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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test 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:
no

Test material

Constituent 1
Reference substance name:
Ammonium bromide
EC Number:
235-183-8
EC Name:
Ammonium bromide
Cas Number:
12124-97-9
IUPAC Name:
ammonium bromide
Details on test material:
- Name of test material (as cited in study report): Ammonium bromide
- Physical state: White powder
- Analytical purity: 99.94%
- Lot/batch No.: 980060
- Storage condition of test material: in the dark at ambient temperature
Sodium bromide is an inorganic salt that dissociates to its composite ions in aqueous solutions at environmental pH and temperature. Comparison of the available data on the various bromide salts have shown that the bromide ion is the relevant ion for determination of the toxicological profile with simple cations such as potassium, sodium or ammonium, that are ubiquitous in nature, having little or no influence on the bromide ion properties. It is therefore justified to read-across data from other inorganic bromide salts to sodium bromide.

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 600 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
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%)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

See attached Results Tables document.

Applicant's summary and conclusion

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

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%).