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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
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:
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 (CAS 12124-97-9)
- Physical state: White granular powder
- Analytical purity: 99.98%
- Lot/batch No.: 610060177
- Expiration date of the lot/batch: 2008-06-30
- Stability under test conditions: Formulations were used within the stability period of eight days
- Storage condition of test material: ambient temperature in the dark.
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
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

Administration / exposure

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)
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

See attached document for Results Tables

Applicant's summary and conclusion

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.