg/kg/day were affected with severity
ranging from mild to moderate; at 175 mg/kg/day, 11/23 males were affected, with the majority showing only minimal changes and only 4 showing epididymal debris.
In females test article-related depletion of corpora lutea was present in the ovaries of 3 females administered 175 mg/kg/day sodium bromide and 10 females treated at 500mg/kg/day surviving to terminal kill. A further high dose female, killed in week 23, also showed depletion of corpora lutea.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of sodium bromide.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

At 350 mg/kg/day there were slightly fewer estrous stages in the 14 day assessment period, owing to some females with extended periods of diestrus.
No corpora lutea were found in the ovaries of 10 females in the 500 mg/kg/day dose group, but overall follicle counts were not affected and 6 of these females had at least one pregnancy. There were no other treatment-related effects on female reproductive organ weights or histopathology.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

At 350 mg/kg/day effects on sperm motility, morphology and sperm count were identified but there were no adverse effects on testicular spermatid counts or reproductive organ weights.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

350/500 mg/kg/day:
Only two males treated did not mate with either a treated or untreated female (mating index 89.5%) but only 11/19 (64.7%) mated females became pregnant. There were no findings in either of the non-mated males which differed from mated males. All males in this group showed minimal/mild tubular spermatid retention and/or minimal- moderate Sertoli cell spermatid retention and 19/20 showed associated cellular debris in the epididymis. As these findings were also observed in males which died or were killed in week 12 they were likely present during the mating period. There was no apparent correlation, however, between the severity of the findings and pregnancy outcome of the pairings with treated or untreated females.
In females treated at 500 mg/kg/day, 20 of 22 females mated with treated or untreated males, and 15 were pregnant. Five females mated but were not pregnant, one of which was paired with an untreated male. Only 3 of these females had no corpora lutea present at follicle count. Two females did not mate or become pregnant with either treated or untreated males, one of which was also recorded as having no corpora lutea. The mating index was 45.5% for females mated with treated males and 90.9% (within the historical control range) including untreated males. The overall female fertility index (with treated/untreated males) was 75% and with treated males was 60%, significantly lower than the control index (p≤ 0.01) for the treated/untreated males. Six pregnant females were recorded as having no corpora lutea, 4 of which mated only with an untreated male. There was also evidence of a delay in mating as only 6/20 matings occurred in the first 5 days of pairing, compared to 20/24 in control females.
There was no adverse effect on the duration of gestation or gestation index but no litters survived after day 5 post-partum. There were reductions in litter size at birth, the number of liveborn pups and pup survival. There was evidence of poor maternal care as pups were thin, cold to touch, not nursing, had no milk band present and had mild to moderate dehydration.
Owing to reduced group size (due to unscheduled deaths/terminations), declining clinical condition, poor reproductive performance and a marked effect on pup viability, animals treated at 350/500 mg/kg/day were not re-paired for a second cohabitation and the high dose group was terminated at the end of the P generation.

175 mg/kg/day
Of 24 males paired with treated females in the first cohabitation period, 22 were confirmed mated and 16 of the mated females were pregnant. Of 2 males re-paired, one mated with the untreated females, so the mating index was 95.8% with all females and 91.7% with treated females, and the fertility index for treated and untreated females was 73.9%. In the second cohabitation period, 22 males were paired, 19 mated and 14 females were pregnant. The mating index was 86.4% and the fertility index was 73.7%, significantly lower than concurrent controls (p≤ 0.01). but only slightly lower than the historical control range.
In females, there was no adverse effect on estrous cycles. In the first cohabitation period, 22/24 females paired with treated males mated and 16 were pregnant. Two females did not mate and 6 females mated but did not deliver. In the second cohabitation period, 19/22 paired females mated and 14 were pregnant. Two of the 5 non-pregnant females had not been pregnant at the first cohabitation period, and one had not mated. Two of the females which did not mate had no corpora lutea at follicle count and histopathology, as did one female not pregnant at the second cohabitation.
At 175 mg/kg/day, other than the lower number of females which delivered, there were no adverse effects on duration of gestation, gestation index, number of pups born, pup viability, sex ratio, anogenital distance, growth or physical development (as assessed by pinna unfolding, hair growth, tooth eruption and eye opening) in either the F1a or F1b litters.

50 mg/kg/day
All males mated, but 2 did not impregnate a female at the first cohabitation. At the second cohabitation, 22 of 23 paired males mated and all mated males impregnated a female.
Of the 24 females paired with treated males at the first cohabitation period, all were confirmed mated and 22 were pregnant. In the second cohabitation period, only one female did not mate and all mated females were pregnant. There were no adverse effects on gestation or littering parameters or on pup survival, growth or physical development.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other:

Remarks:

Administration of 350 mg/kg/day to P males or 500 mg/kg/day for P females elicited severe toxicity, characterised by mortality, adverse clinical signs including ataxia, sedation, lack of motor co-ordination, hunched posture, dehydration, lack of grooming, and consideration should be given to the potential for under-reporting of such behaviours in nocturnal animals. There was also reduced body weight gain and food intake, most severe in males (26% reduction in weight gain over the dosing period). These effects were considered so severe as to preclude proper assessment of reproductive performance. Similar effects were observed at 175 mg/kg/day, but the clinical sings were less severe, effects on food intake were more moderate and male weight gain over the dosing period was 13% lower than controls

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

350 mg/kg bw/day (actual dose received)

System:

female reproductive system

Organ:

lungs

other: Spermatid head retention in P0 Males. Depleted corpora lutea in P0 Females.

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

Haematological findings:

not examined

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 175 mg/kg/day male body weights were significantly reduced (p≤ 0.01) after week 9 and mean body weights were 89.9% of the control group value at the end of the dosing period.
Food consumption was reduced from week 7 onwards. There was no effect on female body weight or food intake.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see above.

Food efficiency:

not examined

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

not examined

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

F1-generation
A total of 23, 22 and 15 pups per sex per litter from Groups 1, 2 and 3 were selected from the F1a litters to form the F1 generation. The following parameters and end points were evaluated in this study for the F1a generation: viability, clinical signs, developmental evaluations, sexual maturation, body weights, body weight changes, food consumption, estrous evaluation, reproductive capacity, maternal behavior, gross necropsy findings, ovarian and uterine examinations, male reproductive assessments, organ weights, and histopathological examinations.

There was no effect on mating or fertility of males and females at 175 mg/kg/day. Although the total count and number of motile sperm in the vas deferens was lower than controls, these values were within the historical control range and an association with treatment was considered questionable. There were no effects on testicular sperm counts, morphology, reproductive organ weights or pathology, with the exception of 3 males showing a minimal/mild spermatid head retention at histopathology (although there was no deficit in vas deferens sperm count or motility and one control male showed the same effect, of mild severity). There were no effects on female reproductive organ weights, although the number of estrous stages in the evaluation period was slightly lower than controls and there were some differences in certain follicle types at ovarian examination for which, in view of effects at the higher dosage, the possibility of an association with treatment could not be discounted.

There was no adverse effect on gestation, littering, litter size or pup survival growth and development of the F1a or F1b litters and effects in the F1 generation were limited to minimal/mild spermatid head retention in 3 males (compared to one control male) or irregularity of estrous cycle/differences in follicle counts, none of which adversely affected mating or fertility. There were no adverse effects on the F2 litters.

Key result

Dose descriptor:

NOAEL

Generation:

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on the clinical observations seen at the 175mg/kg.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no adverse effects on the F2 litters.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no adverse effects on the F2 litters.
There was no adverse effect of treatment on gestation, gestation index, litter size at birth of the F2 pups or pup viability, growth and physical development

The following parameters and end points were evaluated in this study for the F1b and F2 generation: viability, clinical signs, sexual maturation, developmental evaluations, body weights, body weight changes, gross necropsy findings, and organ weights.
There were no adverse effects on the F2 litters.
There was no adverse effect of treatment on gestation, gestation index, litter size at birth of the F2 pups or pup viability, growth and physical development.

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

Conclusions:

In conclusion, significant adverse effects were observed on clinical condition, body weight gain and food intake in the P generation, in males treated at 350 mg/kg/day and, to a lesser extent, in females treated at 500 mg/kg/day. Adverse effects on reproductive capacity were observed at these dose levels, with reduced male and female fertility, adverse effects on sperm count and morphology. All males also showed retained spermatid heads of minimal to moderate severity, and 10 females had depleted corpora lutea (although there was no effect on ovarian follicle counts and six of these females became pregnant). Litter size at birth was lower and pup viability, impaired by poor maternal care, was so poor as to preclude a second generation.

Similar but less marked effects on clinical condition, body weight and food intake were observed in males and females treated at 175 mg/kg/day. Mating performance was unaffected by treatment, and although fertility was reduced in both cohabitation periods, overall male fertility was within the historical control range, which may suggest transient and/or recoverable effects. Fewer males (11/23) showed retention of spermatids, the majority were described as minimal and there was no direct correlation with other effects. Five females were not pregnant at either pairing, but mating and fertility indices were within the historical control range and only 2 of these had no corpora lutea.
There were no indicators of toxicity or adverse effects on reproductive parameters in either generation evaluated at 50 mg/kg/day of sodium bromide.
The NOAEL for parental toxicity, reproductive performance and pre-and postnatal development was therefore established as 50 mg/kg/day

There was no adverse effect on gestation, littering, litter size or pup survival growth and development of the F1a or F1b litters and effects in the F1 generation were limited to minimal/mild spermatid head retention in 3 males (compared to one control male) or irregularity of estrous cycle, neither of which adversely affected mating or fertility. There were no adverse effects on the F2 litters.

Executive summary:

SUMMARY

The study design was as follows and as outlined in the study schematic below.

Experimental Design - P Generation Male Rats

Group No.	Test Material	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	No. of Animals
1	R.O. deionized water	0	5	0	24
2	Sodium Bromide	50	5	10	24
3	Sodium Bromide	175	5	35	24
4	Sodium Bromide	350	5	70	24

Experimental Design - P Generation Female Rats

Group No.	Test Material	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	No. of Animals
1	R.O. deionized water	0	5	0	24
2	Sodium Bromide	50	5	10	24
3	Sodium Bromide	175	5	35	24
5	Sodium Bromide	500	5	100	24

Experimental Design - F1 Generation Male Rats

Group No.	Test Material	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	No. of Animals
Group No.	Test Material	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	F1a	F1b
1	R.O. deionized water	0	5	0	23	21
2	Sodium Bromide	50	5	10	22	22
3	Sodium Bromide	175	5	35	15	14
4	Sodium Bromide	350	5	70	0	-
- = Not applicable

Experimental Design - F1 Generation Female Rats

Group No.	Test Material	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	No. of Animals
Group No.	Test Material	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	F1a	F1b
1	R.O. deionized water	0	5	0	23	21
2	Sodium Bromide	50	5	10	22	22
3	Sodium Bromide	175	5	35	15	14
5	Sodium Bromide	500	5	100	0	-
- = Not applicable

Dose volumes were adjusted based on the most recently recorded body weight. The formulations were stirred continuously during dose administration. Dose administration was completed during the morning of each day. The test or control substance formulations were given using a syringe with attached gavage needle.

Termination of dosing in each generation of animals was scheduled after the data were examined by the Sponsor and the possibility of further investigations had been discounted.

P generation males were given the test or control substance formulations once dailyby oral gavagebeginning 10 weeks beforethe first cohabitation period, during cohabitation(s) and,gestation, littering and post-partum periods until all F1a and for Groups 1 to 3, F1b generation pups were weaned and continuing through to the day before euthanasia (183-186 dosing days).

P generation females were given the test or control substance formulations once daily beginning 10 weeks beforethe first cohabitation, during the cohabitation, gestation, littering and post-partum periods until all F1a and (for Groups 1 to 3) F1b generation pups were weaned and continuing through to the day before euthanasia (>181 days). Any dam in the process of parturition was not given the test or control substance formulations until the following work day. Such events were noted in the raw data and tabulated.

The F1a and F1bgeneration pups were not directly given thetest or control substance formulations prior to weaning, but may have been exposed to thetest or control substance formulationsduring maternal gestation (in utero exposure) or via maternal milkor excreta during the lactation period.

One weaned pup per sex from each available litter in Groups 1 to 3 were selected forthe F1ageneration (Subset A, rearing and mating) were administered the test or control substance formulations once daily beginning on Day 21 postpartum,forat least 10 weeks before cohabitation, during the cohabitation, gestation, littering and post-partum periods until all F2 generation pups were weaned,and continuing through to the day before euthanasia.

The F2ageneration pups were not directly given thetest or control substance formulations, but may have been exposed to thetest or control substance formulationsduring maternal gestation (in utero exposure) or via maternal milk or excreta during the lactation period.

The following parameters and end points were evaluated in this study for the F1a generation: viability, clinical signs, developmental evaluations, sexual maturation, body weights, body weight changes, food consumption, estrous evaluation, reproductive capacity, maternal behavior, gross necropsy findings, ovarian and uterine examinations, male reproductive assessments, organ weights, and histopathological examinations.

P Generation Male and Female Rats

Systemic Toxicity

Administration of 350 mg/kg/day to males and 500 mg/kg/day of sodium bromide to females produced severe toxicity, characterized by increased mortality (4 males and 9 females died or were terminated early) andadverse clinical observations, including dehydration, ungroomed coat, chromodacryorrhea, hunched posture, ptosis, urine-stained abdominal fur, decreased motoractivity, chromorhinorrhea,ataxia, piloerection, low carriage, thin body condition, and bradypnea, with effects generally more severe in males. Reduced body weight gain was observed in males from week 3 onwards and body weight at the end of the dosing period was 75% of control values: food intake was also lower from week 4 onwards. In females, reduced body weight gain and food intake was observed only during late gestation and lactation.

In the 175 mg/kg/daydose group,similar clinical signs occurred but they were less marked and at a lower incidence, especially in females. Effects on body weight were only observed in males and were more moderate, with aterminal mean body weight of 86.8% of the control value, and significantly reduced food intake from week 6 onwards. Female food intake was reduced only in early lactation. In the F1 generation,malebody weightgain was lowerafter week 4 and body weight at the end of the dosing period(PND 147)was87.9%of the control group value. Female body weights were not affected and values at the end of the premating period(PND 91)and the end of gestation were 97% and 94.9% of control group values, respectively. Male food intake was reduced from week 2 onwards, but female food intake was reduced in late gestation and early lactation only.

Administration of 50 mg/kg/day sodium bromide had no adverse effect on body weight gain or food intake in males or females of the P or F1 generation and any other findings were regarded as not adverse.

Effects on male and female reproduction and development of the offspring

350/500 mg/kg/day

At 350mg/kg/day effects on sperm motility, morphology and sperm count were identified but there were no adverse effects on testicular spermatidcountsorreproductive organ weights. No corpora lutea were found in the ovaries of 10 females in the 500 mg/kg/day dose group, but overall follicle counts were not affected and6 of these females had at least one pregnancy. There were no other treatment-related effects on female reproductive organ weights orhistopathology. There were, however, slightly fewerestrous stagesin the14 dayassessment period, owing to some females with extendedperiods of diestrus.

Only two males treated at 350 mg/kg/day did not mate with either a treated or untreated female (mating index 89.5%) but only 11/19 (64.7%) mated females became pregnant. There were no findings in either of the non-mated males which differed from mated males. All males in this group showed minimal/mild tubular spermatid retention and/or minimal- moderate Sertoli cell spermatid retention and 19/20 showed associated cellular debris in the epididymis. As these findings were also observed in males which died or were killed in week 12 they were likely present during the mating period. There was no apparent correlation, however, between the severity of the findings and pregnancy outcome of the pairings with treated or untreated females.

In females treated at 500 mg/kg/day, 20 of 22 females mated with treated or untreated males, and 15 were pregnant. Five females mated but were not pregnant, one of which was paired with an untreated male. Only 3 of these females had no corpora lutea present at follicle count. Two females did not mate or become pregnant with either treated or untreated males, one of which was also recorded as having no corpora lutea. The mating index was 45.5% for females mated with treated males and 90.9% (within the historical control range) including untreated males. The overall female fertility index (with treated/untreated males) was 75% and with treated males was 60%, significantly lower than the control index(p≤ 0.01)for the treated/untreated males. Six pregnant females were recorded as having no corpora lutea, 4 of which mated only with an untreated male. There was also evidence of a delay in mating as only 6/20 matings occurred in the first 5 days of pairing, compared to 20/24 in control females.

There was no adverse effect on the duration of gestation or gestation index but no litters survived after day 5 post-partum. There were reductions in litter size at birth, the number of liveborn pups and pup survival. There was evidence of poor maternal care aspups werethin,cold to touch, not nursing,hadno milk band presentand hadmild to moderate dehydration.

Owing to reduced group size (due to unscheduled deaths/terminations), declining clinical condition, poor reproductive performance and a marked effect on pup viability, animals treated at 350/500 mg/kg/day were not re-paired for a second cohabitation and the high dose group was terminated at the end of the P generation.

175 mg/kg/day

Of 24 males paired with treated females in the first cohabitation period, 22 were confirmed mated and 16 of the mated females were pregnant. Of 2 males re-paired, one mated with the untreated females, so the mating index was 95.8% with all females and 91.7% with treated females, and the fertility index for treated and untreated females was 73.9%. In the second cohabitation period, 22 males were paired, 19 mated and 14 females were pregnant. The mating index was 86.4% and the fertility index was 73.7%, significantly lower than concurrent controls(p≤ 0.01). but only slightly lower than the historical control range.

In females, there was no adverse effect on estrous cycles. In the first cohabitation period, 22/24 females paired with treated males mated and 16 were pregnant. Two females did not mate and 6 females mated but did not deliver. In the second cohabitation period, 19/22 paired females mated and 14 were pregnant. Two of the 5 non-pregnant females had not been pregnant at the first cohabitation period, and one had not mated. Two of the females which did not mate had no corpora lutea at follicle count and histopathology, as did one female not pregnant at the second cohabitation.

At 175 mg/kg/day, other than the lower number of females which delivered, there were no adverse effects on duration of gestation, gestation index, number of pups born, pup viability, sex ratio, anogenital distance, growth or physical development(as assessed bypinna unfolding, hair growth, tooth eruption and eye opening)in either the F1a or F1b litters.

50 mg/kg/day

All males mated, but 2 did not impregnate a female at the first cohabitation. At the second cohabitation, 22 of 23 paired males mated and all mated males impregnated a female.

Of the 24 females paired with treated males at the first cohabitation period, all were confirmed mated and 22 were pregnant. In the second cohabitation period, only one female did not mate and all mated females were pregnant. There were no adverse effects on gestation or littering parameters or on pup survival, growth or physical development.

Control group

The mating index for males in the first cohabitation period was 95.8% and the fertility index was 100%. The mating and fertility indices for males in the second cohabitation period were 100%, and for females were 100% in both cohabitation periods. In comparison, values from historical control ranges for the species and strain at the laboratory were 75-100% for both mating and fertility for males, and 75-100% and 76%-100% for mating and fertility in females, respectively.

Overall mating and fertility performance (first and second cohabitation periods)

In total, over both cohabitation periods, all males in Groups 1, 2 and 3 mated at least one female.

All control males impregnated at least one female and there was only one male in the 50 mg/kg/day group which did not achieve a pregnancy.

At 175 mg/kg/day, although reduced pregnancy rates were observed at both cohabitation periods, unusually, the affected animals differed and in total only 2 males did not impregnate a female, giving an overall male fertility index of 91.7% and 77.3% for males and treated females (within thehistorical controlrange). This was likely a deficit in these males, as neither of the treated females allocated to one male became pregnant at the alternative pairing and the allocated untreated female was not mated, but the females allocated to the other male both became pregnant with alternative males.

At histopathology, minimal/mild spermatid retention was noted in the testes of both these males and both males had >10% abnormal sperm at seminology evaluation : one further male also had minimal lymphocytic infiltration of the epididymis: however, all these findings were observed in several other males in this group which achieved a pregnancy, and their results for other semen parameters were within the range of the remainder of the group. Spermatid retention (Sertoli cell and/or tubular, minimal/mild) was observed in 9 other males in this group but all impregnated at least one female, as did the remainder of the group, suggesting any effect on fertility may have been temporary and/or recoverable.

All females in Groups 1, 2 and 3 mated during either the first or second cohabitation periods. Five females in the 175 mg/kg/day group did not get pregnant from either pairing (with treated males only), giving an overall female fertility index of 77.3% (17/22), below the concurrent control value but within thehistorical controlrange. Only two of these females showed marked depletion of corpora lutea at histopathology and no corpora lutea at ovarian follicle examination. One of these females had also shown extended estrus (9 days) and a further female, showed extended periods of diestrus (which may indicate pseudopregnancy). Another female which had marked depletion of corpora lutea at histopathology and no corpora lutea at follicle counting, was pregnant at the first pairing (but not at the second).

F1-generation

A total of 23, 22 and 15 pups per sex per litter from Groups 1, 2 and 3 were selected from the F1a litters to form the F1 generation

At 175 mg/kg/day malebody weights were significantly reduced(p≤ 0.01)after week 9 and mean body weights were 89.9% of the control group valueat the end of the dosing period.

Food consumption was reduced from week 7 onwards. There was no effect on female body weight or food intake.

There was no effect on mating or fertilityof males and females at 175 mg/kg/day. Althoughthe total count and number of motile sperm in the vas deferens waslower than controls, these values were within thehistorical controlrange and an association with treatment was considered questionable. There were no effects on testicular sperm counts, morphology, reproductive organ weights or pathology, with the exception of 3 males showing a minimal/mildspermatid head retentionat histopathology (although there was no deficit in vas deferens sperm count or motility and one control male showed the same effect, of mild severity). There were no effects on female reproductive organ weights, although the number of estrous stages in the evaluation period was slightly lower than controls and there were some differences in certain follicle types at ovarian examination for which, in view of effects at the higher dosage, the possibility of an association with treatment could not be discounted.

There was no adverse effect of treatment on gestation, gestation index, litter size at birth of the F2 pups or pup viability, growth and physical development

Similar but less marked effects on clinical condition, body weight and food intake were observed in males and females treated at 175 mg/kg/day. Mating performance was unaffected by treatment, and although fertility was reduced in both cohabitation periods, overall male fertility was within thehistorical controlrange, which may suggest transient and/or recoverable effects. Fewer males (11/23) showed retention of spermatids, the majority were described as minimal and there was no direct correlation with other effects. Five females were not pregnant at either pairing, but mating and fertility indices were within thehistorical controlrange and only 2 of these females had no corpora lutea. There was no adverse effect on gestation, littering, litter size or pup survival growth and development of the F1a or F1b litters and effects in the F1 generation were limited to minimal/mild spermatid head retention in 3 males (compared to one control male) or irregularity of estrous cycle/differences in follicle counts, none of which adversely affected mating or fertility. There were no adverse effects on the F2 litters.

There were no indicators of toxicity or adverse effectson reproductive parametersin either generationevaluated at 50mg/kg/day of sodium bromide.

The NOAEL for parental toxicity, reproductive performance and pre- and postnatal development was therefore established as50mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 50 mg/kg bw/day
Study duration:: subchronic
Species:: rat
Quality of whole database:: Klimisch-1 According to appropriate OECD testing guideline and GLP

Effects on developmental toxicity

Description of key information

Treatment with sodium bromide at 100 mg/kg bw/day was not associated with any observable adverse effects on or in utero development of the conceptus.
 LOAEL: 300 mg/kg bw/day based reduced body weight gains in dams and foetal skeletal anomalies and variants (equivalent to 233 mg (Br-)/kg bw/day)
 NOAEL: 100 mg/kg bw/day (equivalent to 77.6 mg (Br-) /kg bw/day)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

other: - 83/571/EEC

Deviations:

Qualifier:

according to guideline

Guideline:

other: JMAFF No.4200

Deviations:

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

other: Crl: CD BR VAF/Plus

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 196-259 g (first batch 201-259 g, second batch 196-239 g)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosage volumes were calculated for individual animals on Day 6 of pregnancy and adjusted according to bodyweight on Day 8, 10, 12 and 14.

VEHICLE
- Concentration in vehicle: 0, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean achieved concentrations of sodium bromide in formulations prepared on the first and last days of treatment were within 8% of nominal concentrations.

Details on mating procedure:

Female rats were time-mated to identified males ofthe same strain. 64 animals were mated on the first day with remaining animals mated a day later. The day of mating, as judged by the appearence of sperm in the vaginal smear or by the presence of a vaginal plug, was considered as Day 0 of pregnancy.

Duration of treatment / exposure:

Days 6-15 post coitum

Frequency of treatment:

daily

Duration of test:

until Day 20 post coitum

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

25 females/group

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20 of pregnancy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- on Days 3, 6, 8, 10, 12, 14, 16, 18 and 20 of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- only two animals were examined; one which died uring the study period and another one which had to be killed for humane reasons.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]

Statistics:

Significance tests, employing analysis of variance followed by an intergroup comparison with the control, were performed on the following parameters and results are presented in relevant table of this report:
bodyweight change, mean food consumption, litter data, sex ratio and foetal abnormalities and variants.
Depending on the heterogeneity of variance between treatment groups, parametric tests, analysis of variance followed by Williams`test or non-parametric tests, Kruskal-Wallis followed by Shirley`s test were used to analyse these data, as appropriate.
For litter data and foetal changes the basic sample unit was the litter, and, due to the preponderance of non-normal distributions, non-parametric analyses were routinely used. Analysis of foetal abnormalities was performed using a trend test on the number of litters affected, followed by a one-tail 2 sample permutation test. All significant (ie p≤0.05) intergroup differences from the control are reported only when supported by a significant analysis of variance (p≤0.05). Where 75% or more of the values for a given variable were the same, a Fisher`s exact test was used.

Indices:

Pre-implantation loss:
(No. corpora lutea - No. implantations)/ No. corpora lute x 100

Post implanation loss:
(No. implantations - No. young) / No implantations x 100

Sex ratio at Day 20:
(No live males) / total no. live young x 100

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment at 1000 mg/kg bw/day was associated with unsteady gait in all animals. This sign was first apparent following administration of the second dose on Day 7 post coitum. Thereafter, all animals showed this sign at daily examination prior to dosing on Day 8-11 inclusive and, after dosing on Days 8-15. From Day 11, although most animals showed unsteady gait prior to dosing on Days 12 and 13, the incidence was lower on Days 14 and 15 with only 12/24 animals showing this signs prior to dosing on Day 15. Although the final dose was administered on Day 15 post coitum, unsteady gait was apparent for 8/24 animals on Day 17.
As the treatment period progressed, additional abnormalities of movement became apparent: all animals showed feet falling through the cage grid floor during ambulation on at least one occasion, and 23 animals showed poorly coordinated movements on at least one occasion. Both of these signs were first apparent after dosing on Day 9 post coitum. In both cases, there was striking difference in the incidence of affected animals prior to dosing as opposed to after dosing: although only 1 or 2 animals showed these signs prior to dosing up to 24 animals showed feet falling through the cage grid floor during ambulation after dosing, and up to 16 animals showed poorly coordinated movements. It was noted that the incidence of poorly coordinated movements was highest towards the end of the treatment period (days 13-15). Following administration of the final dose on Day 15, these signs were not apparent on Days 16-20.
Treatment at 1000 mg/kg bw/day was also associated with reduced bodytone in all animals. This sign was first apparent following administration of the second dose on Day 7 post coitum. During Days 8-15, there was a clear difference in the incidence of affected animals prior to dosing as opposed to after dosing. This difference was most pronounced towards the end of the treatment period (Days 13-15), when only 1 to 4 animals were affected prior to dosing in contrast to 18-24 animals after dosing. Although the final dose was administered on Day 15, reduced bodytone was apparent for 3/24 animals on Day 16 and 1/24 animals on Day 17.
Treatment at 1000 mg/kg bw/day was also associated with hair loss; 22/24 animals showed hair loss compared with 0/25 controls. In all cases, the hair loss was first noted between Day 14 and Day 19 post coitum and in 7 animals was apparent on Day 20 post coitum.
Occasional instances of increased lacrimation, brown staining on fur, periorbital staining and wet staining around the urogenital region were also observed at 1000 mg/kg bw/day.
No clinical signs considered to be attributable to treatment were observed at 100 or 300 mg/kg bw/day.
There was one mortality on the study which was considered to be related to treatment. One animal of the highest dosage group (1000 mg/kg bw/day), was sacrificed for humane reasons prior to dosing on Day 11 of pregnancy. Prior to sacrifice, reduced bodytone, unsteady gait, red periorbital staining, brown staining on fur, poorly coordinated movements, increased lachrymation, wet urogenital staining and bodyweight loss (bodyweight loss of 19 g were recorded between Days 6-8 of pregnancy) were evident. In addition, poorly coordinated movements were observed earlier than for the other animals in the same treatment group. Post mortem examination failed to establish any obvious cause for the physical condition. It is clear evidence that this animal showed a more severe response to treatment compared with the rest of the animals at the same dosage level, all of which survived to termination.
At 1000 mg/kg bw/day, mean bodyweight gain during the first six days of treatment was significantly lower than in controls. Thereafter, mean bodyweight gains during Days 12-16 were comparable to the controls. However, mean bodyweight gains during Days 16-20 were significantly lower than in controls, also after correction of body weight gains for gravid uterus weight.
At 300 mg/kg bw/day, bodyweight gain throughout Days 6-16 was comparable to the controls. However as at 1000 mg/kg bw/day, bodyweight gain during Days 16-20 was significantly lower than in controls. This was also true after correction for gravid uterus weight.
At 100 mg/kg bw/day, there was a slightly increased bodyweight gain compared to controls.
Food consumption of animals treated at 1000 mg/kg bw/day was higher than in controls during the first four days of treatment (differences attained statistical significance for Days 8 and 9), despite the fact that bodyweight gains were significantly lower than in controls during this period. This was reflected in higher food conversion ratios during this period, indicative of impaired efficiency of food utilisation. Food consumption at this dosage was noticeably higher than in controls during Days 14-15 and lower during Days 18-19.
At 100 and 300 mg/kg bw/day, there were no adverse effects on food consumption or food utilisation.
Other than the previously mentioned increased incidence of hair loss in the 1000 mg/kg bw/day group compared with controls, the incidence of findings noted at macroscopic post mortem examination did not indicate any obvious adverse effect of treatment.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
One female receiving 100 mg/kg bw/day showed total litter loss in utero (total resorption). In view of the absence of similar findings at higher dosages which indicates no dose-response relationship this finding is considered to be co-incidental and unrelated to treatment. The following assessment is based on the 23, 21, 24 and 22 females with live young at Day 20 in Groups 1-4, respectively.
Reproductive performance:
No effects on the reproductive performance, fetal deaths, fetal weight as well as on the sex ratio was evident on comparison of treated groups with concurrent controls.
Skeletal and visceral malformations:
There was a higher incidence of foetuses from the 1000 mg/kg bw/day group showing malformations. These malformations were principally visceral, affecting the urogenital system (i.e. absent left kidney and/or ureter, absent or narrow left uterine horn), and thoracic skeletal malformations manifest as abnormalities of the ribs. No similar malformations were observed in the controls.
At 100 and 300 mg/kg bw/day, the type and incidence of malformations did not indicate any adverse effect of treatment.
Skeletal anomalies and variants:
At 1000 mg/kg bw/day, the incidence and distribution within litter of foetuses with skeletal anomalies was significantly different from that of controls. Minimally distorted ribs were seen in 8 foetuses in 7 litters, another 5 foetuses in 4 litters showed more severe rib anomalies and were classified as malformed. There was and increased incidence of foetuses/litters showing irregular ossification of the thoracic vertebral centra, and shortened/absent 13th ribs. The latter finding being corroborated by a complete absence of foetuses showing supernumerary ribs, a highly unusual incidence. The percentage of foetuses/litters with reduced ossification of the cranial centres was statistically significantly higher than in controls.
At 300 mg/kg bw/day, the incidence and distribution within litters of foetuses with skeletal anomalies was also statistically significantly different from that of controls. The difference was principally due to an increased incidence of foetuses with reduced ossification. In addition, there was a slightly higher percentage incidence of foetuses with variant sternebrae, principally due to an increase in unossified sternebrae. It was noted that only one foetus showed supernumerary ribs. In view of the effects observed at 1000 mg/kg bw/day, these differences are considered to be related to treatment.
At 100 mg/kg bw/day, the type, incidence and distribution of skeletal anomalies and the percentage incidence of supernumerary ribs and variant sternebrae did not indicate any obvious adverse effects of treatment.
Visceral anomalies:
The type, distribution and incidence of visceral anomalies did not indicate any obvious adverse effects of treatment.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Basis for effect level:

other: embryotoxicity

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table A6.8.1/05-1: Summary of Adult Performance

Category	Group (Dosage [mg NaBr/kg/day])
Category	1 (0)	2 (100)	3 (300)	4 (1000)
No. of mated	25	25	25	25
No. of killed (Day 11 of pregnancy)	0	0	0	1
No. of non-pregnant	2	3	1	2
Total litter loss in utero	0	1	0	0
No. with live young at Day 20	23	21	24	22

Table A6.8.1/05-2: Bodyweights and bodyweight change during pregnancy - dams with live young, group mean values

Group

( [mg NaBr/kg/day])

No. of animals

Bodyweight [g] at Day of pregnancy

And (bodyweight change [g]) from Day 6 of pregnancy

Corrected bodyweight (gain)

***

(0)

222.7

(-47.0)

249.9

(-19.8)

269.7

(0)

281.8

(12.1)

293.4

(23.7)

310.9

(41.2)

325.5

(55.8)

346.0

(76.3)

376.5

(106.8)

414.9

(145.2)

333.4

(63.7)

(100)

221.1

(-46.8)

248.4

(-19.5)

268.0

(0)

280.5

(12.6)

294.1

(26.2)

312.0

(44.0)

327.9

(59.9)

347.4

(79.4)

379.1

(111.1)

417.9

(149.9)

336

(68)

(300)

224.2

(-45.6)

249.0

(-20.8)

269.8

(0)

282.5

(12.8)

296.2

(26.5)

312.8

(43.0)

325.5

(55.7)

345.3

(75.6)

369.3

(99.5)

403.7

(133.9)

328.2

(58.5)

(1000)

223.4

(-46.3)

248.5

(-21.2)

269.7

(0)

279.6

(9.9)

284.5

(14.9)

298.1

(28.4)

312.9

(43.2)

333.0

(63.3)

360.1

(90.4)

390.7

(121.0)

316.2

(46.5)

* statistically different from control with p ≤ 0.05

** statistically different from control with p ≤0.01

*** bodyweight and bw gain were corrected for gravid uterus weight (bw day 20- gravid uterus weight; bw gain - gravid uterus weight)

Table A6.8.1/05-3: Food consumption during pregnancy - Dams with live young, group mean values [g/rat/day]

Days of pregnancy	Group (Dosage [mg NaBr/kg/day])
Days of pregnancy	1 (0)	2 (100)	3 (300)	4 (1000)
No. of animals observed:	23	21	24	22
3-5	27	26	25	26
6-7	27	28	28	30
8-9	28	28	29	32*
10-11	28	30	29	28
12-13	30	31	30	31
14-15	31	32	32	36**
16-17	34	34	32	34
18-19	33	34	31	30**

* statistically different from control with p ≤ 0.05

** statistically different from control with p ≤ 0.01

Table A6.8.1/05-4: Litter data - Group values

Group Dosage [mg NaBr/kg/day])	1 (0)	2 (100)	3 (300)	4 (1000)
Dams with live young
No. of litters	23	21	24	22
Group mean values
No. of corpora lutea	16.2	15.4	15.3	15.7
No. of implantations	14.9	14.6	14.0	14.1
No. ofin uterodeath - early - late - early and late	0.6 0.2 0.8	0.6 0.0 0.6	0.7 0.1 0.8	0.8 0.2 1.0
No. of live young	14.1	14.0	13.1	13.1
Litter weight [g]	53.69	55.0	50.52	49.0
Foetal weight [g]	3.81	3.92	3.84	3.75
Graivid uterine weight [g]	81.48	81.88	75.35	74.46
Sex ratio [%]	48.0	54.0	47.5	48.0
Litter incidence (´n`)
Number ofin uterofoetal death: early - 0 - 1 - 2 - 3	13 7 2 1	12 6 3	13 6 4 1	10 8 3 1
Number ofin uterofoetal death: late: - 0 - 1 - 2	19 4	21	21 3	19 2 1
Number ofin uterofoetaldeath: early and late: - 0 - 1 - 2 - 3 - 4	10 10 2 1	12 6 3	11 8 4 1	9 7 4 2

Table A6.8.1/05-5: Foetal abnormalities - prevalence and distribution in litters

Category	No. of affected foetuses/litter (n)	Group (Dosage [mg NaBr/kg/day])
		1 (0)	2 (100)	3 (300)	4 (1000)
		No. of litters with ´n` foetuses affected
No. of litters examined		23	21	24	22
Malformation	0	20	21	20	14
	1	2	-	3	5
	2	-	-	1	2
	3	1	-	-	-
	7	-	-	-	1
Visceral anomaly	0	11	10	16	14
	1	11	6	7	3
	2	1	4	1	3
	3	-	1	-	1
	4	-	-	-	1
Skeletal anomaly	0	12	12	6*	4**
	1	6	4	4	3
	2	2	3	6	5
	3	1	2	6	3
	4	2	-	1	5
	5	-	-	1	-
	6	-	-	-	2
Mean foetuses affected per litter [%]
Malformations		1.7	0.0	1.7	5.3
Visceral anomalies		7.8	11.4	5.7	13.2
Skeletal anomalies		13.9	11.9	29.1	40.6

* statistically different from control with p ≤ 0.05

** statistically different from control with p ≤ 0.01

Table A6.8.1/05-6: Skeletal Variants of Foetuses - Group values

Group (Dosage [mg NaBr/kg/day])	Foetuses examined	Foetuses with
		13 ribs		14 ribs		Normal sternebrae		Unossified sternebrae		Reduced sternebrae		Asym./bip. sternebrae		Total variant sternebrae
		No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%
1 (0)	159	146	92.5	13	7.5	94	58.6	45	27.9	26	17.9	1	0.4	65	41.4
2 (100)	149	130	88.1	19	11.9	78	51.9	40	27.1	38	25.9	5	3.7	71	48.1
3 (300)	154	153	99.4	1	0.6	64	42.9	62	40.0	37	23.0	5	3.0	90	57.1
4 (1000)	137	137	100.0*	0	0*	27	20.3**	89	63.3**	51	38.8**	5	2.8	110	79.7**

* statistically different from control with p ≤ 0.05

** statistically different from control with p ≤ 0.01

Conclusions:

Treatment of pregnant rats with sodium bromide through organogenesis caused severe maternal toxicity at the high dose level of 1000 mg/kg bw/day while at the mid dose level of 300 mg/kg bw/day, maternal toxicity was less pronounced as in the high dose group and was characterised by significantly depressed body weight gains. Taking into consideration the results of the fetal examinations it is concluded that sodium bromide treatment with concentrations of 1000 mg/kg bw/day leads to a higher incidence of foetuses/litters showing absent left kidney, absent left ureter and absent/narrow left uterine horn. Foetuses treated at 300 and 1000 mg/kg bw/day showed a higher incidence of reduced ossification of diverse components of the skeleton. There was no observable maternal or foetal effect related to treatment at 100 mg/kg bw/day while at the mid and high dose level, maternal toxicity as a consequence of treatment with sodium bromide was evident. Therefore, the no effect level for the parent female and in utero development of the foetus is determined to be 100 mg/kg bw/day.

Executive summary:

Materials and Methods

The potential developmental toxicity of sodium bromide was investigated in pregnant Crl: CD BR rats. Dosages of 0, 100, 300 and 1000 mg/kg bw/day were administered daily at a constant volume of 10 mL/kg bw in water as the vehicle by intragastric intubation, to groups of 25 rats each from Days 6-15 post coitum inclusive. On Day 20 post coitum, females were sacrificed and subjected to examination, litter values determined and foetuses subsequently sexed. Half of the foetuses were examined for visceral abnormalities; the remainder were observed for skeletal changes. During the study, clinical signs, body weights, body weight gains and food consumption of dams were regularly examined and reproduction parameters were determined after Cesarian section.

Results and Discussion

Treatment of pregnant dams with sodium bromide during gestation days 6 through 15 was associated with clear signs of maternal toxicity principally manifest as a lower rate of bodyweight gain during Days 6-12 of pregnancy, abnormalities of gait, reduced bodytone and poorly coordinated movements at the top dose level of 1000 mg/kg bw/day. No effects on the reproductive performance, fetal deaths, fetal weight as well as on the sex ratio was evident on comparison of treated groups with concurrent controls. Detailed examination of foetal morphology at this dose level revealed a higher incidence of foetuses/litters showing absent left kidney, absent left ureter, absent/narrow left uterine horn, distorted ribs, shortening/absence of 13th ribs, irregular ossification of the thoracic vertebral centra, reduced and/or unossified sternebrae and, reduced ossification of one or more cranial centres, than in the control group. Although it was noted that seven foetuses in one litter had no left kidney and ureter (3 of these foetuses also had an absent or narrow left uterine horn), these abnormalities were also apparent for one foetus in each of two further litters. In addition, one foetus in another litter had a small left kidney, absent left ureter and a markedly narrow left uterine horn. Therefore, an association with treatment of dams is considered likely, since the litter and not the foetus is the principal unit of assessment, but these effects are probably secondary to severe maternal toxicity at this dose level. It is noteworthy that there was no obvious reduction in mean foetal weight. Some of the observed skeletal abnormalities may reflect effects on maternal bodyweight gain and food consumption. In contrast, the defects observed in the urogenital system are extremely rare and considered more likely to reflect a selective effect on embryofoetal development than a secondary effect resulting from toxicity to the parent female. At 300 mg/kg bw/day, no adverse effects on the parent female were observed during the treatment period. However, following the withdrawal of treatment, bodyweight gain was statistically significantly lower than controls. This effect was also recorded at 1000 mg/kg bw/day on the present study. This is circumstantial evidence that the lower rate of bodyweight gain following the withdrawal of treatment reflects an earlier effect during the dosing period, which has been detected within the context of this screening study. Detailed examination of foetal morphology revealed a higher incidence of foetuses showing reduced ossification of various components of the skeleton compared with controls. It is noteworthy that there was no obvious reduction in mean foetal weight. At 100 mg/kg bw/day, there was no observeable maternal response to treatment and no obvious adverse effects on morphological development of the conceptus.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 100 mg/kg bw/day
Study duration:: subacute
Species:: rat

Additional information

Sodium bromide is an inorganic salt that dissociates to its composite ions in aqueous solutions at environmental pH and temperature. Comparison of the available data on the various bromide salts have shown that the bromide ion is the relevant ion for determination of the toxicological profile with simple cations such as potassium, sodium or ammonium, that are ubiquitous in nature, having little or no influence on the bromide ion properties. It is therefore justified to read-across data from other inorganic bromide salts to sodium bromide.

Sodium bromide developmental toxicity in the rat:

In a key study (Myers 1995) The potential developmental toxicity of sodium bromide was investigated in pregnant Crl: CD BR rats. Dosages of 0, 100, 300 and 1000 mg/kg bw/day were administered daily at a constant volume of 10 mL/kg bw in water as the vehicle by intragastric intubation, to groups of 25 rats each from Days 6-15 post coitum inclusive. On Day 20 post coitum, females were sacrificed and subjected to examination, litter values determined and foetuses subsequently sexed. Half of the foetuses were examined for visceral abnormalities; the remainder were observed for skeletal changes. During the study, clinical signs, body weights, body weight gains and food consumption of dams were regularly examined and reproduction parameters were determined after Cesarian section.

Summary of maternal toxic effects:

Treatment at 1000 mg/kg bw/day was associated with unsteady gait, feet falling through cage grid floor, poorly coordinated movements, reduced bodytone and hair loss. There was one mortality at this dose level. Lower bodyweight gains were observed during days 6 to 12 and 16 to 20 of pregnancy. Increased food consumption was observed during days 6-9 and 14-15, followed by lower food consumption during days 18-19.

Treatment at 300 mg/kg bw/day was associated with lower bodyweight gains during days 16-20 of pregnancy.

Treatment at 100 mg/kg bw/day was not associated with any observable maternal responses.

Summary of embryotoxic effects:

There were no adverse effects on any of the litter parameters recorded for any of the dose groups.

Detailed examination of foetal morphology from dams treated at 1000 mg/kg bw/day revealed higher incidences of foetuses/litters showing absent left kidney, absent left ureter, absent/narrow left uterine horn, distorted ribs, shortened/absent 13^thribs, irregular ossification of the thoracic vertebral centra, reduced and/or unossified sternebrae and reduced ossification of one or more cranial centres than in controls.

In the 300 mg/kg bw/day dose group, detailed examination of the foetal morphology revealed a higher incidence of foetuses showing reduced ossification of various components of the skeleton compared with controls.

Treatment at 100 mg/kg bw/day was not associated with any observable adverse effects on or in utero development of the conceptus.

Conclusion

LOAEL: 300 mg/kg bw/day based reduced body weight gains in dams and foetal skeletal anomalies and variants (equivalent to 233 mg (Br^-)/kg bw/day)

NOAEL: 100 mg/kg bw/day (equivalent to 77.6 mg (Br^-) /kg bw/day)

Ammonium bromide developmental toxicity

In a teratogenicity study performed with ammonium bromide pregnant rats were treated once daily by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day during Days 6-19 of gestation. The animals were monitored during gestation for clinical signs of toxicity, bodyweight and food consumption performance and animals were terminated on Day 20 of gestation. The status of each implantation was recorded and the viable foetuses were examined externally and weighed. Foetuses were subjected to macroscopic, visceral and skeletal examination. Clinical signs (neurotoxic effects) and reduced bodyweight gain (18%) were noted in dams at 1000 mg/kg bw/day. Clinical signs (piloerection) were also noted in dams at 300 mg/kg bw/day. The clinical signs noted at the dose level of 1000 mg/kg bw/day consisted of rolling gait, animal limp when handled, hunched posture, subdued behaviour, piloerection, eyes dark and abnormal respiration. One animal of this dosage group was sacrificed on Day 10 of gestation due to the severity of these signs. Foetal effects were noted at all dose levels. There was a dose related increase in the incidence of foetuses with kinked ribs (4.5%, 9% and 25% after treatment with 100, 300 and 1000 mg/kg bw/day, respectively compared to 1.6% in controls). This effect was often associated with incomplete ossification of ribs (2%, 9% and 16% after treatment with 100, 300 and 1000 mg/kg bw/day, respectively compared to 0% in controls). A dose-related increased incidence of displaced testis was noted (4%, 8% and 10% after treatment with 100, 300 and 1000 mg/kg bw/day, respectively compared to 1.6% in controls). This finding at mid and high dose, was outside historical background range of incidences for this strain of rats at the laboratory in question (historical ctr range:0-4.1%) . At 1000 mg/kg bw/day, reduced mean foetal weight (15%), increased incidence of foetuses with slightly kinked ribs (4% compared to 1% in controls) and abnormalities (reduced/absent/displaced/cystic) of the left kidney, often associated with absence of the left adrenal and/or left ureter were noted (12.5% compared to 0% in controls). Some of the affected foetuses of this dose group also had narrowing of the left uterine horn (7% compared to 0% in controls) and flattened/small spleen (9% compared to 0% in controls). Moreover, increased incidence of reduced/absent thyroid was noted at 1000 mg/kg bw/day (3.8% compared to 0.5% in controls), and there was also an increased incidence of small foetus noted at this dose level (24% compared to 2% in controls). Also at 1000 mg/kg bw/day curved scapula (8.7% compared to 0.5% in controls) was observed, this finding generally occurred in foetuses with kinked ribs, and there was a slight increase in the incidence of foetuses with reduction in size of the 13th ribs (6.8% compared to 0% in controls). NOEL for maternal toxicity was determined at 100 mg/kg bw/day. NOAEL for maternal toxicity was determined at 300 mg/kg bw/day based on clinical signs of neurotoxicity and reduced bodyweight gain noted at 1000 mg/kg bw/day.In the 3-generation reproductive study (NaBr Section 8.7.3 - Reproductive toxicity Van Leeuwen et al (1983)) maternal effects on the thyroid hormone was noted at 3800 ppm equivalent to 187 mg bromide/kg bw/day. Therefore, an effect at 300 mg/kg bw/d could be expected.Signs of piloerection noted in dams at 300 mg/kg bw/day were in the absence of other effects not considered adverse.

No NOEL/NOAEL was determined for developmental toxicity (Irvin and Hallmark, 2000).

In another teratogenicity study performed with ammonium bromide pregnant rats were treated once daily by gavage at dose levels of 0, 50, 300, 600 and 800 mg/kg bw/day during Days 6-19 of gestation. In addition two groups were assigned to control and 300 mg/kg bw/day groups to serve as recovery animals (littering phase). Animals were monitored for clinical signs of toxicity, bodyweight and food consumption. Main study animals were killed on Day 20 of gestation and status of each implantation was recorded. Viable foetuses were examined for visceral and skeletal abnormalities, including the state of skeletal ossification. Animals from the recovery group were allowed to litter and rear their young to weaning. Pups were necropsied and skeletons stained and examined for abnormalities with particular emphasis on the changes seen during organogenesis. Clinical signs (neurotoxic effects) were noted in dams at 600 and 800 mg/kg bw/day. The clinical signs consisted of staggering, rolling gait, subdued behaviour, slow/irregular respiration, body held low, hunched posture and piloerection. One animal at 600 mg/kg bw/day was sacrificed on Day 11 of gestation due to the severity of these signs. Bodyweight gain at 800 mg/kg bw/day was reduced (9%) when compared to controls (statistical analysis not performed). Bodyweight gain at 300 and 600 mg/kg bw/day was increased (11% and 28%) when compared to controls (statistical analysis not performed). There were no obvious effects on embryo-foetal mortality or foetal weights at any dose level tested. Increased incidence of abnormalites and variants was noted in foetuses of the three highest dosage groups. At 300, 600 and 800 mg/kg bw/day, there were increased incidences of foetuses with kinked ribs (5.4%, 8.5% and 6.7% of rats showing kinked ribs after treatment with 300, 600 and 800 mg/kg bw/day, respectively, compared to 0.4% in controls), and of foetuses with curved scapulae (1.8%, 2.2% and 5.5% after treatment with 300, 600 and 800 mg/kg bw/day, respectively compared to 0% in controls). There was also an increase at these dose levels of foetuses with incompletely ossified ribs (19%, 29% and 24% after treatment with 300, 600 and 800 mg/kg bw/day, respectively compared to 3% in controls). At 600 and 800 mg/kg bw/day, there were indications of effects on foetal ossification although it was concluded that no statement could be made regarding the influence of ammonium bromide treatment on ossification parameters. At 600 and 800 mg/kg bw/day there were increased numbers of foetuses with fewer than 13 complete ribs (incidence 13 complete ribs was 87% and 76% for the 600 and 800 mg/kg bw/day group, respectively compared to 92% in controls). Among the females treated at 300 mg/kg bw/day that were allowed to litter, the period of gestation was somewhat less than in controls, with a mean duration of 21.3 days for treated rats compared to 21.8 days in control animals. Litter size and survival were not obviously affected. Incidences of abnormalities of the ribs and pelvic girdle for weanlings from these rats were similar to those seen in controls, which indicates that the kinked ribs and curved scapulae seen in the foetueses from rats treated at the same dose level are transient in nature and are reversible effects which resolve after birth. NOAEL for maternal toxicity was determined at 300 mg/kg bw/day (corresponding to 246 mg bromide/kg bw/day) based on clinical signs of neurotoxicity noted at ≥600 mg/kg bw/day.Parental toxicity might have been underestimated since effects on the endocrine system were not investigated. In the 3-generation reproductive study (NaBr Section 8.7.3 - Reproductive toxicity Van Leeuwen et al (1983)) maternal effects on the thyroid hormone was noted at 3800 ppm equivalent to 187 mg bromide/kg bw/day. Therefore, an effect at 300 mg/kg bw/d could be expected.

NOEL for developmental toxicity was determined at 50 mg/kg bw/day. NOAEL for developmental toxicity was determined at 50 mg/kg bw/day (corresponding to 41 mg bromide/kg bw/day) based on abnormalities of the ribs and effects on foetal ossification noted at ≥300 mg/kg bw/day (Barton, 2007).

Justification for classification or non-classification

It is considered appropriate to classify sodium bromide as H361: Repr 2 and explanatory position papers discussing human exposure to bromides and classification are attached in Section 13 of this dossier.

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