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Effects on fertility

Link to relevant study records
Reference
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:
no
Remarks:
GLP was not obligatory at the time of study conduct and study was performed according to good experimental practice
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Not stated
- 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.
Study was not based on standard protocol. Every generation was mated twice (F0 generation mated 4 times): F0 _ F1a, F1b (further mated), F1c (for transplacental Br transport study), F1d (reversibility study) F1b _ F2a, F2b (further mated) F2b _ F3a, F3b Mating of F1 and F2 generations was not performed for the 4800 or 19200 mg/kg (diet)/day dose groups.
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:
Males: 9-10 per group (F0) or 10 per group (F1, F2).
Females: 7-12 per group (F0), 14-19 per group (F1), 10 per group (F2)
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 (Body weight Yes – at termination of each new generation (age 21 days))


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
Organ weights P and F1: uterus, ovaries, testes, prostate, pituitary, thyroid, adrenal glands
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
Mean day 21 bodyweight
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)
Fertility was nil in the 19200 ppm dose group and markedly reduced in the 4800 ppm dose group. In the lower dose groups, no effects related to treatment were found in the breeding results. In the 4800 ppm dose group, the viability of the offspring was lower than in the control group. Organ weights: no pattern of dose related effects was evident except for a dose-dependent decrease in the F0 female adrenals relative weights.
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. Reversibility study (leading to F1d litters): fertility index 62%, viability index 61%, lactation index 90%. Although viability was lower than in control and lower dose groups, the fertility index and lactation index were similar indicating that the effects of bromide on reproduction are reversible. Criss-cross study: treated males with untreated females – 0% pregnant; treated females with untreated males – 20% pregnant. Hence the observed effects were due to infertility of the male as well as female rats
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
Description (incidence and severity):
Body weight examinations at termination of each new generation (age 21 days)
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.
Macroscopic examination of all of the pups after 21 days revealed no evidence of anomalies. From transplacental studies (F1c generation) the results show that foetuses in utero must have been exposed to bromide, since the concentration of bromide in the kidneys of corresponding dams and foetuses is almost equal.
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]

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]

Plasma

[mmol/L]

Placenta

[mmol/kg]

Kidney

[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]

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.Approximately equal numbers of male and female rats were allowed to breed whilst being administered sodium bromide (doses: 0, 75, 300, 1200, 4800 and 19200 ppm) in their feed. The subsequent F1 generation was allowed to breed resulting in an F2 generation which in turn was allowed to produce an F3 generation. Two litters were obtained at each generation (two additional litters at the F1 generation). Fertility index, viability and lactation indexes were evaluated for each generation. Pathological examinations were made including after 21 days for pups measuring body and organ weights. The reversibility of the fertility effects in the highest dose group was also examined along with the effects of mating treated males with untreated females andvice versa. In the 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

No treatment-related effects on the breeding results were observed up to 1200 ppm. Fertility was nil in the 19200 ppm group and was markedly reduced in the 4800 ppm group. These effects were found to be reversible. In the latter group, the viability of the offspring was lower than in the other groups. Microscopic examinations of all pups born during the entire experimental period provided no evidence of anomalies.

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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
90 mg/kg bw/day
Study duration:
chronic
Species:
rat
Additional information

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

A dose-range finding reproduction toxicity study was performed with ammonium bromide in rats in order to select appropriate dose levels for the conduct of further studies. Ammonium bromide was administered in rats at concentrations of 0, 1600, 3200 and 6400 ppm (corresponding to 0, 127, 242 and 503 mg/kg bw/day in males; 0, 228, 454, 651 mg/kg bw/day in females) of the test substance via food. Animals were treated from two weeks prior to mating until the first generation had been weaned. 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. 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 (127 mg ammonium bromide/kg bw/day) 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 (127 mg ammonium bromide/kg bw/day) 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%) (Bartonet al., 2001).

 

In a three-generation reproduction study (publication) dose levels of 75, 300, 1200, 4800 and 19200 ppm sodium bromide (corresponding to 5.6, 22.5, 90, 360 and 1400 mg/kg bw/day using a conversion of 1 ppm=0.075 mg/kg bw/day) were administered to rats via the diet. 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. The transplacental transport of bromide was investigated in the third litter of the first generation of this study. 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 the observed effects. In order to investigate into the cause of the infertility observed a cross-mating procedure was performed in which untreated males and females were mated with females and males of the 19200 ppm group. To study the reversibility of the effects, an additional litter was bred with parental animals of the highest dose group which were fed control diets for a period of 3 months after the 7-month treatment period. In addition to the investigation of the reproductive performance of rats, additional emphases were placed on the thyroid gland. Animals treated at 19200 ppm diet were not fertile, and fertility of the next lower dose level (4800 ppm) was reduced (fertility index: 25%). 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 (viability index: first litter: 32%, second litter: 61%). During the lactation, 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. Only 20% of females treated at 19200 ppm sodium bromide and mated with untreated males and none of the untreated females mated with high dosed males (19200 ppm) became pregnant. Therefore, the observed effects were due to infertility of male as well as female rats. After being three month on the control diet, these animals were mated again and showed fertility index of 62%, viability index of 61% and lactation index of 90%. From this it can be concluded that animals demonstrate recovery from the infertility effects of bromide and the observed impairment of fertility appears to be reversible, although the viability was lower than in the control and lower dose groups. Adrenals of females of the F0 generation showed a dose-dependent decrease in relative weight (statistically significant at 4800 and 19200 ppm only). No macroscopic anomaly in neither pup was observed. Investigation of thyroid hormone levels revealed a dose-dependent statistically significant decrease in T4 levels in serum of parental male animals of the F0 generation. In parental female animals of the F0 generation T4 levels in serum was statistically significant decreased at 4800 and 19200 ppm only. 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. No NOEL parental was determined in this study. NOAEL parental was determined at 1200 ppm (90 mg/kg bw/day) based on reduced infertility noted in both sexes at ≥4800 ppm, reduced relative adrenal weight noted in females at ≥4800 ppm and reduced thyroid hormone (T4) noted in both sexes at ≥4800 ppm. The NOAEL offspring was 1900 ppm based on reduced viability noted at ≥4800 ppm (Van Leeuwenet al., 1983).


Short description of key information:
In a 3-generation reproduction study with sodium bromide the NOAEL parental was determined at 1200 ppm (90 mg/kg bw/day) based on reduced infertility noted in both sexes at ≥4800 ppm, reduced relative adrenal weight noted in females at ≥4800 ppm and reduced thyroid hormone (T4) noted in both sexes at ≥4800 ppm. The NOAEL offspring was 1900 ppm based on reduced viability noted at ≥4800 ppm

Effects on developmental toxicity

Description of key information
Treatment with sodium bromide at 100 mg/kg bw/day was not associated with any observable adverse effects on or in utero development of the conceptus.
 LOAEL: 300 mg/kg bw/day based reduced body weight gains in dams and foetal skeletal anomalies and variants (equivalent to 233 mg (Br-)/kg bw/day)
 NOAEL: 100 mg/kg bw/day (equivalent to 77.6 mg (Br-) /kg bw/day)
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: - 83/571/EEC
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF No.4200
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: CD BR VAF/Plus
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 196-259 g (first batch 201-259 g, second batch 196-239 g)
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosage volumes were calculated for individual animals on Day 6 of pregnancy and adjusted according to bodyweight on Day 8, 10, 12 and 14.

VEHICLE
- Concentration in vehicle: 0, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean achieved concentrations of sodium bromide in formulations prepared on the first and last days of treatment were within 8% of nominal concentrations.
Details on mating procedure:
Female rats were time-mated to identified males ofthe same strain. 64 animals were mated on the first day with remaining animals mated a day later. The day of mating, as judged by the appearence of sperm in the vaginal smear or by the presence of a vaginal plug, was considered as Day 0 of pregnancy.
Duration of treatment / exposure:
Days 6-15 post coitum
Frequency of treatment:
daily
Duration of test:
until Day 20 post coitum
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females/group
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

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

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

POST-MORTEM EXAMINATIONS: Yes
- only two animals were examined; one which died uring the study period and another one which had to be killed for humane reasons.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
Statistics:
Significance tests, employing analysis of variance followed by an intergroup comparison with the control, were performed on the following parameters and results are presented in relevant table of this report:
bodyweight change, mean food consumption, litter data, sex ratio and foetal abnormalities and variants.
Depending on the heterogeneity of variance between treatment groups, parametric tests, analysis of variance followed by Williams`test or non-parametric tests, Kruskal-Wallis followed by Shirley`s test were used to analyse these data, as appropriate.
For litter data and foetal changes the basic sample unit was the litter, and, due to the preponderance of non-normal distributions, non-parametric analyses were routinely used. Analysis of foetal abnormalities was performed using a trend test on the number of litters affected, followed by a one-tail 2 sample permutation test. All significant (ie p≤0.05) intergroup differences from the control are reported only when supported by a significant analysis of variance (p≤0.05). Where 75% or more of the values for a given variable were the same, a Fisher`s exact test was used.
Indices:
Pre-implantation loss:
(No. corpora lutea - No. implantations)/ No. corpora lute x 100

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

Sex ratio at Day 20:
(No live males) / total no. live young x 100
Details on maternal toxic effects:
Maternal toxic effects:yes

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

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

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

Category

Group (Dosage [mg NaBr/kg/day])

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

3

6

8

10

12

14

16

18

20

1

(0)

23

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)

2

(100)

21

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)

3

(300)

24

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)

4

(1000)

22

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])

1

(0)

2

(100)

3

(300)

4

(1000)

No. of animals observed:

23

21

24

22

3-5

27

26

25

26

6-7

27

28

28

30

8-9

28

28

29

32*

10-11

28

30

29

28

12-13

30

31

30

31

14-15

31

32

32

36**

16-17

34

34

32

34

18-19

33

34

31

30**

*   statistically different from control with p ≤ 0.05

** statistically different from control with p ≤ 0.01

 

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

Group

Dosage [mg NaBr/kg/day])

1

(0)

2

(100)

3

(300)

4

(1000)

Dams with live young

No. of litters

23

21

24

22

Group mean values

No. of corpora lutea

16.2

15.4

15.3

15.7

No. of implantations

14.9

14.6

14.0

14.1

No. ofin uterodeath

-                     early

-                     late

-                     early and late

 

0.6

0.2

0.8

 

0.6

0.0

0.6

 

0.7

0.1

0.8

 

0.8

0.2

1.0

No. of live young

14.1

14.0

13.1

13.1

Litter weight [g]

53.69

55.0

50.52

49.0

Foetal weight [g]

3.81

3.92

3.84

3.75

Graivid uterine weight [g]

81.48

81.88

75.35

74.46

Sex ratio [%]

48.0

54.0

47.5

48.0

Litter incidence (´n`)

Number ofin uterofoetal death: early

-                     0

-                     1

-                     2

-                     3

 

13

7

2

1

 

12

6

3

 

 

13

6

4

1

 

10

8

3

1

Number ofin uterofoetal death: late:

-                     0

-                     1

-                     2

 

19

4

 

 

21

 

 

 

21

3

 

 

19

2

1

Number ofin uterofoetaldeath: early and late:

-                     0

-                     1

-                     2

-                     3

-                     4

 

10

10

2

 

1

 

12

6

3

 

 

 

11

8

4

 

1

 

9

7

4

2

 

 

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

Category

No. of affected foetuses/litter (n)

Group (Dosage [mg NaBr/kg/day])

1

(0)

2

(100)

3

(300)

4

(1000)

No. of litters with ´n` foetuses affected

No. of litters examined

23

21

24

22

Malformation

0

20

21

20

14

1

2

-

3

5

2

-

-

1

2

3

1

-

-

-

7

-

-

-

1

Visceral anomaly

0

11

10

16

14

1

11

6

7

3

2

1

4

1

3

3

-

1

-

1

4

-

-

-

1

Skeletal anomaly

0

12

12

6*

4**

1

6

4

4

3

2

2

3

6

5

3

1

2

6

3

4

2

-

1

5

5

-

-

1

-

6

-

-

-

2

Mean foetuses affected per litter [%]

Malformations

1.7

0.0

1.7

5.3

Visceral anomalies

7.8

11.4

5.7

13.2

Skeletal anomalies

13.9

11.9

29.1

40.6

*   statistically different from control with p ≤ 0.05

** statistically different from control with p ≤ 0.01

 

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

Group

(Dosage [mg NaBr/kg/day])

Foetuses examined

Foetuses with

13 ribs

14 ribs

Normal sternebrae

Unossified sternebrae

Reduced sternebrae

Asym./bip. sternebrae

Total variant sternebrae

No.

%

No.

%

No.

%

No.

%

No.

%

No.

%

No.

%

1 (0)

159

146

92.5

13

7.5

94

58.6

45

27.9

26

17.9

1

0.4

65

41.4

2 (100)

149

130

88.1

19

11.9

78

51.9

40

27.1

38

25.9

5

3.7

71

48.1

3 (300)

154

153

99.4

1

0.6

64

42.9

62

40.0

37

23.0

5

3.0

90

57.1

4 (1000)

137

137

100.0*

0

0*

27

20.3**

89

63.3**

51

38.8**

5

2.8

110

79.7**

*   statistically different from control with p ≤ 0.05

** statistically different from control with p ≤ 0.01

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

Materials and Methods

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

Results and Discussion

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

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

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

Sodium bromide developmental toxicity in the rat:

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

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.

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 13thribs, 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

The NOEL for teratogenic effects is 100 mg/kg bw/day, comparable to the NOAEL for maternal toxicity (100 mg/kg bw/day). Therefore, sodium bromide should not be classified to reproduction, based on effects in the range of maternal toxicity.

For further information regarding human data, see attached file.

Additional information