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REACH

Ammonium bromide

EC number: 235-183-8 | CAS number: 12124-97-9

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

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 at 50mg/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

Referenceopen all close all

Reference 1

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-02-15 to 1999-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP dose range-finding study conducted under conditions similar to OECD Guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

Dose range-finder for OECD 416; 2-generation reproduction study. Performed to similar standard but with limited animal numbers and observations.
Parental animals only dosed.

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: (P) 7 weeks
- Weight at study initiation: (P) Males: 172-184 g ; Females: 126-142 g
- Fasting period before study: None
- Housing: Mating and males: 2 per cage, in propylene cages with stainless steel grid bottoms and mesh tops. Mated females: individually in solid bottom cages with sterilsed wood shaving bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C±2°C
- Humidity (%): 23-55%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 1999-02-15 To: 1999-04-28

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The diet contained a constant concentration of test material. Fresh batches of treated diets were prepared at weekly intervals during the study. A dry powder premix (at 40000 ppm) was prepared by adding a requisite quantity of untreated diet to an appropriate quantity of test material followed by mixing for approximately 2 hours. Formulated diets for the high and intermediate dose group (6400 ppm and 3200 ppm respectively) were prepared by adding a requisite quantity of untreated diet to an appropriate quantity of previously formulated premix followed by mixing for approximately one hour. The formulated diet for the low dose group (1600 ppm) was prepared by adding a requisite quantity of untreated diet to an appropriate quantity of previously formulated high dose diet (6400 ppm) followed by mixing for approximately one hour.

Details on mating procedure:

Animals were paired on a one male to one female basis, with both animals being in the same treatment group. Mating was judged to have occurred if sperm was present in a vaginal lavage or if a copulatory plug was in situ. Vaginal lavage was examined early each morning. The Day of mating was designated Day 0 of gestation.
Seven days were allowed for mating. If no mating sign was observed during that time, the female was allowed a ´rest` period of two days before placed with a second male for additional seven days. If no mating considered to have occurred at the end of the seconnd mating period the female was transfer to an individual solid bottomed cage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulated diets undertaken with regard to concentration and homogeneity. Triplicate samples were taken from each formulation during Week 1 and Week 6 of the study.
Analysed concentrations were within ± 7% of nominal. The coefficients of variation were low indicating homogeneity.

Duration of treatment / exposure:

F0 animals were treated for 2 weeks prior to mating, throughout the mating, gestation and lactation periods until the first generation had been weaned.

Frequency of treatment:

Animals had access to formulated diet ad libitum

Details on study schedule:

Dose range-finding study - parental animals only treated

Remarks:

Doses / Concentrations:
1600 ppm
Basis:
nominal in diet
corresponds to 127 mg.kg bw/day for males and 228 mg/kg bw/day for females

Remarks:

Doses / Concentrations:
3200 ppm
Basis:
nominal in diet
corresponds to 242 mg.kg bw/day for males and 454 mg/kg bw/day for females

Remarks:

Doses / Concentrations:
6400 ppm
Basis:
nominal in diet
corresponds to 503 mg.kg bw/day for males and 651 mg/kg bw/day for females

No. of animals per sex per dose:

10 animals/sex/dosage group

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:all post-weaning animals were examined for reaction to treatment on each day. Observations included appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta. In addition, all animals were checked for viability twice daily

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: weekly, commencing one week prior to first administration of treated diets until termination.
F0 females: once a week prior to the first day of dosing then weekly until the start of the mating period; during gestation and lactation, weights were recorded on Days 0, 7, 14 and 20 of gestation, then on Days 1, 7, 14 and 21 of lactation. (Day 0 of gestation was the day of detection of a positive mating sign and Day 0 of lactation was the day of birth of the litter.)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Animals had access to water and diet ad libitum.
Food consumption was recorded weekly for each cage of F0 animals, commencing one week prior to first administration of treated diets, until mating. Food consumption monitoring was suspended during the mating period and then, for males, recommenced as before. For mated females, consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-7, 7-14 and 14-21 of lactation

WATER CONSUMPTION: No data

Oestrous cyclicity (parental animals):

Not examined; study is a dose range finder

Sperm parameters (parental animals):

Not examined; study is a dose range finder

Litter observations:

Number and sex of pups, stillbirths, live births, presence of gross anomalies, examination for presence of milk in stomach
Bodyweights: Pre-weaning F1 pups: by litter, en masse (sexes separate), on Days 1, 4, 7 and 14 of lactation, and individually by sex on Day 21 of lactation.

Postmortem examinations (parental animals):

Parental animals were sacrificed after their litter had reached Day 21 of lacatation An external examination, macroscopic examination of tissues and organs of thoracic and abdominal cavities in situ, reproductive tract examined for signs of pregnancy, number of visible implantation sites

Postmortem examinations (offspring):

Examinations at pre-weaning comprised: external examination of anomalies; presence of milk in stomach; gross necropsy of cranial, thoracic and abdominal cavities in situ (for pups found dead or killed on or after Day 14 of lactation)
Examinations at weaning comprised: external examination of anomalies, macroscopic examination of tissues and organs of cranial, thoracic and abdominal cavities in situ (animals killed on Day 21 of lactation)

Statistics:

Not performed

Reproductive indices:

See Tables 8, 9 and 10 in attached Results document

Offspring viability indices:

Birth Index:
Total number of pups born (live and dead)/Number of implantation scars

Live Birth Index:
Number of pups live on Day 0 of lactation/ Total number born (live and dead)

Viability Index:
Number of pups live on Day 4 of lactation/Number live on Day 0

Lactation Index:
Number of pups live on Day 21 of lactation/ Number Live on Day 4

Overall Survival Index:
Number of pups live on Day 21 of lactation/Total number of pups born (live and dead)

Also see Tables 8, 9 and 10 in attached Results document

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See details below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See details below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See details below

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: See details below

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

See details below

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortalities:
There were no premature deaths during the study.
Clinical signs:
At 6400 ppm, rolling gait was noted in all animals and was generally noted following the first few days of treatment and persisted throughout the treatment period. In addition, piloerection and hunched posture accompanied this finding. In females, approximately half of the animals also showed hyperactivity. As a result of the generally ill condition of these animals, most developed staining on their body and an unkempt appearance to their coat.
At 3200 ppm clinical effects of treatment were similar to those noted for animals at 6400 ppm, but were of a lesser severity. Nine males and six females showed rolling gait, but the onset of this was around the fifth week of treatment and was generally evident throughout the treatment period. None of the animals at this level showed an unkempt coat.
At 1600 ppm, three females showed transient piloerection but it was uncertain if this finding was related to treatment due to the lack of rolling gait at this dose level.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights/body weight gain:
In males, a reduction in bodyweight gain throughout the first week of treatment was noted at 6400 ppm when compared to control. Thereafter, the bodyweight gain for these animals was essentially similar to controls; consequently there was a reduction in the overall bodyweight gain throughout the treatment period (-7.5% at week 8). At 3200 ppm, the mean bodyweight gain in males was slightly reduced (-6.7% at week 8) and at 1600 ppm there was an increase in bodyweight gain throughout the first weeks of treatment, which could not positively be attributed to treatment.
For females there was no obvious treatment related effect on bodyweight performance prior to mating at any dose level, and during gestation for animals treated at 1600 and 3200 ppm when compared with control. At 6400 ppm, the calculations during gestation for females were based on one animal as a result of poor pregnancy rate. For this animal, the bodyweight gain throughout the gestation period was less than any individual animal in the control group. The group mean bodyweights at the start of lactation were greater than control at 3200 and 1600 ppm. However, by Day 14 of lactation, the absolute weights were essentially similar in all dose groups.
Food consumption:
A reduction in group mean food consumption, compared with control, was noted for males towards the latter part of the treatment period at 6400 and 3200 ppm. Food consumption in males treated at 1600 ppm was similar to controls.
In females, food consumption during the pre-mating and gestation periods was similar in all dose groups. Slight differences in the food consumption during lactation were not obviously a result of treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
At 6400 ppm, although seven females showed a positive mating sign, only one female became pregnant. Fertility indices in males and females were 10%.
At 3200 ppm, small differences in the mating performance, as assessed by the number of nights to positive mating sign and by the number of animals passing oestrus, and small differences in the fertility indices were considered too small to be positively attributable to treatment.
Mating performance and fertility indices at 1600 ppm were similar to control

The mean duration of gestation was similar in all groups.
At 6400 ppm, the only litter produced did not survive to Day 4 of lactation.
At 3200 ppm, there was a reduced lactation index noted, but largely reflects the litter where all pups died and events leading to pup mortality had probably been established by Day 4 of lactation.
At 1600 ppm, no treatment-related effects on pup survival and lactation index was noted.

Mean duration of gestation was 21.6, 21.4, 22.1 and 22 days for animals treated with 0, 1600, 3200 and 6400 ppm ammonium bromide respectively, showing slight increase of duration of gestation with increasing amounts of test substance. The mean number of implant sites per pregnancy did not differ between the groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Necropsy findings of F0 females were limited to four animals, all treated at 6400 ppm, which showed a dilated uterus at necropsy; none of these animals were pregnant. In addition, one of these animals had dark foci on all lung lobes. These findings were not considered to be related to treatment. No adverse findings during gross necropsy were seen in F0 females dosed with 1600 and 3200 ppm.

Dose descriptor:

NOAEL

Effect level:

1 600 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See details below

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

See details below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See details below

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
At 6400 ppm, the only litter produced did not survive to Day 4 of lactation.
At 3200 ppm, all pups in 4 out of 9 litters died before Day 21 of lactation and included one litter where all pups were born dead, resulting in a reduced gestation index at this level. Considering the litters where some pups survived, there was a slight increase in pup mortality compared to the other dose groups.
At 1600 ppm, there were no obvious effects on litter size or survival.
The viability index (number of pups live on Day 4 of lactation/number live on Day 0) and overall survival index (number of pups live on Day 21 of lactation/total number of pups born) for the control group was lower than might have been expected.

Three pups from two litters treated at 3200 ppm and one pup from one litter treated at 1600 ppm were killed due to their condition (cold, subdued behaviour, abnormal breathing) on or before Day 12 of lactation. It is not possible to indicate positively if these findings were attributable to treatment.

CLINICAL SIGNS (OFFSPRING)
For three pups of the 3200 ppm group and one pup from the 1600 ppm group, cold, subdued behaviour and abnormal breathing were recorded but these findings could not be positively related to treatment.
Other occasional observations on the pups and litters were considered to be incidental and consistent with those usually seen in this type of study.

BODY WEIGHT (OFFSPRING)
Slight differences in pup weights at birth did not indicate any obvious adverse effect of treatment.
At 3200 ppm, mean weights of litter and pups were lower than control by Day 21 of lactation.
Slight differences in litter and pup weights at 1600 ppm compared to control were not positively related to treatment.
Since none of the pups of the only litter produced at 6400 ppm survived until Day 21 of lactation, pup body weights could not be determined in this dose group.

Dose descriptor:

NOAEL

Generation:

Effect level:

1 600 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increased mortality noted in pups at ≥3200 ppm and reduced mean of litter mean pup weight noted at 3200 ppm (M: 5%; F: 11%)

Reproductive effects observed:

not specified

See attached Results Tables document.

Conclusions:

Rats of the highest dosage group (6400 ppm) showed rolling gait, piloerection, hunched posture and unkempt coat in both sexes and hyperactive activity in females only. The clinical effects observed for the group treated at 3200 ppm were the same, however no unkempt coat was noted and the effects were less severe. At 1600 ppm, animals showed transient piloerection only. Reduced bodyweight gain was noted in males at 3200 (13%) and 6400 (16%) ppm. Reduced food consumption was also noted in males at ≥3200 ppm. A slight increase of duration of gestation was noted at 3200 and 6400 ppm (mean duration: 22.1 and 22 days, respectively, compared to 21.6 days in controls). The mean number of implant sites per pregnancy did not differ between the groups. Mating performance and female fertility index was reduced at 3200 ppm (female fertility index: 90%) and 6400 ppm (female fertility index: 10%). From the rats treated at 6400 ppm, only one became pregnant and the litter produced was dead before Day 4 of lactation. At 3200 ppm, there was an increased incidence of total litter loss and a slight increase in pup mortality for surviving litters. There were no obvious effects on litter size or survival in the lowest dosage group (1600 ppm). No NOEL parental was determined. The NOAEL parental was 1600 ppm based on clinical signs of neurotoxicity noted in both sexes at ≥3200 ppm, reduced bodyweight gain noted in males at ≥3200 ppm and reduced mating performance and female fertility index noted at ≥3200 ppm. Clinical signs of piloerection noted at the dosage level of 1600 ppm was in the absence of other clinical signs not considered as an adverse effect. The NOEL offspring was 1600 ppm. The NOAEL offspring was 1600 ppm based on increased mortality noted in pups at ≥3200 ppm and reduced mean of litter mean pup weight noted at 3200 ppm (M: 5%; F: 11%).

Executive summary:

Materials and method

The study was performed in rats to determine a maximum tolerated dose of ammonium bromide which can be used as the highest dose in subsequent reproduction studies, and to provide guidance in the selection of the lower dose levels.

Rats were treated via the diet with 0, 1600, 3200 or 6400 ppm of ammonium bromide (corresponding to 0, 120, 240, and 480 mg/kg bw/day with 1ppm=0 0.075 mg/kg bw/day) for two weeks prior to mating, throughout the mating, gestation and lactation periods until termination after the first generation had been weaned.

Results and discussion

There were no premature parental deaths during the study.

Parental toxicity at 6400 ppm was demonstrated in males by reduced bodyweight gain over the treatment period and reduced food consumption after cohabitation in males. Clinical signs of reaction to treatment of both sexes at this dose level included rolling gait, piloerection, hunched posture and unkempt coat. In addition, females showed hyperactive behaviour. These clinical effects were consistent with those previously seen with this test material.

At 3200 ppm, slightly reduced bodyweight gain in males over the treatment period was noted. Clinical signs of reaction to treatment in both sexes were similar to those described for animals at the highest treatment group (6400 ppm), however no unkempt coat was noted and the effects were generally less severe.

Mating performance and fertility were obviously affected by treatment at 6400 ppm. For the one female which produced a litter, the litter was dead before Day 4 of lactation.

At 3200 ppm, there was an increased incidence of total litter loss in 4 out of 9 litter and a slight increase in pup mortality for surviving litters. Mean litter and pup weights were reduced by Day 21 of lactation when compared to control.

At the low dose level of 1600 ppm, no effects or findings which could be directly related to treatment with ammonium bromide were observed

Reference 2

Endpoint:

multi-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No food consumption, pup body weights and pup litter sizes determined.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

and study was performed according to good experimental practice

Deviations:

yes

Remarks:

No food consumption, pup body weights and litter size determination

GLP compliance:

Remarks:

GLP was not obligatory at the time of study conduct and study was performed according to good experimental practice

Limit test:

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Not applicable
- Age at study initiation: Male rats were mated for the first time at the age of 4 months
- Weight at study initiation: no data

Route of administration:

oral: feed

Vehicle:

other: No vehicle used; test substance was applied via food

Details on exposure:

Dosing regime followed that of van Logten et al (1974) 90-day repeat oral dose study: NaBr Section 8.6.2 - subchronic repeat dose Van Logten et al (1974) Supp

Details on mating procedure:

Male rats of proven fertility were mated with females for the first time at the age of 4 months. In three successive generations, at least two litters per female rat were raised. In the first generation a third litter was raised for the investigation of transplacental transport of bromide. Furthermore, an additional litter was bred with parent animals of the highest dose group which were changed to the control diet in order to investigate the reversibility of observed effects.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Not indicated

Frequency of treatment:

Not indicated

Details on study schedule:

No additional details on study schedule given.

Remarks:

Doses / Concentrations:
75, 300, 1200, 4800 and 19200 mg NaBr/kg diet
Basis:
nominal in diet
(corresponding to 5.6, 22.5, 90, 360 and 1400 mg/kg bw/day with 1ppm=0.075 mg/kg bw/day)

No. of animals per sex per dose:

7-19 animals/sex/group

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: animals were observed for adverse clinical signs during the treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: at start and termination of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
not examined

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

PARAMETERS EXAMINED
fertility, viability, body weight

GROSS EXAMINATION OF DEAD PUPS:
organ weights were determined for adrenals, thyroid, pituitary, testes, prostate, ovaries and uterus
PARAMETERS EXAMINED
fertility, viability, body weight

Postmortem examinations (parental animals):

GROSS NECROPSY
no data

Postmortem examinations (offspring):

HISTOPATHOLOGY / ORGAN WEIGTHS
organ weights were determined for adrenals, thyroid, pituitary, testes, prostate, ovaries and uterus.

Statistics:

No data

Reproductive indices:

Fertility Index = no. of pregnancies x 100/no. of matings

Offspring viability indices:

Viability Index = no. of pups alive at Day 5 x 100/no. of pups born alive.
Lactation Index = no. of pups alive at Day 21 x 100/no. of pups alive at Day 5

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, treatment-related

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

not specified

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0 animals: A dose-dependent decrease in thyroxine (T4) concentration was observed in the serum. Sodium bromide concentrations in the range of 125-2000 mg/kg diet in combination with ´chloride-free` diet in addition revealed decreased thyroxine concentrations in serum in animals treated at 500 and 2000 mg/kg. Uptake of radiolabelled iodide was measured within this experiment and showed significatly increased uptake after 500, but only slight increase after 125 and 2000 mg NaBr/kg diet.
F1 parents: No effects were described in the investigation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)/REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)/REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Animals treated at 19200 mg/kg diet were not fertile, and fertility of the next lower dose level (4800 mg/kg diet) was reduced. Because of the diminished fertility in these dosage groups, second and third generations were bred only from the groups dosed with sodium bromide concentrations up to 1200 mg/kg diet. In these groups no effects related to treatment were found in the breeding results.
To investigate whether infertility occurred in males or in females, untreated males and females were mated with females and males of the highest dosage group. Of the treated females with untreated males only 20% became pregnant, and none of the untreated females mated with treated males became pregnant.
Reversibility of the observed effects were studied in animals fed a diet containing 19200 mg NaBr/kg for 7 month followed by a control diet for 3 months before mating. In contrast to the infertility of these animals observed before, fertility index was 62%, viability index 61% and lactation index 90%.

OTHER FINDINGS (PARENTAL ANIMALS)
The lactation index was comparable among all groups investigated.

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

300 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL for systemic toxicity: 300 mg NaBr/kg diet, corresponding to 30 mg/kg bw/day NaBr (23.3 mg/kg bw/day “bromide”) and 15 mg /kg bw/day NaBr (11.7 mg/kg bw/day “bromide”) for young and older rats, respectively

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

1 200 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL for reproductive toxicity: 1200 mg NaBr/kg diet, corresponding to 120 mg/kg bw/day NaBr (93.2 mg/kg bw/day “bromide”) and 60 mg /kg bw/day NaBr (46.6 mg/kg bw/day “bromide”) for young and older rats, respectively.

Remarks on result:

other: Generation: P and F1 (migrated information)

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

1 200 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL for developmental toxicity: 1200 mg NaBr/kg diet, corresponding to 120 mg/kg bw/day NaBr (93.2 mg/kg bw/day “bromide”) and 60 mg /kg bw/day NaBr (46.6 mg/kg bw/day “bromide”) for young and older rats, respectively

Remarks on result:

other: Generation: F1 and F2 (migrated information)

Dose descriptor:

LOAEL

Remarks:

Systemic

Effect level:

1 200 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: P and F1 (migrated information)

Dose descriptor:

LOAEL

Remarks:

Reproductive

Effect level:

4 800 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: P and F1 (migrated information)

Dose descriptor:

LOAEL

Remarks:

Development

Effect level:

4 800 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: F1 and F2 (migrated information)

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
Pup viability at the 4800 ppm dose levels was significantly reduced but survival was shown to be greater in the second when compared to the first litter. All the young of the first litter alive on post-natal day 5 died before day 21 while all young alive on post-natal day 5 were still alive on day 21 in the second litter. No effects on breeding were observed at dose levels of 1200 ppm and below

BODY/ORGAN WEIGHTS (OFFSPRING)
Body- and organ-weight determination did not reveal a clear pattern of dose-related effects in neither of the three generations. Only the adrenals of females of the F0 generation showed a dose-dependent decrease in relative weight which could not be observed in later generations.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

other: F1 and F2

Effect level:

1 200 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

Reproductive effects observed:

not specified

Litter observations:

Breeding results with fertility index, viability index and lactation index are given in table 6.8.2/02-1. No data for the dose groups on litter size and sex ratio are given.

Pup mean bodyweights on Day 21 did not differ between control and treatment groups (see table 6.8.1/02-1).

Viability of the young was greater in the second litter than in the first. Furthermore, during the lactation of the first litter all of the young alive at Day 5 died before Day 21. In the second litter, all animals alive at Day 5 were still alive at Day 21.

Macroscopic examination of all pups born during the entire experimental period provided no evidence of anomalies.

Table 6.8.2/02-1:Breeding results in reproduction study on sodium bromide - fertility index, vability index, lactation index and mean bodyweight

Generation	Values for groups fed NaBr at dietary levels [mg/kg diet]
Generation	0	75	300	1200	4800	19200
	Fertility index*
F₀	70	70	72	65	25	0
F₁	62	54	44	53	-	-
F₂	52	67	30	45	-	-
	Viability index**
F₀	90	98	96	92	32/61^¥	-
F₁	92	88	80	97	-	-
F₂	96	98	93	98	-	-
	Lactation index***
F₀	95	96	95	94	0/100^¥	-
F₁	93	85	72	80	-	-
F₂	99	99	99	99	-	-
	Mean bodyweight at Day 21
F₀	40	45	43	43	-/38^¥	-
F₁	41	43	40	38	-	-
F₂	36	38	38	36	-	-

* Fertility index: No. of pregnancies x 100/No. of matings

** Viability index: No. of pups alive at Day 5 x 100/No. of pups born alive

*** Lactation index: No. of pups alive at Day 21 x 100/No. of pups alive at Day 5

^¥data are given separately for first and second litter

Table 6.8.2/02-2:Bromide concentration in plasma, placenta and kidneys of dams fed 75-4800 mg NaBr/kg diet for 7 months and in foetal kidneys

Dietary concentration of NaBr [mg/kg]	Maternal bromide levels			Bromide level in foetal kidneys [mmol/kg]
Dietary concentration of NaBr [mg/kg]	Plasma [mmol/L]	Placenta [mmol/kg]	Kidney [mmol/kg]	Bromide level in foetal kidneys [mmol/kg]
75	0.5 ± 0.1	0.4 ± 0.1	0.3 ± 0.1	0.3 ± 0.1
300	2.2 ± 0.1	1.4 ± 0.1	1.4 ± 0.3	0.9 ± 0.1
1200	7.8 ± 0.9	6.3 ± 1.5	4.4 ± 1.1	3.2 ± 0.8
4800	27.6 ± 2.8	16.7 ± 1.5	15.3 ± 1.4	11.0 ± 0.6

Values are means ± SD for groups of seven animals

Table 6.8.2/02-3:Bodyweights and relative organ weights

Generation	Parameter	Values for groups fed NaBr at dietary levels [mg/kg]
Generation	Parameter	0	75	300	1200	4800
MALES
F₀	No./group	9	9	9	10	10
	Bodyweight	422	398	381	391	362
	Adrenals	0.011	0.011	0.011	0.011	0.012
	Thyroid	0.0060	0.0057	0.0056	0.006	0.006
	Pituitary	0.0029	0.0029	0.0029	0.003	0.0033
	Testes	9.68	0.745	0.776**	0.744	0.712
	Prostate	0.119	0.13	0.121	0.135	0.134
F₁	No./group	10	10	10	10	-
	Bodyweight	409	391	388	395	-
	Adrenals	0.01	0.01	0.011	0.012	-
	Thyroid	0.0063	0.0064	0.006	0.0067	-
	Pituitary	0.0026	0.0026	0.0027	0.0028	-
	Testes	0.771	0.759	0.769	0.763	-
	Prostate	0.077	0.093	0.093	0.102*	-
F₂	No./group	10	10	10	10	-
	Bodyweight	438	373**	397*	378**	--
	Adrenals	0.01	0.01	0.009	0.01	-
	Thyroid	0.0076	0.0074	0.007	0.0081	-
	Pituitary	0.0032	0.0031	0.0027**	0.0029	-
	Testes	0.787	0.821	0.679	0.793	-
	Prostate	0.103	0.12	0.109	0.104	-
FEMALES
F₀	No./group	7	11	9	12	11
	Bodyweight	254	256	249	243	249
	Adrenals	0.02	0.019	0.019	0.017*	0.017**
	Thyroid	0.0062	0.0066	0.0066	0.0073	0.0073
	Pituitary	0.0056	0.0055	0.0052	0.0052	0.0046*
	Ovaries	0.022	0.021	0.022	0.025	.024
	Uterus	0.171	0.166	0.18	0.15	0.143
F₁	No./group	19	15	14	16	-
	Bodyweight	244	254	252	241	-
	Adrenals	0.018	0.018	0.017	0.017	-
	Thyroid	0.0073	0.007	0.0074	0.0083	-
	Pituitary	0.0047	0.0052	0.0049	0.0053*	-
	Ovaries	0.026	0.029	0.027	0.027	-
	Uterus	0.167	0.159	0.15	0.14*	-
F₂	No./group	10	10	10	10	-
	Bodyweight	267	244	259	241**	-
	Adrenals	0.019	0.018	0.017	0.018	-
	Thyroid	0.0096	0.0083	0.0094	0.0103	-
	Pituitary	0.0053	0.0048	0.005	0.0056	-
	Ovaries	0.027	0.024	0.027	0.027	-
	Uterus	0.188	0.16	0.179	0.164	-

Bodyweights are expressed in g.

Organ weights are expressed as g/100 g bodyweight.

* Significantly different from the control with 0.01≤ P < 005

** Significantly different from the control with 0.001≤ P < 0.01

Conclusions:

In this three-generation reproductive toxicity study it was demonstrated that administration of 4800 and 19200 ppm of NaBr caused a reduction in the fertility of both sexes of rats, an increased litter loss and pup mortality. A cross-mating experiment revealed that the effects of bromide on reproduction system are reversible as could be shown by one group of animals of the highest dosage level which were changed to control diet and mated again.

Executive summary:

Materials and Methods

The investigations addressed results from bromide studies present in the literature and provided additional data about the effects of bromide in the endocrine and reproductive systems. Estimation of an ADI is discussed in relation to the present residue situation. A three-generation reproduction study in rats was performed using sodium bromide concentrations within a range of 75-19200 mg/kg diet, examining the effects on reproductive system. In three successive generations, at least two litters per female rat were raised. In the first generation, a third litter was raised for the investigation of transplacental transport of bromide. Furthermore, an additional litter was bred with parent animals of the highest dosage group which were changed to control diet in order to investigate reversibility of effects.

Results and Discussion

Formerly performed studies demonstrated that administration of sodium bromide for four weeks at dietary concentrations in the range of 300-19200 mg/kg diet showed that bromide was readily absorbed and within three weeks bromide concentration in plasma reached plateau level (van Logten et al, 1973; please refer to section 6.4.1/03). Anomalies were observed in the highest dose group only. These rats showed signs of motor incoordination of the hind legs and depressed grooming; organ weight determination revealed increased relative weight of the kidneys. No histopathological changes of organs or differences in food consumption and water intake were observed. This was surprising since with replacement of about 50% of chloride through bromide impair of electrolyte balance was expected. These results were further confirmed by a 90-day experiment with the same dose regimen with the addition of a 75 mg NaBr/kg diet group (van Logten et al, 1974; please refer to section 6.4.1/04). In contrast to the short term experiment the investigation over 90 days showed growth retardation in the highest dosage group and decreased food conversion. In addition, a slight decrease in concentration of lymphocytes and doubling of neutrophilic granulocytes were observed. The most prominent effects were seen on the thyroid and gonads: Relative weight of thyroid was increased and relative prostate weight was decreased with both decreased spermatogenesis and vacuolisation of the zona fasciculata; females showed a decrease in the number of corpora lutea. These effects which strongly suggest an impairment of the endocrine system by bromide, have been confirmed in another 90-day experiment studying the effects of a low-chloride intake on bromide toxicity (van Logten et al, 1976; please refer to section 6.4.1/05). In a previous study it had been shown that elimination of bromide from the circulation was strongly dependent upon chloride intake. Omission of chloride from the diet caused an increase in bromide half-life. Low-chloride diet in combination with bromide intake showed effects on the same target organs as the investigations on bromide performed before. In addition, corticosterone concentration in plasma was determined and revealed a decrease at the two highest dosage levels used. This fits well with the histopathological changes in adrenals indicative of a decreased synthesis of glucocorticosteroids. Since this process is regulated by adrenocorticotropic hormone (ACTH) released by the pituitary, the origin of the observed effect might be a dysfunction of the hypothalamus-pituitary axis. Also other observed morphological changes, like a decrease in corpora lutea and impairment of spermatogenesis, and the observed growth retardation could be attributed to a decrease in pituitary function. The former might be due to a decreased secretion of gonadotropic hormone, the latter to a decreased secretion of somatotropic hormone by the pituitary gland. However, it can not be excluded that bromide has a direct effect on the other organs on the endocrine system. In particular, activation of the thyroid can not be explained by a decreased pituitary function. In the present three-generation reproductive toxicity study it was shown that of the high dosed (19200 mg NaBr/kg diet) females mated with untreated males only 20% became pregnant, and none of the untreated females mated with high dosed males became pregnant. Therefore, the observed effect of reduced and absent fertility in the 4800 and 19200 mg/kg diet groups, respectively, were due to infertility of male as well as female rats. This conclusion is in accordance with the histopathological lesions found in the testes as well as in the ovaries in the 90-day studies. In addition, pup viability at the 4800 ppm dose levels was significantly reduced but survival was shown to be greater in the second when compared to the first litter. All the young of the first litter alive on post-natal day 5 died before day 21 while all young alive on post-natal day 5 were still alive on day 21 in the second litter. No effects on breeding were observed at dose levels of 1200 ppm and below. For the group of animals treated at 19200 mg/kg diet, infertility was observed. After 7 month on the high dose level, the diet was changed to control diet for 3 month and rats were mated again. In contrast to the infertility observed before, animals showed fertility index of 62%, viability index of 61% and lactation index of 90%. From these results it is clear that the effects of bromide on reproduction system are reversible but could not be entirely compensated. No macroscopic anomaly in neither pup was observed throughout the investigation, although it is known that bromide easily crosses the placenta. Dams and F1 rats examined for bromide concentrations in internal organs showed equal amounts of bromide in kidney which provides that rats had been exposed to bromide in utero. Body and organ weight determinations did not reveal a clear dose-related pattern. A dose-dependent decrease in T4 levels in serum of parent animals of the F0 generation was observed. This finding is indicative of an inhibitory action of bromide on the synthesis of thyroid hormones, resulting in a physiological feedback mechanism of increased thyrotropic hormone (TSH) secretion by the pituitary gland causing an increased stimulation of the thyroid. This is in good agreement with the activation of the thyroid found by histological examination in the previous 90-day studies mentioned above. The decrease in thyroid hormones in animals treated at high dose levels was confirmed in an experiment on the time dependency of the effect of bromide on the thyroid. Using standard diets containing 4800 and 19200 mg NaBr/kg, significantly decreased thyroxine concentrations were found in both groups. From this experiment, it appeared that after only three days the thyroxine concentration in serum was significantly decreased and that it remained constant during an experimental period of 12 weeks. Experiments using a ´chloride-free`diet revealed reduced T4 levels in serum in animals treated at 500 and 2000 mg NaBr/kg diet. Using radiolabeled iodide, significantly increased iodide uptake for treatment with 500 mg NaBr, but only slight increase for treatment with 125 and 2000 mg NaBr/kg ´chloride-free`diet was observed. For an activated thyroid increased uptake and release of iodide is expected. However, the effect appeared to be biphasic. At 500 mg/kg the uptake was greater than with 2000 mg/kg, in the latter group the uptake was less and the release measured between 24 and 48 hours seemed to be enhanced. This can probably be explained by two opposite effects of bromide on the thyroid. Bromide, as a halogen, competes with iodide for the uptake in the thyroid gland and can replace iodide in thyroid hormones; thus, the synthesis of T4 might be decreased. This would lead to an enhanced stimulation of the thyroid by the pituitary gland. At 2000 mg/kg diet, the bromide concentration in the serum is probably so high in relation to that of iodide that even an activated thyroid takes up relatively more bromide than iodide. Therefore, in this dose group the iodide uptake by the thyroid might be less than in the 500 mg/kg group, although the release is faster. In this case, however, the additional possibility of a diminished stimulatory action of the pituitary at the highest dose level cannot be excluded. Although bromide has a very low acute oral toxicity, a striking complex of presumably related changes in the endocrine system was observed on subchronic administration. The most prominent alteration appeared to be the effect on the thyroid, found histopathologically and by the measurement of circulating thyroid hormones. Also in the present three-generation toxicity study, the decrease in thyroid hormones was the most sensitive criterion. On the basis of the effect on the thyroid in the 90-day study, a NOAEL of 300 mg/kg diet can be determined in the rat. For bromide ion this value is corresponding to 11.7 mg/kg bw/day for older rats (23.4 mg/kg bw/day for young rats). In an experiment with human volunteers dosed daily with 1 mg/kg bw for 8 weeks no changes in haematological, biochemical or endocrinological parameters were found. Plasma bromide levels determined in the volunteers were about 0.9 mmol/L, approximately 10% of the bromide concentration of 8 mmol/L found to induce alterations in the thyroid of rats. Therapeutic range of bromide is 6-12 mmol/L in man.

Reference 3

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 parameters in either generation evaluated at 50mg/kg/day of sodium bromide.

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

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

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

Referenceopen all close all

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-01-08 to 2007-10-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

The objectives of this study were to indicate whether doses of 600 and 800 mg/kg/day were unequivocally maternally toxic and whether those doses produced the irreversible foetal malformations that were seen at 1000 mg/kg/day in the previous developmental toxicity study in rats. This study was also designed to demonstrate whether or not the kinked ribs observed at 300 mg/kg day were no longer present at weaning, and to attempt to identify a No Observed Effect Level.
In addition to the requirements of OECD guideline 414 two recovery groups were included in the study. Animals of these additional groups were allowed to litter and rear newborns until weaning.

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: Not stated (One hundred and fifty seven time-mated females were obtained in one delivery to provide 154 females for the study. The delivery consisted of 3 sub-batches, mated over 3 successive days. On delivery (12 January 2007), one batch was on Day 1 of gestation, the second on
Day 2 and the third on Day 3 (day of detection of a vaginal plug or sperm in a vaginal smear = Day 0 of gestation)
- Weight at study initiation: 190-291 g (at detection of mating)
- Fasting period before study: No
- Housing: individually in solid-bottomed propylene cages (42 x 27 x 20 cm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 3-5 days (until commencement of treament on Day 6 of gestation)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 40 - 74%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 2007-01-12 To: 2007-06-20

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations of each dose level were prepared independently. The required weight of test substance was weighed, and the appropriate volume of vehicle (water for irrigation) added. Solutions were mixed until visibly homogeneous.

VEHICLE
- Concentration in vehicle: 5 mg/mL, 30 mg/mL, 60 mg/mL and 80 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:

Samples of dosing formulations were analyzed for concentration and homogeneity.
The dosing solutions were analysed on 2 occasions, from those prepared for dosing on the first and second weeks of dosing.
On each occasion, triplicate samples of approximately 1 mL were taken from each level containing the test item, and triplicate 2 mL samples from the Control.
Analysis of dosing formulations revealed concentrations within 2% of nominal and the low coefficients of variation (< 2%) indicated satisfactory homogeneity.

Details on mating procedure:

The objectives of this study were to indicate whether doses of 600 and 800 mg/kg/day were unequivocally maternally toxic and whether those doses produced the irreversible foetal malformations that were seen at 1000 mg/kg/day in the previous developmental toxicity study in rats. This study was also designed to demonstrate whether or not the kinked ribs observed at 300 mg/kg day were no longer present at weaning, and to attempt to identify a No Observed Effect Level.
Females were mated prior to acceptance onto the study. Mating procedure in accordance with OECD Guidelines for developmental toxicity.
No more than 2 females used on the study were inseminated by any one male

Duration of treatment / exposure:

Mating period: 3 consecutive days
Duration of exposure: Days 6-19 of gestation
On Day 20 of gestation main study animals were sacrificed and Caesarean section was performed. Rats from the recovery group were allowed to litter; maternal animals and pups were killed for examination at weaning (Day 21 of lactation).

Frequency of treatment:

Once daily

Duration of test:

Main test: 20 days
Recovery groups: 41 days (The day on which parturition commenced was designated Day 0 of lactation)

Remarks:

Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
600 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
800 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

22 females/group for both the main study and the additional two recovery groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for Groups 4 and 5 are between the original Intermediate and High dose levels used in a previous developmental toxicity study, Irving and Hallmark (2000). The level for Group 3 was the original Intermediate dose level and was used again to allow comparisons between the 2 studies; it was also an appropriate level for the littering phase. The dose level for Group 2 is below the previous Low dose level, and was intended to be a No
Observed Effect Level.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 4 and 6-20 of gestation. Only those recorded on Days 4, 6, 12, 15, 17 and 20 are presented in this report.
In addition, for the animals that littered, maternal bodyweights were recorded on Days 1, 7, 14 and 21 of lactation (where the day of littering is Day 0 of lactation).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: daily, commencing on Day 4 of gestation until Day 20 of gestation. For animals that littered, consumption was recorded over Days 0-7, 7-14 and 14-21 of lactation

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes (females allowed to litter)
- Sacrifice on lactation day 21
- Organs examined: Dams were necropsied; external examination followed by macroscopic examination of the tissues and organs of cranial, thoracic and abdominal cavities in situ. Reproductive tracts were examined for signs of implantation; numbers of implantation sites were recorded.

OTHER: Observations on females with litters during lactation:
Females from the recovery groups (0 and 300 mg/kg bw/day) were allowed to litter normally. The day on which parturition commenced was designated Day 0 of lactation. The duration of gestation in days was evaluated. The number of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation.

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
- Head examinations: No data

OTHER:
In addition to the requirements of OECD guideline 414 two additional groups were included in the study. Animals were either treated daily over Days 6-19 of gestation with 300 mg ammonium bromide/kg bw/day or left untreated. These additional groups of rats were allowed to litter and rear newborns until weaning. Pups were necropsied and examined for abnormalities.

Pre-weanlings found dead or which had to be killed before Day 14 of lactation were sexed, checked for the presence of milk in the stomach and for any externally visible abnormalities. Pups were fixed for optional further examination.
Weanlings at scheduled termination were necropsied, macroscopic examination of the tissues and organs of the thoracic and abdominal cavities in situ was performed. Pups were weighed prior to necropsy. Following examination, pups were fixed and stained for skeletal examination confined to the ribs and alignment of the pelvic girdle.

Statistics:

None

Indices:

See Table 4 in attached Results document

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: See details below

Details on maternal toxic effects:
All animals receiving 600 or 800 mg/kg bw/day showed treatment-related clinical signs, which included staggering, rolling gait, subdued behaviour, slow/irregular respiration, body held low, hunched posture, piloerection, eyelids encrusted, stained eyes and stained fur. These signs are indicative for neurological effects. Other findings noted at these levels were considered to be either associated with the above findings or to have been incidental. One animal of the 600 mg/kg bw/day group was killed for humane reasons on Day 11 of gestation with signs that were similar but more marked than others at this level or at 800 mg/kg bw/day. Necropsy did not indicate a cause of death, but death was probably attributable to treatment. At 300 mg/kg bw/day one animal was subdued, with body held low on one occasion, and a second animal had rolling gait on one occasion. Although these signs were also observed at 600 and 800 mg/kg bw/day, the low incidence at 300 mg/kg bw/day could not be conclusively attributed to treatment. The other clinical signs observed at this level were considered to be incidental.
One animal of the recovery group that was treated with 300 mg/kg bw/day, was found dead on Day 1 of lactation. There were no clinical signs recorded prior to death, and it was considered that the death was incidental, possibly associated with parturition.
No clinical signs were observed after treatment with 50 mg/kg bw/day.
There were no necropsy findings at any dose level that were associated with treatment.
At 800 mg/kg bw/day, mean weight gain over Days 6-17 of gestation was lower than in control animals (-9.8%). Weight gain over Days 6-20 appeared similar to control when the comparison was made with those control animals sacrificed at Day 20 of gestation; however, when the weight gain was adjusted for gravid uterus weight or when compared with those control animals allowed to litter, it was considered that gain over Days 6-20 had been lower than control. At 600 mg/kg bw/day, mean gain over Days 6-20 of gestation was slightly greater than control (+ 14.8%), particularly after Day 15 of gestation; the difference became more noticeable when the gain over Day 6-20 was adjusted for gravid uterus weight. At 300 mg/kg bw/day, mean weight gain over Days 6-20 was slightly greater than control (+ 13.9%), including the gain after adjustment for gravid uterus weight. On Day 1 of lactation, recovery animals that had been treated at 300 mg/kg bw/day before, showed mean weight slightly greater than control, but on Days 7 and 14 of lactation, mean weights were similar to control rats. Over Days 14-21 of lactation, these animals had a slightly greater weight loss compared with control. In the 50 mg/kg bw/day dose group, mean bodyweight values were similar to control.
In the 800 and 50 mg/kg bw/day dose groups, mean food consumption of dams was similar to control and in the 600 mg/kg bw/day dose group, mean food consumption was slightly greater than control. Animals that had been treated with 300 mg/kg bw/day during the gestation period consumed slightly more food than control during gestation, but showed slightly lower food consumption than control rats during lactation.
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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: See details below

Details on embryotoxic / teratogenic effects:
At 300, 600 and 800 mg/kg bw/day, there was a increased incidence of foetuses with kinked ribs, with the incidence being similar in all three groups. There was also an increase at these levels of foetuses with the minor abnormalities of slightly kinked and incompletely ossified ribs. Additionally, 14 foetuses at 800 mg/kg bw/day, and 5 foetuses each at 300 and 600 mg/kg bw/day had curved scapulae of which one foetus at 800 mg/kg bw/day also had incomplete ossified scapula.
At 600 and 800 mg/kg bw/day, there were increased numbers of foetuses with fewer than 13 complete ribs. At 300 mg/kg bw/day, the incidence of foetuses with fewer than 13 complete ribs was considered too small to be attributed to treatment.
Incidences of other major and minor abnormalities at 300, 600 and 800 mg/kg bw/day were considered to be essentially similar to controls.
There was no indication of any treatment related effect on the incidence of foetal abnormalities at 50 mg/kg bw/day.
At 300, 600 and 800 mg/kg bw/day, there was an increased incidence of foetuses with many parameters that tend to indicate delayed ossification of skull bones and lumbar vertebral arches, unossified 5th metacarpals and the number of sternebrae retarded. However, there was also an increase in ossification of cervical vertebral centra, a parameter that tends to indicate advanced ossification. Other parameters such as ossified anterior arch of atlas (increased at 300 and 800 mg/kg bw/day but not at 600 mg/kg bw/day) and incomplete ossification of pubes (increased at 300 and 600 mg/kg bw/day but not at 800 mg/kg bw/day) showed no clear relationship to dose. Taking the parameters together, it was considered that there had been some effect on foetal ossification at these dose levels.
Animals treated with 50 mg/kg bw/day showed skeletal ossification parameters similar to control.

Pregnancy data
There were no obvious effects on embryo-foetal mortality at any level tested. Slight intergroup differences in pregnancy performance and foetal weight were considered too small to be attributed to treatment.
Females that were allowed to litter and had been treated with 300 mg/kg bw/day showed a mean duration of gestation less than control, with a higher proportion of females having a 21 day gestation period, compared to controls.

Littering phase:
Mean values for litter size and survival at 300 mg/kg bw/day were similar to control. There was no indication of a treatment-related effect on maternal care or feeding. Mean pup weights were slightly lower in pups from treated rats compared with controls, particularly on Day 21 of lactation and more noticeably among males. Mean litter weights at 300 mg/kg bw/day were also lower than control. It was considered that these effects were related, to some extent, to the shorter duration of gestation after treatment.
Among weanlings, the incidences of abnormalities and variants at 300 mg/kg bw/day were similar to control. With the exception of one pup which had minimally kinked ribs, there were no pups with kinked ribs at 300 mg/kg bw/day.
Other minor findings such as mild bruising, swelling or cold, that are typical for pre-weaning pups were observed but have not been reported

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

See attached document for Results Tables

Conclusions:

In this study, 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 due to the severity of these signs. Bodyweight gain at 800 mg/kg bw/day was reduced (9%) when compared to controls (no statisticaly analysis was performed). Bodyweight gain at 300 and 600 mg/kg bw/day was increased (11% and 28%) when compared to controls (no statistically analysis was performed). There were no obvious effects on embryo-foetal mortality or foetal weights at any dose level tested. 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. NOEL for maternal toxicity was determined at 300 mg/kg bw/day. 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. 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 scapulae and effects on foetal ossification noted at ≥300 mg/kg bw/day.

Executive summary:

A developmental toxicity study was performed with ammonium bromide in rats, applying concentrations of 50-800 mg/kg bw/day of the substance by gavage during the period of organogenesis, to detect effects on pregnancy (in accordance with OECD guideline 414). The study was also designed to demonstrate whether or not the kinked ribs observed at 300 mg/kg bw/day in a previous study and also in this study, were no longer present and were, thus, reversible at weaning and to attempt to identify a NOAEL for embryotoxic/teratogenic effects.

Mated female rats were randomised into 4 test groups and one vehicle group. Two additional groups were assigned to control and 300 mg/kg bw/day groups to serve as recovery animals (littering phase). Rats were treated once daily over Days 6-19 of gestation, where the day of detection of mating was assigned Day 0.

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

Maternal toxicity at 600 and 800 mg/kg bw/day was characterized by clinical signs of toxicity (including staggering, rolling gait, subdued behaviour, slow/irregular respiration, body held low, hunched posture and piloerection). One animal at 600 mg/kg bw/day was killed for humane reasons. Necropsy findings did not indicate a cause of death, but the death was probably attributable to treatment. Bodyweight gain at 800 mg/kg bw/day was lower than control, but gain at 600 mg/kg bw/day was slightly greater than control. Food consumption at 800 mg/kg bw/day was similar to control, but consumption at 600 mg/kg bw/day was slightly greater than control.

Maternal effects at 300 mg/kg bw/day during gestation were limited to a slightly greater bodyweight gain and slightly greater food consumption. Among females with litters, there was a slightly greater weight loss than control during the third week of lactation, and lower food consumption during lactation.

There were no indications of maternal toxicity at 50 mg/kg bw/day.

There were no obvious effects on embryo-foetal mortality or foetal weights at any level tested.

At 300, 600 and 800 mg/kg bw/day, there were increased incidences of foetuses with kinked and/or slightly kinked ribs (5.4%, 8.5% and 6.6% 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). At 600 and 800 mg/kg bw/day, there were increased numbers of foetuses with fewer than 13 complete ribs. At 300, 600 and 800 mg/kg bw/day, there were indications of effects on foetal ossification, although some parameters tended to indicate delayed ossification whilst others would be expected to show advanced ossification. In a previous teratogenicity study with ammonium bromide (dose levels: 100, 300 and 1000 mg/kg bw/day), there was a similar pattern of findings amongst the ossification parameters and it was concluded that no statement can be made regarding the influence of ammonium bromide treatment on ossification parameters.

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, and the mean pup weights were slightly lower suggesting a consequence of the shorter gestation. Litter size and survival were not obviously affected.

The most important finding is, that among weanlings from rats treated at 300 mg/kg bw/day that were allowed to litter, the incidences of abnormalities of the ribs and pelvic girdle were similar to controls. This indicates that the kinked ribs and curved scapulae seen in the foetuses from rats treated at the same dose level are reversible effects which resolve after birth and are, thus, considered to be a transient change only.

The present study was designed to supplement the information from the standard developmental toxicity study in rats performed previously (Section 8.7.2 – Developmental toxicity AmBr Irvine and Hallmark (2000) Key). In that study, a dose of 1000 mg/kg bw/day was associated with maternal toxicity (clinical signs and reduced weight gain) and irreversible foetal kidney malformations which were observed in 7 out of 9 females that had shown the lowest weight gains over the first days of dosing. In the present study, doses of 600 and 800 mg/kg bw/day showed unequivocal maternal toxicity but the irreversible foetal malformations were not seen. From the findings in these two studies, it was considered that the threshold dose for unequivocal maternal toxicity was lower than the threshold dose for the irreversible foetal malformations in soft tissue, because maternal toxicity was seen at dose levels where no adverse effects on soft tissue had been noted. The previous study also indicated an increased incidence of foetuses with kinked ribs at 100, 300 and 1000 mg/kg bw/day, and with curved scapulae at 1000 mg/kg bw/day. Based on a review by Kast (1994) it was considered that the kinked ribs and the curved scapulae would have completely reversed within 3 weeks after birth and should therefore be considered as a reversible pathological finding. In the present study examination of weanlings treated at 300 mg/kg/day (dosing discontinued after Day 19 of gestation) showed that the kinked ribs were no longer present at weaning and confirmed this hypothesis also for ammonium bromide that the kinked ribs and curved scapulae should be regarded as a reversible pathological finding.

In the previous report it was considered by the original author that there were no indications of maternal effects at 100 and 300 mg/kg/day which could be confirmed in the present study. A review of the data from the previously performed teratology study indicated a slightly increased weight gain over Days 6-20 of gestation at 300 mg/kg/day when the effect of gravid uterine weight was included in the consideration which cannot be considered to be a toxicologically relevant nor adverse effect. However, a review of the data from that study indicated a slightly increased weight gain over Days 6-20 of gestation at 300 mg/kg/day when the effect of gravid uterine weight was included in the consideration.

Since the time of that study, a 13 week toxicity study and this current study have been conducted, and both of these studies have indicated a tendency for increased weight gain at lower doses (increased weight gain over Days 6-20 of gestation at 300 and 600 mg/kg bw/day in this study; increased weight gain over early weeks of treatment in females at 225 mg/kg/day in a 13-week study but a decreased weight gain at higher levels (decreased weight gain over Days 6-12 and 6-20 of gestation at 1000 mg/kg bw/day in the previous teratogenicity study). There has also been a tendency, at some dose levels, for an increased weight gain in the early part of the dosing period, then decreased weight gain in the latter part of the dosing period (increased weight gain over first week of treatment, followed by overall reduced weight gain in females at 750 mg/kg/day during a 13 week toxicity study).

The authors of the present study have set the maternal and foetal NOAEL at 50 mg/kg bw/day. However, the maternal effects seen at 300 mg/kg bw/day are increased weight gain and increased food consumption which usually are not determined as adverse or toxic effects. Therefore, the maternal NOAEL should be derived at 300 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. Since for the foetus effects (e.g. kinked ribs, curved scapula) were detected after dams had been treated at 300 mg/kg bw/day, foetal NOAEL should be at 50 mg/kg bw/day for the period of organogenesis and at 300 mg/kg bw/day post-weaning.

In this study, 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 due to the severity of these signs. Bodyweight gain at 800 mg/kg bw/day was reduced (9%) when compared to controls (no statisticaly analysis was performed). Bodyweight gain at 300 and 600 mg/kg bw/day was increased (11% and 28%) when compared to controls (no statistically analysis was performed). There were no obvious effects on embryo-foetal mortality or foetal weights at any dose level tested. 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. NOEL for maternal toxicity was determined at 300 mg/kg bw/day. 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 scapulae and effects on foetal ossification noted at ≥300 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Limited, Margate, Kent, England
- Age at study initiation: about 9 weeks
- Weight at study initiation: mean weight of 220 g
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was formulated as a solution in water for irrigation. Fresh suspensions were prepared daily on the day prior to use. For all formulations containing test material, the requisite quantity of test material was weighed, the necessary volume of vehicle added and the formulation mixed by manual inversion

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of 5 mL-samples of dosing formulations revealed differences from nominal concentrations of less than 2% for all dose levels of ammonium bromide, and low coefficients of variations indicated that the formulations were homogeneous

Duration of treatment / exposure:

Mating procedure: 3 consecutive days
Duration of exposure: Days 6-19 inclusive of gestation (Day 0 being the day of detecting mating)
On Day 20 Caesarean Section was performed.

Frequency of treatment:

once daily

Duration of test:

20 days

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

24 females/group

Control animals:

yes, concurrent vehicle

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Days 4 and 6-20 of gestation. Only those recorded on Days 4, 6, 9, 12, 15, 17 and 20 of gestation are presented in the report

FOOD CONSUMPTION: Yes
The weight of food consumed by each animal was recorded daily, commencing on Day 4 of gestation

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: No
- Sacrifice on gestation day 20
During necropsy of the first batch of animals, large variation in the size of placentae between dams was noticed. Therefore, for the necropsy of the second and third batch of rats, the placental weights were taken, where appropriate, for each animal. These data are not included in the report, but are maintained in the study files

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
- Head examinations: No data

Statistics:

Litter mean foetal weight data (sexes combined) were analyzed using the Kruskal-Wallis ANOVA.
Maternal bodyweight gain over Days 6-12 and 6-20 of gestation, and food consumption over Days 7-20 of gestation were analysed by analysis of variance. Where significant heterogeneity occurred, log and/or square root transformations were applied but variations remained heterogeneous, and so the Kruskal-Wallis ANOVA was used.

Indices:

See Table 3 in attached Results Document

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: See details below

Details on maternal toxic effects:
Treatment related clinical observations were noted for all animals treated at 1000 mg/kg bw/day and included rolling gait, animal limp when handled, hunched posture, subdued behaviour, piloerection, eyes dark and abnormal respiration indicative for neurological effects. These signs were noted following one day of treatment and generally persisted throughout the treatment period. One animal of this dosage group was sacrificed on Day 10 of gestation due to the severity of these signs. One animal of the medium dose group (300 mg/kg bw/day) showed piloerection on Days 8-12 of gestation, however in the absence of any of the other treatment related findings noted for the high dose group (1000 mg/kg bw/day) this was not considered to be directly related to treatment. Other occasional clinical signs such as hair loss and staining were considered to be unrelated to treatment. No clinical signs of toxicity were noted in the 100 mg/kg bw/day dose group.
The group mean bodyweight gain over the dosing period was significantly reduced at 1000 mg/kg bw/day (-18%) when compared with the control group. This reduction was a consequence of the reduced body weight gain during the first six days of treatment (Days 6-12 of gestation). There was a notable weight loss for two of the animals of this dosage group during this period. Thereafter, bodyweight gain for animals treated at 1000 mg/kg bw/day was similar to control. Bodyweight performance at 300 and 100 mg/kg bw/day was essentially similar to control. The slightly but significantly increased weight over Days 6-20 was attributed to the increased mean uterus weight at that level.
Slight differences in the group mean food consumption of the high dose group during the first half of the treatment, particularly around Days 10-13 of gestation, were considered to be related to the low food consumption of two of the animals for which a weight loss during the first six days of treatment was noted. Food consumption of the medium and low dose group was slightly but significantly greater than control, 107% and 105% of control values, respectively, when comparing food consumption from Days 7-20 in total (467, 457 and 435 g for medium, low and control group, respectively). These differences were considered too small to be positively attributed to treatment.
At necropsy for one animal of the high dose group uterus filled with red fluid was noted. Other occasional maternal necropsy findings noted for the remaining animals were considered to be unrelated to treatment.
There was no evidence of any effect of treatment with ammonium bromide on the pregnancies, in terms of intra-uterine mortality. The level of pre-implantation loss was higher than normally expected, with values of 24, 16, 26 and 21% for control, low, medium and high dose group, respectively. There was no indication of a treatment-related effect as it reflected events occurring prior to treatment.
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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: See details below

Details on embryotoxic / teratogenic effects:
Major foetal abnormalities:
Significantly reduced mean foetal weight at 1000 mg/kg bw/day (P<0.001; -14.5%) was noted when compared with control. At this dose level, 26 foetuses from 7 litters (out of 208 foetuses from 22 litters investigated) had abnormalities of the left kidney (absent/small/displaced/cystic); one of these foetuses also had an absent right kidney and right ureter. In 20 of these 26 foetuses the left ureter and/or left adrenal was also absent. Some of the affected foetuses also had narrowing of the left uterine horn (19 foetuses from 7 litters), and/or flattened/small spleen (18 foetuses from 6 litters). There was a dose-related increase in the incidence of foetuses with kinked ribs, with foetal incidence of 25% in the high dose group compared to 1.6% in controls and 4.5% and 9% in low and medium dose groups, respectively. The litter incidence was 82% in high dose group compared to 14% in controls and 32% in low and 41% in medium dose, respectively. Kinked ribs were often associated with incomplete ossification of ribs (foetal incidence of 16% in high dose and 0% in control group). At the high dose level, there was an increase in the number of foetuses with slightly kinked ribs (foetal incidence of 4.3% compared to 1.1% in controls) and curved scapula was observed in 18 foetuses from 8 litters (foetal incidence of 8.7% compared to 0.5% in control group). The latter finding generally occurred in foetuses with kinked ribs. Three foetuses from one litter had a short tail, but since this occurred in one litter this may have been an incidental finding. Other major foetal abnormalities were considered to have been incidental. For one small foetus of the low dose level a reduced spleen was seen, but there were no associated abnormalities of the kidneys, adrenals, ureters or uterus. For this reason this spleen finding was considered not to be of toxicological significance.
Minor foetal abnormalities:
The minor visceral abnormalities did not provide any obvious indication of an adverse effect of treatment. The incidences of foetuses with dilated ureter, dilated renal pelvis and displaced testis were considered to be within the expected background frequency, although the possibility that the incidence at 1000 mg/kg bw/day represented a marginal increase could not be entirely discounted: 4.4% from the high dose group exhibited dilated renal pelvis compared to 1.6% in control group. There were 6.3% foetuses with dilated ureter in high dose, 5.9% in medium dose, 3.2% in low dose and 3.1% in control group. Displaced testis were seen with an incidence of 9.6% in the high dose group (1000 mg/kg bw/day) compared to 1.6% in the controls and 4.5% and 8% in low and medium dose groups (100 and 300 mg/kg bw/day), respectively. There was a slight increase in the incidence of foetuses at 1000 mg/kg bw/day with reduction in size of the 13th rib(s). The incidence of findings at 100 and 300 mg/kg bw/day were not obviously associated with treatment.
Skeletal ossification:
The ossification parameters provided no clear indication of any effects on skeletal ossification Although parameters like skull bones, sacral vertebral arches and unossified 5th metacarpals indicated treatment related decrease in ossification, other parameters (lumbar centra, 2nd and/or 4th metacarpal/metatarsal) showed no effect and some parameters indicated advanced ossification (anterior arch of atlas, cervical vertebral centra). Taking all these parameters into consideration, it was not possible to state that skeletal ossification had been affected by treatment with ammonium bromide.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: Foetal toxicity observed at all dose levels

Abnormalities:

not specified

Developmental effects observed:

not specified

See attached document for Tables of Results.

Conclusions:

In this study, 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 least at low and mid dose, was within the background range of incidences for this strain of rats at the laboratory in question according to the study author. No historical control data is submitted to confirm this statement. 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. 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

Executive summary:

Ammonium bromide was tested in rats to detect effects of pregnancy when maternal exposure to the test material was confined to the period of major organogenesis. The study was performed in accordance with OECD guideline 414 and OPPTS 870.3700.

Mated rats were randomized into 4 treatment groups, each containing 24 animals. Rats were orally dosed by gavage daily over Day 6-19 inclusive of gestation, where Day 0 was the day of detecting mating. Ammonium bromide dose levels were 0, 100, 300 and 1000 mg/kg/day which were administered in water for irrigation as the vehicle at a constant volume of 10 mL/kg bw. Animals were monitored for clinical signs of toxicity, body weight and food consumption. The study was terminated on Day 20 of gestation. Status of each implantation was recorded and viable foetuses were examined. Half the foetuses from each uterus were fixed in methylated ethyl alcohol and examined by open dissection for abnormalities of the thoracic and abdominal viscera; kidneys and heart were cut and skeletons stained. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification. The other half of the foetuses were fixed in Bouin´s fluid for detailed analysis of soft tissue abnormalities using a freehand sectioning technique.

Maternal toxicity at 1000 mg/kg bw/day included abnormal clinical observations such as rolling gait, animal limp when handled, piloerection, subdued behaviour, hunched posture, eyes dark and abnormal respiration which were generally noted throughout the treatment period. These effects are consistent with those already described by other research groups after treatment with ammonium bromide. A reduction in bodyweight gain and slightly reduced mean food consumption was noted at 1000 mg/kg bw/day throughout the first 6 days of treatment.

There were no maternal effects at dose levels of 100 and 300 mg/kg bw/day.

Intra-uterine mortality was unaffected at all dose levels tested, but there was a statistically significant reduction in mean foetal weight (with P<0.001) at 1000 mg/kg bw/day.

Foetal abnormalities at 1000 mg/kg bw/day included reduced/absent left kidney: 26 from 208 investigated foetuses, corresponding to a foetal incidence of 12.5% and a litter incidence of 31.8% (7 from 22 litters investigated) compared to a foetal incidence of 0% in controls. Kidney abnormalities were often associated with absence of the left adrenal and/or left ureter. 6.7% of foetuses treated with 1000 mg/kg bw/day also had narrowing of the left uterine horn and 9% showed abnormalities of the spleen. There was a slight increase in the foetuses with reduction in size of the 13^thribs (6.8% in high dose group compared to 0% in controls).

A dose-related increase in the incidence of foetuses with kinked ribs was observed; foetal incidence of 1.6% in control, 4.5% in low dose, 9% in medium dose and 25% in high dose group animals were reported. The litter incidence of this abnormality was 14%, 32%, 41% and 82% for control, low, medium and high dose group, respectively. Kinked ribs were often associated with the appearance of incomplete ossification of ribs (foetal incidence of 0, 2, 9 and 16% for control, low, medium and high dose group, respectively). Treatment with 1000 mg/kg bw/day resulted in an increased incidence of foetuses with slightly kinked ribs (4.3% compared to 1.1% in controls). Within this dose group, 8.7% of foetuses showed curved scapula (0.5% in controls), 9% had abnormalities in spleen and 6,7% narrow uterine horn (in each case control values were 0%). Curved scapula generally appeared in animals which had also kinked ribs.

Kinked ribs, or wavy ribs, have been reported in rats by many researchers. Although kinked ribs have been classified as major abnormalities in this investigation, many researchers classify them as foetal variations. These findings have been reviewed by Kast (1994) who indicated that wavy ribs are often combined with fexures of limb bones, including the scapula. Wavy ribs are caused by numerous compounds with a large variety in their chemical structure and biological activity, and several modes of aetiopathology are discussed. Kast concluded that the wavy ribs developed in late gestation, and would have completely reversed within three weeks after birth, and therefore should be considered as a reversible pathological finding.

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.

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

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting 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.

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.

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.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1995

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

No investigation of maternal parameters such as body weight gain, corrected body weight gain, food consumption and clinical signs; only one dose applied; no skeletal and visceral examinations performed in pups. Therefore, the publication serves as supportive information as the investigations performed are not in compliance with OECD guideline 414 for the conduct of developmental toxicity studies in rats.

Qualifier:

no guideline available

Deviations:

not applicable

Principles of method if other than guideline:

study was performed according to good experimental practice

GLP compliance:

Remarks:

but study was performed according to good experimental practice

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Not indicated
- Age at study initiation: Not applicable
- Weight at study initiation: Not applicable

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 250 mg % (corresponding to 0.25 g/100 ml or 200 mg/kg bw)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Days 5-15 of gestation

Frequency of treatment:

daily

Duration of test:

Until postnatal Day 90

Remarks:

Doses / Concentrations:
250 mg %
Basis:
other: corresponding to 0.25 g/100 ml or 200 mg/kg bw assuming a bodyweight of 250 g per rat throughout the study period with water consumption of 20 ml per day

No. of animals per sex per dose:

18 per group in treated groups, 14 in the control group

Control animals:

other: Tap water or 250 mg % NaCl ( probably corresponding to 0.25 g/100 ml or 200 mg/kg bw)

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: not applicable

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

POST-MORTEM EXAMINATIONS: No data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter] brain was examined only.
- Skeletal examinations: No

Statistics:

Statistical differences were tested by ranking data and using the U-test to avoid dependency of test results on the frequency distribution of actual values.

Details on maternal toxic effects:

Maternal toxic effects:not examined

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Compared to controls, a smaller bodyweight was observed in bromide-treated rats from birth onwards (statistically significant from PD19 onwards). Similarly, brain weights were consistently lower in the bromide-treated group than in controls (statistically significant from PD8 onwards).
To estimate the deficits in development, measurements were performed and compared between bromide-treated and control rats of the same age. The delay in development was expressed as the difference between ages at which equal values were recorded in controls and bromide treated rats and secondly, bodyweight and brain weight difference of rats of the same age give incidence if treatment effects development. Analysis of these data revealed that bromide does not monotonously interfere with development. Three phases have been identified for the parameters measured, although the separation was less obvious for the bodyweight than for the brain parameters. During PD1-10 the developmental delay was small; bromide contents of blood and brain decreased towards control values. The next phase (PD10-40) started with a marked increase in deficit until PD20. During the following weeks the developmental deficit did not further increase. From PD 40 on, bromide-treated rats asymptotically reached maximal values (bodyweight, brain weight, protein content in brain) which however remained at subadult levels.
Bromide application specifically interfered with the development of olfactory system. In both control and bromide-treated rats, increasing numbers of olfactory glomeruli were established before and after birth.
Compared to controls, bromide caused a delay of a few days for the time required at which acid phosphatase activity first appeared in glomeruli after the latter had been formed as shown with synaptophysin immunohistochemistry. Thereafter, the acid phosphatase-staining pattern soon became similar to that of untreated controls or saline-treated animals.
The protein content was consistently lower in brains of bromide-treated animals than in controls from birth onwards. The difference was 11% of the mean control onwards.
Synaptophysin immunohistochemistry revealed, however, that the size of glomerular profiles was on average 30% larger in treated than in control rats. A quantitative evaluation confirmed the visual impression of the existence of a subpopulation of large glomeruli. Additionally, this figure shows that a small shift to larger diameters was already present at PD8 and that the size difference increased for more than one month after birth, although the average size of glomerular profiles increased further in both experimental and control animals for at least three months after birth

Abnormalities:

not specified

Developmental effects observed:

not specified

Blood samples of control pups showed approximately 1 mM bromide from the first postnatal day onward, whereas in bromide-treated rats values about 4.5 higher than in controls were determined indicative for a blood-placental in utero transfer of bromide from the dams to the offspring. During the following postnatal days, bromide concentration continuously decreased in blood as a consequence of its elimination in weaning rats and reached control level by about postnatal Day 20.

In brain homogenates prepared from control rats, bromide content was always lower than in blood of the same animals again indicative for a blood-placental in utero transfer of bromide from the dams to the offspring.. Immediately after birth, the concentration of bromide was three-fold higher in treated rats than in controls. At postnatal Days (PD) 2, 4, 6 and 8 bromide content decreased continuously and reached control values by PD 10. During the postnatal period, bromide concentration in brain decreased in absolute values, but the ratio brain/blood increased from 30 to about 50%.

Conclusions:

Treatment with bromide during Days 5-15 of gestation resulted in slightly lower bodyweight gain, brain weight and protein content of brain in the offspring, but the differences compared to control were only minor in degree and should therefore not be regarded as an adverse effect. During onset of development of pups of dams which have been treated through gestation, bromide levels continuously declined and were comparable to controls at later time points. Furthermore, the results of this investigation demonstrated that bromide-mediated developmental delays were completely compensated.

Executive summary:

Materials and Methods

The study was designed to investigate the potential effects of sodium bromide. Rat embryos were exposed to sodium bromide by providing an aqueous solution of 250 mg % NaBr (corresponding to 200 mg/kg bw) in the drinking water, ad libitum to their dams. Controls received either tap water or 250 mg % NaCl (corresponding to 200 mg/kg bw). Application was restricted to Days 5-15 of gestation.

Results and Discussion

It had been demonstrated previously that enteral administration of sodium bromide may mimic some of the neurotrophic or neuroplastic effects of locally applied γ-aminobutyric acid (GABA). In adult rats, prolonged NaBr administration produced essentially the same dendritic changes in principal ganglion cells of the superior cervical ganglion as GABA. This effect included the formation of paramembraneous densities. NaBr was found to inhibit acetylcholine release and synaptic transmission in this ganglion. Corresponding changes in acetylcholinesterase were noted after long-lasting NaBr treatment indicating a differential modulation of various molecular forms of this enzyme. Like GABA, NaBr finally promoted synapse formation of principal ganglion cells with foreign cholinergic nerves implanted into the superior cervical ganglion. The data presented in this investigation suggest that intake of sodium bromide by pregnant rats might interfere with general growth and brain development in the offspring. This effect apparently depends on bromide ions, because sodium chloride treatment did not induce any comparable changes. The present experiments provide further evidence that bromide ions may influence neuroplasticity under variable conditions. For example NaBr has turned out to affect different parts of the nervous system (sympathetic ganglia, visual system, and cerebral cortex) at various ages. For the dams, the period of bromide administration was restricted to the 5th and 15th day of pregnancy. This period was apparently not identical with the time of bromide exposure to the offspring. The beginning of action may have been delayed, because blood concentration of bromide rises slowly and may not have reached sufficient levels until the dams fat stores were possibly saturated with bromide although other studies demonstrated that bromide is preferentially distributed in the extracellular space. After the end of treatment, the bromide content in blood and brain tissue remained significantly higher than in control pups until 10 and 20 days after birth, respectively. Thus, bromide may have directly influenced the offspring’s development during a period of three to four weeks, i.e. at some time between the last 6-9 days of gestation and 10-20 days after birth. The higher blood bromide content in pups during post-natal days 10 – 20 may be explainable by an exposure of the pups during the lactation period via breast milk of the maternal animals. Bromide-dependent retardation of pup development is apparently not based on a uniform mechanism of interaction with developmental process. The kinetics of retardation rather suggests a distinction of three phases (PD1-10, PD10-40, PD40-90). Developmental delays were small and were more or less completely compensated, while bromide concentrations decreased to control levels in blood and brain. Surprisingly, developmental deficits strongly increased thereafter. This finding indicates that that direct bromide actions on body growth and brain development if existing at all, were almost completely compensated, while bromide decreased and approached control level in blood. That developmental retardation occurred thereafter, suggests that bromide must have affected some mechanisms involved in the regulation of postnatal development. Thus, indirect influences of bromide on development have to be taken into account. Also during the following phases (PD10-40 and PD40-90), development was not uniformly influenced. Developmental retardation became prominent during two periods of development (PD10-17 and PD40-60), while deficits became smaller during intervening time. What appeared as a transient improvement indicates that there might have been a second trial to compensate deficits in development between PD20 and PD40 that is after weaning. Since the time course was similar for bodyweight and brain parameters, partial recovery was probably based on regulatory mechanism which do not act specifically on the organ level but more generally, e.g. like any hormonal mechanism. In view of the complex set of deficits found after bromide treatment, one hardly expects bromide to have a uniform action mechanism or to modify a single type of molecular process indirectly influencing various other aspects of development. However, the fact that the reduction in brain weight is accompanied by a general deficit in body growth indicates that bromide may generally affect some metabolic regulation. One suggestion how bromide could interfere with general metabolism has been given by Buchberger (Buchberger et al, 1990; please refer to section 6.4.1/12). The authors investigated the effect of bromide on the thyroid gland in rats and showed that with increasing serum bromide concentrations T4 and T3 levels decreased and that there was an increased thyroid weight in the treated groups compared to controls. Treatment with 200 mg sodium bromide/kg bw/day already showed these effects, possibly via iodide replacement in the thyroid gland and with that induction of changes regarding the thyroid hormones. Further on, in a review by Kast (Kast, 1994; please refer to section 6.8.1/04) it is supposed that endocrine modulations might affect the rats foetus. Another possibility would be that bromide caused a general intoxication. In the present experiments, however, the application time was probably too short to provide enough bromide for intoxication. After providing adult rats for 10 days with 250 mg% of bromide in drinking water (which is corresponding to 200 mg/kg bw), the maximum blood concentration reached a level of 10 mM bromide in blood, which is approximately 1/7th of those blood levels found in mice during bromism. Not all bromide effects seem to cause a deficiency in development. In the olfactory bulb, the glomerular synaptic complexes were on average larger in bromide-treated rats than in controls, although in both groups of animals the size of glomeruli increased with postnatal age. Reasons for this discrepancy are at present unknown. On the one hand, some partial processes of brain development may be promoted rather than retarded by bromide and by its direct effects. On the other hand, bromide might also inhibit regressive processes, which are continually ongoing during replacement of olfactory receptor cells and their synapses in glomeruli. Bromide might have affected such degrading functions in neurons, e.g. by interfering with acidification in endosomes or lysosomes. Apart from interfering with general regulatory mechanisms of development, bromide may also exert some specific action on neural systems. As already mentioned, some GABA effects can be mimicked by sodium bromide. Another research group found that both GABA and NaBr can induce the formation of low affinity GABA receptors on cultured cerebellar neurons. Therefore, bromide-dependent hyperpolarisation may also interfere with some steps in the cascade of development reactions characterizing the trophic action of GABA. Bromide ions may hyperpolarize cells in various ways either by activating chloride influxes or by binding to synaptic membranes and thereby modifying their surface charge. By whatever mechanism, sodium bromide can reduce the release of certain but not all neurotransmitter substances from presynaptic nerve endings. Some reports indicate that strengthening GABA-like effects during development, by benzodiazepines, may affect brain development including olfactory functions. In addition, bioelectrical activity exerts trophic influences on normal brain development. As far as developmental deficits are concerned, future search for mechanisms will therefore have to distinguish among the several different possible modes of bromide action. Summarizing the results of this investigation, the effects seen in the pups from bromide-treated rats compared to control animals regarding bodyweight performance, brain weight and protein content in brain are only minor in degree. Bodyweights from bromide-treated dams between postnatal Day 60 and 90 were about 40 g less compared to controls (200-240 g compared to 240-280 g in treated and control animals, respectively), but there was a high standard deviation within the bromide-treated group. The differences in brain weight were negligible and the mentioned reduction in protein content of brain of about 11% can be explained by the slightly lower brain weight. It is important to note that in a study by Buchberger and colleagues (Buchberger et al, 1990; please refer to section 6.4.1/12) it was shown, that treatment with 200 mg sodium bromide/kg bw/day induces changes in the thyroid gland in the absence of adverse clinical signs. Changes of parameters of the endocrine system of dams were discussed to be the cause of modifications within the foetus, when treatment was performed during pregnancy (review by Kast, 1994; please refer to Document IIIA, Section 6, point 6.10/06). Since approx. the same amount of sodium bromide was applied to the rats in the present investigation, it is justified to assume that the same effects on the thyroid were induced by treatment and that these effects influenced foetal development. However, the effects seen within the present investigation were only minor in degree and should therefore not be regarded as an adverse effect.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008

Reliability:

1 (reliable without restriction)

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

Remarks:

study is a dose range finder

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

Remarks:

study is a dose range finder

GLP compliance:

yes

Limit test:

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd, Blackthorn, Bicester, Oxon
- Age at study initiation: animals for the non-pregnant part of the study were approximately 21 weeks of age; pregnant animals were approximately 19-22 weeks of age
- Weight at study initiation: 3.84-4.16 kg (non-pregnant part); 2.98-3.73 kg (pregnant animals)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Samples of the test material formulation were analysed for concentration of sodium bromide. The accepted range was ± 10% of nominal concentration and the results indicated that the performed formulations were within ± 7% of the nominal concentration

VEHICLE
- Concentration in vehicle: 31.3, 62.5, 125 and 250 mg/mL for the non-pregnant phase and 25, 50 and 100 mg/mL for the pregnant phase of the study.
- Amount of vehicle (if gavage): 4 mL/animal/day

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Samples of the test material formulation were analysed for concentration of sodium bromide. The accepted range was ± 10% of nominal concentration and the results indicated that the performed formulations were within ± 7% of the nominal concentration.

Duration of treatment / exposure:

please refer to "any other information on materials and methods".

Frequency of treatment:

Escalating dose animals: 3 consecutive days
Fixed dose animals: 13 consecutive days
Pregnant phase: Day 3 - Day 28 of gestation

Remarks:

Doses / Concentrations:
125, 250, 500 and 1000 mg/kg bw/day for the escalating dose regime and 500 mg/kg bw/day for the fixed dose for 13 days during the non-pregnant phase of the study. 100, 200 and 400 mg/kg bw/day for the pregnant phase of the study.
Basis:
actual ingested

No. of animals per sex per dose:

6 females for the non-pregnant part of the study: 3 for the escalating dose part, 3 for the fixed dosage.
24 time-mated female rabbits were used for investigations on potential developmental effects of the test substance: 6 animals/dosage group and 5 control animals receiving vehicle only

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, immediately post dosing and in addition one and five hours after dosing during the working week.

BODY WEIGHT: Yes
- Time schedule for examinations: Non-pregnant animals were weighed daily, pregnant animals were weighed on Days 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Individual food consumption was recorded for non-pregnant phase animals daily and for pregnant phase animals for the periods Days 3-6, 6-9, 9-11, 12-15, 15-18, 18-21, 21-23, 24-27 and 27-29 of gestation.

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 / No / No data
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: No data

Statistics:

None performed due to the variability of the data and the small group size used in this dose range finding study.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Escalating dose animals:
Treatment of three animals at dose levels of 125, 250 and 500 mg/kg bw/day for three consecutive days did not reveal any overt signs of toxicity as assessed by clinical condition, bodyweight and bodyweight change or food intake. Treatment of the same animals at 1000 mg/kg bw/day on a single occasion resulted in notable clinical signs (decreased respiration and ataxia) and the following day this dosage was terminated. Necropsy did not show any cause for the clinical conditions.

Fixed dose animals:
Following treatment at 500 mg/kg bw/day for 13 days, one animal showed decreased respiration rate and ataxia on Day 8 and was euthanized for animal welfare reasons. There was an indication of reduced food intake in the days preceding this occurrence, but no corresponding effect on bodyweight gain. Necropsy finding of this animal did not reveal any cause for its condition. Treatment of the other two animals was not associated with evidence of toxicity as assessed by clinical condition, bodyweight, bodyweight change, food intake and necropsy findings were considered to be unremarkable.

Pregnant phase:
Treatment of pregnant rabbits at 400 mg/kg bw/day from day 3 through 28 of gestation demonstrated ataxia in two animals from Day 25 and 28 of gestation, respectively, and resulted in early termination of the first affected animal on Day 27. When ataxia was observed, the signs persisted to the next dosing occasion on the following day. Lower food intake was apparent for both of these animals from Day 24 of gestation but there was no obvious corresponding effect on bodyweight gain. Necropsy findings of either animal did not reveal any obvious cause for the clinical observations. Treatment of the other four animals at 400 mg/kg bw/day was not associated with marked evidence of toxicity as assessed by clinical condition, bodyweight, bodyweight change, food intake or necropsy findings. There were no obvious adverse effects observed at dosages of 100 and 200 mg/kg bw/day. Neither the type, incidence nor distribution of necropsy findings indicated any adverse effect of treatment with sodium bromide.

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Only a small number of litters were available for the evaluation of treatment related effects. The numbers of implantations, pre and post implantation losses, live litter size, sex ratio, litter, placental and foetal weights and external foetal morphology did not indicate any adverse effect of treatment when administering sodium bromide doses of 100, 200 and 400 mg/kg bw/day during Days 3-28 of gestation.

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: fetotoxicity

Dose descriptor:

LOAEL

Effect level:

> 400 mg/kg bw/day (actual dose received)

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

One female dosed at 100 mg/kg bw/day gave birth to two of the offspring before necropsy on Day 29 of gestation. In the absence of any signs of early delivery for other animals at this or higher dosages, this single event was considered incidental and not related to treatment.

Table A6.8.1/01-1: Bodyweight, Gravid Uterus Weight and Bodyweight Performance during Gestation - Group Mean Values

Dose Level [mg/kg bw/day]	Bodyweight [kg]		Bodyweight Change Days 3-29 [kg]	Gravid UterusWeight	Adjusted Bodyweight Day 29 [kg]	Adjusted Bodyweight Change Days 3-29[kg]
Dose Level [mg/kg bw/day]	Day 3	Day 29	Bodyweight Change Days 3-29 [kg]	Gravid UterusWeight	Adjusted Bodyweight Day 29 [kg]	Adjusted Bodyweight Change Days 3-29[kg]
0	3.83	3.68	0.3	0.445	3.24	-0.14
100	3.21	3.84	0.63	0.53	3.31	0.1
200	3.2	3.83	0.62	0.495	3.33	0.13
400	3.17	3.76	0.59	0.499	3.26	0.09

Table A6.8.1/01-2: Food Consumption during Gestation - Group Mean Values

Dose level [mg/kg bw/day]	Food Intake [g/animal/day] during day of gestation:
Dose level [mg/kg bw/day]	3-6	6-9	9-12	12-15	15-18	18-21	21-24	24-27	27-29
0	102	121	110	129	132	143	149	124	117
100	151	190	173	160	167	151	116	96	115
200	161	173	158	160	171	168	147	117	109
400	121	153	151	144	161	160	140	99	124

Table A 6.8.1/01-3: Litter Data on Day 29 of Gestation - Group Mean Values

Dose Level [mg/kg bw/day]	No. of corpora lutea	No. of implants	No. embryonic/foetal deaths			Implantation loss [%]		No. live foetuses			% male foetus	Male foetal weight [g]	Female foetal weight [g]	Mean foetal weight [g]	Litter weight [g]	Mean placental weight [g]	Total placental weight [g]
Dose Level [mg/kg bw/day]	No. of corpora lutea	No. of implants	E	L	T	Pre	Post	M	F	T	% male foetus	Male foetal weight [g]	Female foetal weight [g]	Mean foetal weight [g]	Litter weight [g]	Mean placental weight [g]	Total placental weight [g]
0	13.3	7.8	0.3	0.5	0.8	42.1	11.3	3.5	3.5	7	53	43.7	39.7	42.6	285.9	5	33.7
100	11.6	8.8	0.2	0	0.2	23.8	2.5	3.8	4.8	8.6	43.3	48.3	4	47.7	400.2	5.6	47.8
200	11.5	8.5	0.2	0.5	0.7	24.1	7	3.8	4	7.8	48.7	42.2	41.3	42	329.3	5.5	43.3
400	12	7.8	0.3	0	0.3	35.4	2.8	4.3	3.3	7.5	54.4	48	44.4	46.2	337.8	5.8	43

E early

L late

T total

M male

F female

Conclusions:

Treatment of non-pregnant animals at 500 or 1000 mg/kg bw/day or pregnant animals at 400g/kg bw/day was associated with adverse clinical signs (ataxia and decreased respiration rate) in the absence of clear effects on bodyweight or food intake during the observation period. In the pregnant phase of the study, no treatment-related effects on the fetal parameters examined were noted up to the highest dose level studied.
The study author concluded that the observed clinical signs were resulting from cumulative toxicity with some animals being more susceptible than others. For the main developmental toxicity study on sodium bromide dose levels of 25, 75 and 250 mg/kg bw/day were selected and, in addition, the start of application was set on Day 6 of gestation. The main study was performed using the dosages recommended by the study author.
From the results obtained in this dose range finding study, the applicant, however, concludes that the same dosage levels than those applied in the dose-range-finding study should have been used for the main study since no severe maternal toxicity was seen at 400 mg/kg bw/day and no signs attributable to treatment were noticed at the lower dosage levels used (100 and 200 mg/kg bw/day).

Executive summary:

Materials and Methods:

The purpose of the study was to provide information for the selection of suitable dose levels for a pre-natal developmental toxicity study on sodium bromide in the rabbit. The study was designed to investigate effects of sodium bromide on the progress and maintenance of pregnancy in the rabbit, including early indication of potential effects on embryonic and foetal development. The test substance was administered orally by gavage, prior to implantation and throughout the period of organogenesis until the day prior to expected delivery. The study was divided in two parts: at first, the maximum tolerated dose was established using non-mated animals and applying an escalating dosing regime; then the potential effects on pregnancy were investigated in time-mated animals at a fixed dose level following administration for 13 days and at three dose levels during days 3 through 28 of gestation. Sodium bromide was administered once daily to three non-pregnant rabbits using an escalating dosing regime. An initial dosage of 125 mg/kg bw/day was investigated, followed by 250, 500 and 1000 mg/kg bw/day at intervals of 3 days. A further three non-pregnant animals were dosed once per day for 13 consecutive days at 500 mg/kg bw/day which were euthanized on Day 14. For investigation on the pre-natal development, two groups of six time-mated rabbits were dosed daily on Days 3-28 of gestation at 100, 200 and 400 mg/kg bw/day. A control group of 5 time-mated rabbits received vehicle only. All animals were euthanized on Day 29 of gestation. Clinical signs, bodyweights and food consumption were monitored during the investigation period and all animals were subjected to gross necropsy including examination of the uterus. In the offspring, foetal sex, external foetal appearance and foetal weight were determined.

Results and Diskussion:

Treatment of non-pregnant animals at 1000 mg/kg bw/day on the escalating dose regime was associated with ataxia and decreased respiration rate following a single day of treatment which precluded this dosage from further investigation. While this effect may have been directly attributable to this dosage, it may also, in part, be a result of cumulative toxicity from the previous dosages applied (125, 250 and 500 mg/kg bw/day for three consecutive days each). Treatment of non-pregnant animals at 500 mg/kg bw/day for 13 consecutive days resulted in one animal being euthanized after showing similar signs following seven days treatment, although the remaining two animals survived termination (Day 14) without showing any adverse effects of treatment. Treatment of pregnant animals at 100, 200 and 400 mg/kg bw/day on Days 3-28 of gestation was not associated with any clear adverse effects on bodyweight, bodyweight gain or food consumption during gestation. There were no clinical signs observed after treatment with 100 and 200 mg/kg bw/day. At 400 mg/kg bw/day, two out of six animals showed ataxia towards the end of the study, with one animal being euthanized due to animal welfare reasons. Macroscopic necropsy observation did not indicate any adverse effects of treatment in the dams. No treatment-related effects were noted in the remaining 4 animals treated at 400 mg/kg bw/day. There was no pre- or post-implantation loss noticed and no adverse effects were observed regarding foetal weight, sex ratio or macroscopic findings. Although ataxia and decreased respiration as the only adverse clinical signs in dams were observed after treatment at 400 mg/kg bw/day and no adverse effects appeared at 100 and 200 mg/kg bw/day, the study author suggests to use 250 mg/kg bw/day as a high dose for the main teratology study in rabbits, since treatment at 400 mg/kg bw/day could lead to early termination for some animals. In addition, it was argued that the observed ataxia is a result of cumulative toxicity and therefore treatment should start at Day 6 of gestation to reduce the risk of early termination of animals. However, the applicant concludes that there were no adverse effects observed in four out of six animals dosed at 400 mg/kg bw/day and there were no treatment related changes in the foetal parameters examined (foetal weight, sex ratio, implantation loss) at this dose level. In addition, clinical signs observed in two rabbits treated at 400 mg/kg bw/day were not indicative for severe toxicity, were seen only towards the end of the study and the application period started already on day 3 vs. day 6 according to the guideline. Since dams did not show any treatment-related effects when dosed at 100 and 200 mg/kg bw/day, the dosing regime used for this dose range finding study (concentrations and duration of treatment) should have been used for the main study as well.

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

equivalent or similar to guideline

Guideline:

other: 12 NohSan No 8147 (Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies) Deviations no

Deviations:

GLP compliance:

yes

Limit test:

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd., Blackthorn, Bicester, Oxon, United Kingdom;
and Harlan France, Gannat, France.
The animals (72 obtained from the UK, 48 from France) were of the same strain and the same genetic lineage.
- Age at study initiation: not indicated in the report
- Weight at study initiation: 2.63-4.76 kg

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PPREPARATION OF DOSING SOLUTIONS:
Satisfactory homogeneity and stability of the test substance in aqueous formulations at concentrations of 1 and 250 mg/ml following storage at +4°C for up to 14 days had been confirmed. Formulations of test material used to dose the animals in this study were routinely analysed for achieved concentration and the results revealed that concentrations were within the acceptable range of ± 10%. Only on one analysis occasion, formulations at 6.25 mg/ml (25 mg/kg bw/day group) and 62.5 mg/ml (250 mg/kg bw/day group) formulation were ±12% of nominal concentration.

VEHICLE
- Concentration in vehicle: 6.25, 18.8 and 62.5 mg/mL
- Amount of vehicle (if gavage): Approximately 4 mL;
The dose volume given was calculated using the most current recorded bodyweight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Satisfactory homogeneity and stability of the test substance in aqueous formulations at concentrations of 1 and 250 mg/ml following storage at +4°C for up to 14 days had been confirmed. Formulations of test material used to dose the animals in this study were routinely analysed for achieved concentration and the results revealed that concentrations were within the acceptable range of ± 10%. Only on one analysis occasion, formulations at 6.25 mg/ml (25 mg/kg bw/day group) and 62.5 mg/ml (250 mg/kg bw/day group) formulation were ±12% of nominal concentration.

Duration of treatment / exposure:

Day 6-28 of gestation

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
25, 75 or 250 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

30 time-mated animals for the test groups and 29 time-mated animals as controls. One animal (number 101), allocated to the Control group, was rejected from the study prior to the commencement of dosing due to animal welfare considerations. The data for this animal are not presented within this report but is retained with the raw data for the study.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels had been chosen based on the result of a dose-range finding study (please refer to Document IIIA, Section 6, Point 6.8.1/01). Because of ataxia being observed at treatment with 400 mg/kg bw/day which could have potentially led to the early termination of animals, thereby reducing the number of litters available for assessment, this precluded the use of 400 mg/kg/day as the high dosage for this main study and it was therefore necessary to choose a dosage where no maternal toxicity could be expected. Therefore, the study author decided to use 250 mg/kg bw/day as the high dose level to prevent potential early termination of animals.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing immediately post dosing and one hour after dosing every day and in addition, five hours after dosing during the working week.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighted on Days 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was recorded for the periods Days 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 of gestation.

WATER CONSUMPTION:
Formal gravimetric measurement of daily water intake was instigated after visual inspection of water bottle residues indicated a possible increase in water consumption for treated animals.

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:

The following parameters were analysed statistically, using the test methods outlined below:
Bodyweight and bodyweight change, food consumption, foetal, placental and litter weights: Bartlett´s test for homogeneity of variance and one way analysis of variance, followed by Dunnet´s multiple comparison test or, if unequal variances were observed, an alternative multiple comparison test.
All litter data parameters: Kruskal-Wallis non-parametric analysis of variance, and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney U-test, where significance was seen.
Foetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis parametric analysis of variance and Mann-Whitney U-test.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
Clinical observations did not indicate any obvious effects of treatment at any dose level used in the investigation. There were no unscheduled deaths during the study.

BODY WEIGHT AND FOOD CONSUMPTION
Bodyweight and bodyweight change during gestation did not indicate any adverse effects of treatment at the dosages investigated. At 250 mg/kg bw/day, overall bodyweight change was slightly superior to controls, due to higher bodyweight gains between Day 8 to 12 of gestation. At the lower dosages (25 and 75 mg/kg bw/day) bodyweight change was essentially similar to controls, although overall bodyweight gain at 75 mg/kg bw/day was superior to control following adjustment for the contribution of the gravid uterus.
Food intakes during gestation did not indicate any obvious adverse effects of treatment at any of the dosages investigated. At 250 mg/kg bw/day, food intake was slightly lower than control prior to treatment with sodium bromide. However, following the commencement on Day 6, food consumption was slightly higher than controls up to Day 24 of gestation. A similar pattern of higher food consumption, although to a lesser extent, was apparent for females receiving 75 mg/kg bw/day from Day 6 to Day 21 of gestation.

GROSS PATHOLOGY
The incidence of necropsy findings among adult animals on Day 29 of gestation did not indicate any obvious adverse effects of treatment.

OTHER FINDINGS
Animals treated at 75 and 250 mg/kg bw/day showed increased water intake. Water consumption of the lowest dosage group (25 mg/kg bw/day) was considered to be unaffected by treatment.

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

> 250 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:
VIABILITY
Intergroup differences in litter data did not indicate any adverse effect of maternal treatment on foetal survival at any of the dosages investigated.

BODY WEIGHT
Placental, litter and foetal weights were unaffected by maternal treatment at any of the dosages used in the investigation.

GROSS PATHOLOGY
Neither the type, incidence or distribution of findings observed for foetuses during necropsy, nor visceral head or skeletal examinations indicated any adverse effect of maternal treatment on foetal growth or morphological development. Similarly, the assessment of skeletal development parameters did not reveal any treatment-related trends in the proportion of foetuses (or litters) that indicated any effect of maternal treatment. At 250 mg/kg bw/day, there was a significant difference in the proportion of foetuses with 13 rib pairs. At 75 mg/kg bw/day, there was a significant difference in irregular ossification of more than one cranial bone.

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: fetotoxicity

Dose descriptor:

LOAEL

Effect level:

> 250 mg/kg bw/day (actual dose received)

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

At 25 mg/kg bw/day one female showed total litter lossin utero. In the absence of any increased post-implantation loss in remaining litters at this dosage or any similar incidences at the higher dosages applied (75 and 250 mg/kg bw/day) the total litter loss in on low dose female only was not considered to be of any toxicological relevance.

Table A6.8.1/02-1: Summary of female performance

Category	Dose Level [mg/kg bw/day]
Category	0	24	75	250
Initial group size	29	30	30	30
Non-pregnant	3	4	4	3
Total litter loss in utero	0	1	0	0
With live young at Day 29	26	25	26	27

Table A6.8.1/02-2: Clinical Observations in Dams - Group Incidence

Parameter investigated	Number of animals affected at Dose Level [mg/kg bw/day]
Parameter investigated	0	25	75	250
Number of females	29	30	30	30
Prior to dosing
Generalised fur loss	4	3	3	8
Generalised scabbing	-	-	2	-
Staining around ano-genital region	-	1	-	1
Scab on tail	1	-	-	-
Crooked gait	1	-	-	-
Broken tail	-	1	-	-
Days 6-12 #
Generalised fur loss	3	4	2	7
Generalised scabbing	-	-	2	1
Staining around ano-genital region	-	1	-	1
Broken tail	-	1	-	-
Days 13-21
Generalised fur loss	2	1	2	6
Generalised scabbing	-	-	1	1
Staining around ano-genital region	-	1	-	2
Red staining of ano-genital region and cage tray	1	-	-	-
Diarrhoea	-	-	1	2
Decreased respiration rate and noisy respiration	1	-	-	-
Broken tail	-	1	-	-
Days 22-29
Generalised fur loss	1	-	-	3
Staining around snout	-	-	-	1
Fur staining	-	-	-	1
Staining around ano-genital region	1	1	-	5
Diarrhoea	-	-	1	3
Wound on upper lip	-	-	1	-
Broken tail	-	1	-	-

Table A6.8.1/02-3: Bodyweight Change - Group Mean Values

Dose level [mg/kg bw/day]	Bodyweight Change [kg] during days of gestation
Dose level [mg/kg bw/day]	3-6	6-7	7-8	8-9	9-12	12-15	15-18	18-21	21-24	24-27	27-29
0	0.07	0	0.02	0.01	0.05	0.08	-0.02	0.05	0.06	0.07	0.05
25	0.08	0.01	0	0.01	0.04	0.07	0.02*	0.01	0.07	0.05	0.05
75	0.04	0.02	0.01	0.02	0.06	0.1	0	0.04	0.06	0.05	0.05
250	0.05	0.03	0.02	0.07	0.09	0.07	0.02**	-0.01	0.05	0.06	0.05

* p0.05

** p0.001

Table A6.8.1/02-4: Gravid Uterus Weight and Adjusted Bodyweight Performance - Group Mean Values

Dose Level [mg/kg bw/day]	Bodyweight [kg]		Bodyweight Change Days 6-29 [kg]	Gravid UterusWeight [kg]	Adjusted Bodyweight Day 29 [kg]	Adjusted Bodyweight Change Days 6-29 [kg]
Dose Level [mg/kg bw/day]	Day 6	Day 29	Bodyweight Change Days 6-29 [kg]	Gravid UterusWeight [kg]	Adjusted Bodyweight Day 29 [kg]	Adjusted Bodyweight Change Days 6-29 [kg]
0	3.45	3.82	0.37	0.5	3.32	-0.13
25	3.52	3.84	0.32	0.47	3.37	-0.15
75	3.5	3.89	0.39	0.47	3.42	-0.08
250	3.47	3.91	0.44	0.51	3.4	-0.08

Table A6.8.1/02-5: Food consumption - Group Mean Values

Dose level [mg/kg bw/day]	Food Intake [g/animal/day] during day of gestation:
Dose level [mg/kg bw/day]	3-6	6-9	9-12	12-15	15-18	18-21	21-24	24-27	27-29
0	129	135	129	116	115	128	119	108	112
25	130	133	127	106	112	124	114	100	99
75	129	141	140	128	125	137	119	109	108
250	122	143	151	138	143*	142	126	109	109

* p0.05

Table A 6.8.1/02-6: Litter Data on Day 29 of Gestation - Group Mean Values

Dose Level [mg/kg bw/day]	No. of corpora lutea	No. of implants	No. embryonic/foetal deaths			Implantation loss [%]		No. live foetuses			% male foetus	Male foetal weight [g]	Female foetal weight [g]	Mean foetal weight [g]	Litter weight [g]	Mean placental weight [g]	Total placental weight [g]
Dose Level [mg/kg bw/day]	No. of corpora lutea	No. of implants	E	L	T	Pre	Post	M	F	T	% male foetus	Male foetal weight [g]	Female foetal weight [g]	Mean foetal weight [g]	Litter weight [g]	Mean placental weight [g]	Total placental weight [g]
0	10.8	8.6	0.1	0.6	0.7	20.2	7.6	4.4	3.5	7.9	56.1	43.2	42.2	43.0	334.7	5.3	41.4
25	9.8	7.8	0.4	0.2	0.6	20.4	6.9	3.8	3.5	7.3	50.9	44.7	45.2	45.4	326.1	5.6	40.1
75	10.4	7.5	0.3	0.2	0.5	27.2	5.6	3.9	3.0	7.0	56.4	46.9	46.4	46.8	315.1	5.9	38.6
250	11.0	8.3	0.1	0.6	0.7	26.7	7.4	4.0	3.6	7.6	53.9	45.9	45.0	46.1	331.6	6.1	41.7

E early

L late

T total

M male

F female

Table A6.8.1/02-7: Foetal Skeletal Development - Group Incidence

Skeletal Development	Dose Level [mg/ml]
	0			25			75			250
	Number of Foetuses (litters) affected
	NF	NL	%	NF	NL	%	NF	NL	%	NF	NL	%
Number examined	205	26	-	182	25	-	181	26	-	204	27	-
Number of ribs: 11/11 11/12 12/11 12/12 12/13 13/11 13/12 13/13	1 1 - 116 15 - 13 59	1 1 - 25 11 - 9 17	0.4 0.5 - 56.1 7.5 - 5.8 29.7	1 - 1 117 9 1 9 43	1 - 1 23 8 1 6 18	0.8 - 0.6 61.6 5.8 0.4 4.5 26.2	- - - 120 5 - 11 45	- - - 24 4 - 10 15	- - - 65.4 2.2 - 6.8 25.6	- - 1 160 10 - 11 21	- - 1 27 7 - 9 10	- - 0.5 80 4 - 4.9 10.6*
Number of fully ossified sternebrae: 4 4 >4	1 7 197	1 5 26	0.5 3.2 96.3	- 13 169	- 9 25	- 8.3 91.7	4 12 165	4 6 26	1.8 5 93.2	10 12 182	6 7 26	3.4 7.7 88.9
Incomlete ossification of more than 1 cranial bone	1	1	1.3	8	4	10.4	5	4	4.0	2	2	1.4
Irregular ossification of more than 1 cranial bone	23	14	25.9	27	18	32.7	42	20	53.2**	31	19	35.1
Asymmetrical ossification of more than 1 sternebrae	1	1	0.5	2	2	1.4	3	3	1.3	10	6	3.5
Two or more sternabrae fused	-	-	-	3	2	1.6	2	2	0.8	5	5	2.1
Total number of affected	163	26	80.3	132	25	75.7	148	26	83.4	148	27	72.2

NF number of foetus

NL number of litters

% group mean %

- not applicable; no foetuses affected

* p0.05

** p0.01

Conclusions:

The oral administration of sodium bromide during Days 6-28 of gestation at dose levels of 25, 75 and 250 mg/kg bw/day did not produce any adverse toxicological changes in the parameters investigated in neither maternal animals nor in foetuses. Therefore, the highest dosage used (250 mg/kg bw/day) can be regarded as a NOAEL both for maternal and embryotoxicity.

Executive summary:

Materials and Methods:

The study was designed to investigate the effects of sodium bromide on embryonic and foetal development of the rabbit. The test substance was therefore administered orally by gavage from implantation throughout the period of organogenesis until the day prior to expected delivery. Time-mated animals were dosed at 25, 75 or 250 mg/kg bw/day of sodium bromide or received distilled water as a control during Days 6-28 of gestation. Clinical signs, bodyweight and food consumption were monitored throughout the study and water consumption was instigated as the study progressed. All animals were euthanized on Day 29 of gestation and subjected to gross necropsy including examination of the uterine contents. Foetuses were subjected to macroscopic necropsy examination. From half of the foetuses from each litter the head was removed and preserved in Bouins fixative for visceral examination and the other half of the foetuses were eviscerated, skinned and processed for skeletal examination. The dose levels used in this main teratology study in rabbit were selected on the basis of a dose-range-finding teratology study where dose levels of 0, 100, 200 and 400 mg/kg bw/day were administered to pregnant rabbits from day 3 through 28 of gestation.

Results and Diskussion:

Treatment of rabbits on Days 6-28 of gestation at dosages of 25, 75 and 250 mg/kg bw/day was well tolerated by the rabbit and was not associated with any adverse toxicological changes. Increased water intake at 75 and 250 mg/kg bw/day was apparent throughout the treatment period and might be due to the high salinity of the dose formulations. While no adverse effects on bodyweight gain or food consumption was apparent, administration of 250 mg/kg bw/day resulted in higher bodyweight gain and food consumption compared to controls during the early part of the treatment period. At 75 mg/kg bw/day, a similar pattern of slightly higher food intake during the early dosing period was apparent, without bodyweight gains being obviously increased. Considering the higher bodyweight change normally observed for rabbits, the observed difference in the early treatment period might reflect normal biological variation. However, the increased food consumption was already seen during the first four days in a teratology study on rats, which were treated at 1000 mg/kg bw/day (please refer to Document IIIA, Section 6, Point 6.8.1/05). There was no obvious effect of maternal treatment on the survival, growth or development of the offspring at dosages up to 250 mg/kg bw/day. There was a decrease in the proportion of foetuses with 13 ribs in the highest dosage group (250 mg/kg bw/day) with 10.6% of animals having 13 ribs compared to 29.7% in controls due to an increase of foetuses with 12 rib pairs. As the lower incidence of 13 rib pairs was not associated with any decrease in rib pairs (i.e. there were no foetuses with 11 rib pairs at this dosage) or any trend in other developmental parameters, the lower proportion of foetuses with 13 rib pairs at 250 mg/kg bw/day can not be considered to be a treatment-related effect and is therefore of no toxicological significance. Furthermore, this effect was seen in a small number of females in this group that had a relatively low number of implantations, with the number of animals with unilateral implantation being also higher at this dosage. As treatment did not start until Day 6 of gestation, it is considered that implantation had already been established and thus, no treatment-related effect for these incidences is taken into account. Likewise, the irregular ossification at 75 mg/kg bw/day of cranial bones was considered to be of no toxicological relevance in the absence of a dose-related response. In addition, the same as described for the high dose group is true, since reduced ossification of cranial bones was seen in litters from a few dams only with a low number of implantations.

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1995

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

No guideline available for the special investigations performed; results reported serve as supportive information as the investigations performed are not in compliance with OECD guideline 414 for the conduct of developmental toxicity studies in rats. In addition, the high dose level administered is in a dose range, where severe maternal toxicity was evident in other teratology studies in rats which were conducted in compliance with current guidelines.

Qualifier:

no guideline available

Deviations:

not applicable

Principles of method if other than guideline:

study was performed according to good experimental practice

GLP compliance:

Limit test:

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Not indicated
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: not indicated

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 1 or 5 g/L

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mean compound intake was about 230 mg/dam/day in the high dose group (5 g/L) with a consumption of 46 mL/dam/day drinking water per day and 73 mg/dam/day for the low-dose group (1 g/L) with a consumption of drinking water of approximately 73 mL/dam/day each day.

Duration of treatment / exposure:

Dams were treated with bromide via drinking water during the whole lactation period, after and during which pups and dams were examined for effects.
For the Br-82 experiment (time course of bromide transfer) a single exposure by subcutaneous injection on Day 12 of lactation.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
Mean compound intake was about 230 mg/dam/day in the high dose group (5 g/L) with a consumption of 46 mL/dam/day drinking water per day and 73 mg/dam/day for the low-dose group (1 g/L) with a consumption of drinking water of approximately 73 mL/dam/day.
Basis:
other: calculated concentration based on substance concentration in vehicle (water) and daily water consumption.

No. of animals per sex per dose:

5 dams/group; 8 pups/dam

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: not indicated

BODY WEIGHT: Yes
- Time schedule for examinations: not indicated

FOOD CONSUMPTION: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

POST-MORTEM EXAMINATIONS: No data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No

Fetal examinations:

- External examinations: Yes: (size and weight were examined for each pup)

Statistics:

All statistical evaluation of the data was performed by nonparametric Kruskal-Wallis analysis of variance, followed by the Mann-Whitney test for pairwise post hoc comparison. The difference is considered significant, if p< 0.05.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
One dam of the low dose and one dam of the high dose group died. There was a gradual increase in food consumption of dams in the course of lactation in both the control and the low dose group. There were no significant differences between these groups with regard to the amount of food consumed. However, in the case of the high dose group, there were no changes in food consumption during the whole lactation period as is expected for lactating rats. Starting from the second week of the nursing period, the amount of food consumed by these rats was markedly lower compared to that consumed by the control rats and the rats of the low-dose group. The same was true for the consumption of drinking water.
The enhanced intake of bromide in the course of the lactation period did not influence significantly the bodyweight of the lactating rats in the low-bromide group; changes were comparable to the control group. Dams of the high-bromide group showed a mild but gradual decrease in bodyweight (see table 6.8.1/07-1).
A very marked effect is seen after high bromide intake in dams on their production of milk (please refer to table A6.8.1/07-2). Throughout the whole lactation period, the production rate of mother’s milk in the dams of the high-bromide group was significantly lower in comparison with the control dams (p<0.002) and the dams of the low-bromide group (p<0.005).

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

900 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
All pups of the control group survived, whereas in the low-bromide and in the high-bromide groups, only 94.8% and 56.3% of the young survived, respectively.
There was a marked effect on the bodyweight of the pups whose mothers received 5 g bromide/L drinking water starting on about Day 4 of life, the bodyweight increments in these suckling were progressively lower in comparison with the control young and the pups from the low-bromide group. This can be causally related to a lower production rate of mother’s milk in the dams of the high-bromide group during the lactation period resulting in a lower pup body weight as a consequence of malnutrition.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Dose descriptor:

LOAEL

Effect level:

900 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Enhanced intake of bromide increased the sum of concentrations of both bromide and chloride, as well as the concentration of sodium in the rat milk (please refer to table 6.8.1/07-3). However, although the chloride concentration in the milk of the dams’ drinking water with the addition of 1 g bromide/L slightly increased, in the milk of dams treated at 5 g/L a considerable part of chloride was replaced by bromide, resulting in lower chloride concentrations compared to control.

Following single exposure with⁸²Br on Day 12 of lactation, radioactive bromide appeared in the body of the young already in the course of the first 3 hours after application to the dams.

Table A6.8.1/07-1: Bodyweight and bodyweight change of the dams

Group (g bromide/L)	Bodyweight on postpartum Day [g]			Bodyweight change [g]
Group (g bromide/L)	2	15	28	Bodyweight change [g]
Control (0)	278.0 ± 16.9	302.8 ± 19.1	279.8 ± 20.5	1.8 ± 7.9
Low-bromide (1)	248.0 ± 11.1	278.6 ± 8.0	262.6 ± 7.9	14.6 ± 11.2
High-bromide (5)	286.2 ± 17.9	264.0 ± 40.3	229.0 ± 74.9	-57.2 ± 67.7*

* values significantly different from the control group and from the low-bromide group (p= 0.008).

Table A6.8.1/07-2: Production Rate of Mothers Milk on different days of the Lactation Period, in Dependence on Bromide Intake of the Dams

Group (g bromide/L)	Mean intake of bromide [mg/day/dam]	Milk on Day [g/10 hours]
Group (g bromide/L)	Mean intake of bromide [mg/day/dam]	10	15
Control (0)	0.24 ± 0.02	20.9 ± 3.4	21.8 ± 6.0
Low-bromide (1)	72.8 ± 7.6	17.6 ± 1.6	23.1 ± 1.8
High-bromide (5)	222.7 ± 28.7	7.4 ± 2.3*	8.4 ± 5.3*

* significantly different from the control and the low-bromide group.

Table A6.8.1/07-3: Concentrations of Bromine, Chlorine and Sodium in Full Milk of Rats, in Dependence on Bromine Content in the Drinking Water

Group (g bromide/L)	Bromide [µmol/g]	Chloride [µmol/g]	Bromide + Chloride [µmol/g]	Sodium [µmol/g]
Control (0)	0.11 ± 0.01	48.9 ± 10.5	49.0 ± 10.5	74.6 ± 10.3
Low-bromide (1)	12.6 ± 0.8	52.6 ± 7.3	65.2 ± 7.5	84.7 ± 11.8
High-bromide (5)	34.1 ± 5.5	29.3 ± 1.7*	63.4 ± 7.1	87.2 ± 5.3

* significantly different from the control and low-bromide group

Conclusions:

Treatment of rats with 5 g bromide/L drinking water resulted in lower bodyweight performance and less milk production in dams. Bodyweight and survival of the pups from this group were also reduced which is likely a consequence of reduced milk production in dams resulting in a state of malnutrition and lowered viability in pups.
The observed effects in the dams and pups might be not only related to a direct effect of bromide in rats, but also a secondary effect from endocrine modulations within the dam. Most importantly, the high dose level in this study is comparable to a dosage which caused severe maternal toxicity in other developmental toxicity studies performed in rats and, thus, effects observable in the offspring are considered to be secondary to maternal toxicity.

Executive summary:

Materials and Methods

The investigation was performed to examine effects of low and high bromide intake in lactating rats on the performance of the dams and on the prosperity of their pups. Rats were given 1 and 5 g bromide per liter drinking water during the lactation period, providing a mean daily bromide dose of 73 and 230 mg/animal?. Animals were observed for clinical signs and bodyweight performance. In addition, food consumption and compound intake were recorded regularly and chloride, bromide and sodium content determined in mothers milk. Pups were observed for survival and body weight during this period. In another experimental series, lactating rats drinking tap water were given approximately 2.3 MBq 82Br in the form of potassium bromide (173 µg bromide) in 0.3 ml saline by subcutaneous injection on Day 12 after delivery. Radioactivity in dams and litter was measured in vivo by means of a γ-spectrometric system.

Results and Discussion

The experiments show that excessive bromide intake in lactating rat dams affected both their own organisms and the organisms of their suckling young. However, marked effects were found only in dams treated at 5 g/L of bromide corresponding to 230 mg/rat/day or about 900 mg/kg bw/day for a rat weighing 250 g. In these dams, two distinct consequences undoubtly caused by a high bromide intake were observed: stagnation in the extent of the consumption of diet and water in the course of the nursing period and a drop in the production rate of mother’s milk. It should be stressed, however, that a decline of the food consumption did not occur immediately after the first contact of the dams with water containing bromide, but in the course of the following 2-3 days. It is therefore evident that the reason for this eating behaviour of the animals was not a change in the taste of the drinking water. One of the possible explanations of this effect supposes that under conditions of high bromide levels in the organism, a disturbance in the physiological function of the stomach could arise. It is known that the biological behaviour of bromide ions in organs and tissues of mammals is analogous to the behaviour of chloride ions, with the exception of the thyroid gland, where the behaviour of bromide is more similar to that of iodide. Under the conditions of enhanced bromide intake, bromide replaces extracellular chloride, the molar sum of both halogens remaining constant. Because bromide is also concentrated in gastric mucosa and secreted into the stomach lumen possible changes in the composition of the digestive juice could disturb the digestive process or could produce an organic disorder of the gastrointestinal system. As a consequence of an excessive intake of bromide in the course of the lactation period in the dams of the high-bromide group, a gradual decrease in their average bodyweight was observed. However, a marked decrease in the bodyweight was noticed only in two of the five rats of this treatment group. Changes in bodyweight of the other rats from this group were negligible and comparable with those in control rats. Very different susceptibility of lactating rats to a high bromide intake is also reflected in a rather large standard deviation of the differences in the bodyweight gain. Very pronounced effects of high bromide levels in the organism of the mothers were observed in the young of the high-bromide group: Only 56% of the pups survived; their mean total bodyweight on Day 27 of life was less than 40% of the bodyweight of the control pups and their general condition was very poor, although the amount of bromide in relation to their bodyweight was lower than compared to their mothers. For instance, an Day 11 the mean amount of bromide received by one suckling rat was 263 mg/kg bw/day, whereas that of the dams was 822 mg/kg bw/day. The approximate dose of bromide obtained by the young was calculated as the product of the amount of received mother´s milk (1.42 g), inferred from the mean daily bodyweight increment (0.95 g) and the mean concentration of bromide in the milk of the dams of the high-bromide group (2722 µg/g). A characteristic property of mammary function is that it is controlled by a number of hormones and local factors. Studies in a number of species have shown that milk composition is markedly resistant to environmental changes, including nutritional changes, whereas the quantity of milk secreted shows considerable plasticity. The rate of milk secretion is lowered, for instance, by deficiencies of thyroxine, adrenocorticosteroids and parathormone. The frequency or completeness of milk removal from the lactating mammary gland regulates the rate of milk secretion by a mechanism that is local, chemical, and inhibitory in nature. The evidence indicates that local regulation of milk secretion by milk removal is through autocrine feedback inhibition by a single milk protein. Rats normally nurse their young very frequently and, depending on litter size, can produce relatively large quantities of milk. The accumulation of milk in the mammary gland does not mechanically restrict the flow of blood through the tissue, and in the rat, mammary blood flow and milk secretion are strongly dependent on a continually applied suckling stimulus. The observed drop in the production of mother’s milk and the stagnation or gradual decrease in the consumption of diet and water by the lactating dams of the high dose group could be caused, therefore, paradoxically by their own young. Provided that a high concentration of bromide in mother’s milk constituted a serious obstacle for the young to receive the milk, a decline in the intensity of lactation and, consequently, in the consumption of food and water by the dams would follow. Indeed, it was found that bromide ions ingested by the dams readily moved into the milk, and via that, they were transferred in a large extent to the suckling. Analysis of mother’s milk showed that the amount of bromide in the samples depended on the bromide concentration in the drinking water taken by the dams. The mean daily consumption of water by the dams of the low-bromide group was 73 mL/dam/day in the course of the second week of the nursing period. Hence, the mean daily intake of bromide in these animals was 73 mg/rat/day. In the case of the rats in the high-bromide group, the mean daily consumption of water was 46 mL/dam/day and thus, the mean daily dose of bromide was 230 mg/rat/day. With the mean daily bromide dose of 73 mg, the chloride concentration in mother´s milk remained practically unchanged and only the sum of the molecular concentrations of both halogens increased, the part of bromide being 19%. However, with the mean daily bromide dose of 230 mg, about 54% of chloride ions were replaced by bromide, the sum of the molecular concentrations of both halogens being unchanged. A rise in the concentration of both halogens caused also an increase in the sum of the concentrations of sodium in the milk. Whereas the increase in the sum of the concentrations of bromide plus chloride was about 30%, the increase in sodium concentration was lower (less than 20%). It is necessary, however, to note that the concentration of sodium was estimated only approximately. Although bromide passed easily through mother´s milk into the body of the young, this transfer occurred at a much slower rate than the transfer of iodide. In the case of iodide, nearly 30% of 131I applied to the mother appeared in the body of the young already in the course of the first three hours. In contrast, only 3% of bromide was detectable in the pups three hours after application to the dam. The observations made in this investigation of a massive transfer of bromide ingested by the dam via mother´s milk to the suckling were not unexpected with regard to the results from previous studies. Another research group (Disse et al, 1996; please refer to Document IIIA, Section 6, point 6.8.1/06) found that bromide applied to pregnant rats in drinking water (2500 ppm NaBr) in the period between Day 5 and 15 of gestation was transferred to embryos via placenta and later, up to Day 10 after birth even via the milk to the offspring. These authors also observed significant delays in postnatal development in all bromide-treated animals. Permanent deficits were recorded for bodyweight, brain weight and protein content in brain tissue, but effects of delayed learning ability were reversible. These results and the observed effects in the present investigation suggest that prenatal and perinatal exposure of rats to moderate concentrations of bromide might interfere with postnatal development including that of brain. To further discuss this point, a study performed by Buchberger and colleagues showed that treatment of rats with 200 mg NaBr/kg bw/day, corresponding to approximately 50 mg NaBr/rat, already resulted in effects on the thyroid gland with decreased T3 and T4 levels and increased thyroid weight in the absence of any adverse clinical signs (Buchberger et al, 1990; please refer to Document IIIA, Section 6.10/14). In a review by Kast possible causes of foetal malformations are discussed; one of them being endocrine modulation (Kast, 1994; please refer to Document IIIA, Section 6, point 6.10/06). Taking these observations together, bromide-treatment leads to changes in the thyroid or rats already at concentrations where no adverse clinical signs are observed. This endocrine modulation has an effect on the unborn rats and probably also via the milk on the sucklings. As mentioned above, reduced thyroxine levels lead to lowered milk secretion. The observed smaller amount of mothers milk seen in the high-bromide dams in this investigation could therefore also be explained by the influence of bromide on the thyroid gland, which leads to reduced amounts of thyroxine (T4). In conclusion, not only bromide could be the reason for the changes observed, but in addition secondary effects of bromide initiated by changes in the endocrine system which might (1) influence the amount of mothers milk produced and (2) be transferred via mothers milk to the pups.

Reference 8

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

Method: other

GLP compliance:

Species:

rat

Strain:

Wistar

Route of administration:

oral: drinking water

Duration of treatment / exposure:

Series 1 and 2: days 2 to 28 postpartum. Series 3: day 12 only

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 1 and 5 g Br-/L
Basis:
nominal in water

No. of animals per sex per dose:

Series 1 and 2: 30 female and 30 male rats were mated. For dosing postpartum: 5 dams per group – all 15 dams gave birth on same day. Number of pups adjusted to 8 per nest.
Series 3: 6 lactating dams, each kept with a nest of 8 young.

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: Series 1 and 2: 27 days. Series 3: 1 day

Dose descriptor:

NOAEL

Effect level:

277 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

277 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Result is stated in units of mg (Br-)/kg bw/day

Conclusions:

Maternal
LOAEL Dose level: 5 g Br-/L (220 mg Br-/day according to author)  847 mg Br-/kg bw/day (equivalent to 1091 mg/kg bw/day NaBr).
Effects: stagnation in the extent of the consumption of diet and water in the course of the nursing period, and a drop in the production rate of mother’s milk.
No NOAEL stated: 1 g Br-/L implied as producing minimal adverse effects (277 mg Br-/kg bw/day equivalent to 356 mg/kg bw/day NaBr)
Teratogenic effects
LOAEL Dose level: 5 g Br-/L (220 mg Br-/day according to author)  847 mg Br-/kg bw/day (equivalent to 1091 mg/kg bw/day NaBr).
44% of the young died. Their mean body weight was 40% of the body weight of the control young and their general condition was very poor
No NOAEL stated: 1 g Br-/L implied as producing minimal adverse effects (277 mg Br-/kg bw/day equivalent to 356 mg/kg bw/day NaBr)

Executive summary:

Female Wistar rats were mated and dosed with sodium bromide in their drinking water from the second day post partum. In an additional experiment, dams were injected with radiolabelled potassium bromide in order to observe the transfer to the pups via the dams’ milk.

For the dams food and water consumption and body weights were determined regularly. The number of surviving foetuses and their weights were also determined along with the total amount of radiolabelled bromide in their bodies.

Adverse effects were only noted in the highest dose group. These included: a stagnation in the extent of the consumption of diet and water in the course of the nursing period, and a drop in the production rate of mother’s milk. Significantly more of the young in the highest dose group died and their mean body weights were lower.

Maternal

LOAEL Dose level: 5 g Br^-/L (220 mg Br^-/day according to author)à847 mg Br^-/kg bw/day (equivalent to 1091 mg/kg bw/day NaBr).

Effects: stagnation in the extent of the consumption of diet and water in the course of the nursing period, and a drop in the production rate of mother’s milk.

No NOAEL stated: 1 g Br^-/L implied as producing minimal adverse effects (277 mg Br^-/kg bw/day equivalent to 356 mg/kg bw/day NaBr)

Teratogenic effects

LOAEL Dose level: 5 g Br^-/L (220 mg Br^-/day according to author)à847 mg Br^-/kg bw/day (equivalent to 1091 mg/kg bw/day NaBr).

44% of the young died. Their mean body weight was 40% of the body weight of the control young and their general condition was very poor

No NOAEL stated: 1 g Br^-/L implied as producing minimal adverse effects (277 mg Br^-/kg bw/day equivalent to 356 mg/kg bw/day NaBr)

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

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

Ammonium 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 ammonium 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.

Additional information

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Diss Factsheets

Ammonium bromide

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

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SUMMARY

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Effects on developmental toxicity

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Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

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