Sodium bromide

Endpoint summary

Description of key information

NOAEL=60mg/kg/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

no

Remarks:

with the relevant sections relating to oestrous cycles, sperm evaluation and histopathology

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

EPA Health Effects Test Guideline OPPTS 870.3100: 90-Day Oral Toxicity in Rodents

Principles of method if other than guideline:

Also OECD Guideline 416, 2001. Two-Generation Reproductive Toxicity Study for testing reproductive toxicity elements in addition to repeated dose toxicity.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: Sodium Bromide
Batch (Lot) No.: 710120333
CAS Number: 7647-15-6
Expiration Date: 31 May 2016
Physical Description: White crystalline solid
Purity: 100.0%
Storage Conditions: Kept at room temperature, protected from light

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
The total number of rats used in this study was considered to be the minimum required to properly characterize the effects of the test substance.

Sex:

male/female

Details on test animals or test system and environmental conditions:

One hundred seventy-six (88 male and 88 female) Crl:CD(SD) rats were received from the Charles River Laboratories, Inc. facility.
The body weight range for the male rats was 146 g to 186 g on the day after arrival and was 165 g to 210 g at randomization and study assignment.
The male rats were approximately 47 days of age at arrival to the Testing Facility.
The body weight range for the female rats was 103 g to 145 g on the day after arrival and was 117 g to 160 g at randomization and study assignment.
The female rats were ap3.7.5. Selection, Assignment, Replacement, and Disposition of Rats
Upon arrival, males and females were assigned to individual housing. Before initiation of dose administration, male and female rats were selected for study on the basis of physical condition and body weights recorded during acclimation. The rats were assigned to dose groups, by sex, based on computer-generated (weight-ordered) randomization procedures. The weight variation of rats used on study did not exceed ±20% of the mean body weight of each sex. Rats were assigned to 5 replicates in order to accommodate the laboratory schedule (with the same number of rats/sex from each group in each replicate). Replicates were assigned to behavioral tests on consecutive days. The first 10 rats in Groups 1, 2 and 5 were assigned to the main study and the remaining 10 rats in Groups 1, 2 and 5 were assigned to the recovery study.
No rats were replaced during this study.
The disposition of all rats was documented in the study records.

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

Housing: The rats were individually housed in solid-bottomed cages during the course of the study except during urinalysis collection when the rats were housed in stainless steel wire bottom metabolism cages.

Environmental Conditions: The study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of 10 changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters. Room temperature and humidity were monitored constantly throughout the study. Room temperature was targeted at 19-25oC);relative humidity was targeted at 30% to 70%. On 5 separate days on numerous occasions, the relative humidity was out of range (up to 75.5% lasting up to 4 hours) during the study. On multiple occasions over 2 days, the relative temperature was out of range (up to 78.2oF lasting up to 6 hours) during the study. These deviations did not impact the outcome of the study because they were all minor and corrected themselves.
An automatically controlled 12-hour light:12-hour dark fluorescent light cycle was maintained. Each dark period began at 1900 hours (± 30 minutes).

Bedding: (Bed-o'Cobs®) was provided. Bedding was changed as often as necessary to keep the animals dry and clean. Analyses for possible contamination were conducted on each lot of bedding; the results of these analyses are on file at the Testing Facility.
There were no known contaminants in the bedding considered to interfere with the objectives of the study. Therefore, no analyses other than those routinely performed by the supplier were conducted.

Food: Rats were given Certified Rodent Diet® #5002 (PMI® Nutrition International, lot number APR25133A) available ad libitum from individual feeders, except during urine collection. Chloride content was analyzed by Covance Laboratories, Madison, WI, to ensure it was not less than 0.5% (value was 0.548%). Only the lot of the food analyzed for chlorine content was used. Results of this analysis were provided by the supplier and were maintained in the raw data.
The food was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Testing Facility.
There were no known contaminants in the food considered to interfere with the objectives of the study. Therefore, no analyses other than those routinely performed by the food supplier or those mentioned in this section were conducted.

Watre: Water was available ad libitum from individual bottles attached to the cages. All water was from a local source and passed through a reverse osmosis membrane before use. Chlorine was added to the processed water as a bacteriostat; processed water was expected to contain no more than 1.2 ppm chlorine at the time of analysis. Water was analyzed monthly for possible bacterial contamination and twice annually for possible chemical contamination. Chlorine levels in the drinking water were documented weekly and maintained in the raw data (see Appendix 1, Protocol, Amendments, and Deviations). There were no known contaminants in the water considered to interfere with the objectives of the study. Therefore, no analyses other than those routinely performed or those mentioned in this section were conducted.

Animal Enrichment- For psychological enrichment, male and female rats were provided with items such as a chewing object. Analyses for possible contamination were conducted on each lot of enrichment device. There were no known contaminants in the chewable enrichment devices considered to interfere with the objectives of the study. Therefore, no analyses other than those routinely performed by the enrichment device suppliers were conducted.

Veterinary care was available throughout the course of the study, and rats were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments (use of a warming pad, moistened meal, bunny block pieces, and cold compresses) were documented in the study records.
proximately 41 days of age at arrival to the Testing Facility.

Route of administration:

oral: gavage

Details on route of administration:

The test and control substances were administered to the appropriate rats by once daily oral gavage from DSs 1 to 90. The dose volume for each rat was based on the most recent body weight measurement. The doses were given using a syringe with attached gavage needle. The first day of dosing was designated as DS 1. The dosing formulations were stirred continuously during dose administration.

The oral (gavage) route was selected for use because: 1) the exact dose can be accurately administered via gavage; and 2) the oral route is a potential route of human exposure.

Vehicle:

water

Details on oral exposure:

The test and control substances were administered to the appropriate rats by once daily oral gavage from DSs 1 to 90. The dose volume for each rat was based on the most recent body weight measurement. The doses were given using a syringe with attached gavage needle. The first day of dosing was designated as DS 1. The dosing formulations were stirred continuously during dose administration.

The oral (gavage) route was selected for use because: 1) the exact dose can be accurately administered via gavage; and 2) the oral route is a potential route of human exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected and analyzed as indicated in Text Table 2, which is presented below in section:"Any other information on

Samples to be analyzed were transferred at ambient conditions to the analytical laboratory at the Testing Facility within 1 week of preparation, when possible.

Analytical Method
Analyses were performed by ion selective electrode measurement using a validated analytical procedure (BRAA00 under Charles River Study No. 20041699) in addition to osmolarity measurement of the Group 2 and 5 samples.

Concentration Analysis
Duplicate sets of samples (5 mL) from the middle of the test and control substance formulations were sent to the analytical laboratory; the remaining triplicate sets of samples from the middle of the test and control substance formulations were retained maintained in a refrigerator set to maintain 4°C at the Testing Facility asbackup samples. Additional duplicate sets of samples (5 mL) from the middle of the Group 3 and 4 test substance formulations were taken for analysis as well astriplicate sets of samples from the middle of the Group 3 and 4 test substance formulations as backup samples were taken on one occasion; however, these samples were not analyzed and were discarded.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10%. Each individual sample concentration
result was considered acceptable if it was within or equal to ± 15%. The average osmolarity results of Groups 1 and 5 agreed within or equal to ± 10%. Remaining backup samples were discarded.
One sample (10 mL) from Groups 2 and 5 were analyzed for density.

Stability Analysis
Duplicate sets of samples (5 mL) from the middle of the Groups 2, 3, and 5 test and control substance formulations were sent to the analytical laboratory. Stability analysis was performed during the last week of study to confirm the stability of the test substance.

Duration of treatment / exposure:

Once daily oral gavage from Days 1 to 90 of Study (DSs 1 to 90).

Frequency of treatment:

Once daily.

Dose / conc.:

60 mg/kg bw/day (nominal)

Dose / conc.:

175 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

deionized water

Dose / conc.:

284 mg/kg bw/day (nominal)

Remarks:

Sodium Chloride

No. of animals per sex per dose:

10 animals per sex per day.

Control animals:

yes, concurrent no treatment

other: Sodium Chloride

Details on study design:

The study design is described in Text Table 1which is presented below in the section of "Any other informtion on materials and methods incl. tables"
The test or control substances were administered to the appropriate rats by once daily oral gavage from Days 1 to 90 of Study (DSs 1 to 90). The sodium chloride group was included to determine the effect of a sodium dose of equivalent osmolarity to that of the high dose sodium bromide group, and hence to distinguish effects of the sodium and bromide components of the test material.
All formulations were considered appropriate for use. The sodium chloride formulation was confirmed to be of equivalent osmolarity to the high dose sodium bromide formulation.

Positive control:

No

Observations and examinations performed and frequency:

In-life Procedures, Observations, and Measurements:
Food consumption was not measured during the acclimation period; however, food was monitored and replenished as necessary to monitor the health and well-being of the rats.
Viability Checks- Rats were observed for general health/mortality and moribundity at least twice daily

Clinical Observations
# General Appearance- Clinical observations were recorded once during the acclimation period, on the day of randomization, daily immediately prior to dose administration, at least weekly during the recovery period and on the day of scheduled euthanasia.

#Postdose Observations- Postdose observations were recorded at approximately hourly intervals for the first 4 hours and at the end of the normal working day for the first week of administration. Beginning with the 8th dose, postdose observations were recorded between 1 and 2 hours after dose administration and at the end of the normal working day.

#Detailed Clinical Observations- were performed prior to daily dosage administration beginning prior to the first dosage and weekly thereafter for all rats (with the exception of the rats selected for FOB on the weeks in which the FOB was conducted, since detailed clinical observations are subsumed within the FOB examination). Detailed clinical observations included, but not limited to, identification of clinical signs related to: general appearance (e.g., skin, fur, changes in eyes, eyeballs and mucous membranes; presence or absence of discharge), body position and posture (e.g., hunchback posture), autonomic nervous system function (e.g., lacrimation, piloerection, pupil diameter, respiration, excretion), motor coordination, ambulatory abnormalities, reaction to being handled and to environmental stimulation, nervous system (e.g., tremor, convulsion, muscular contractions), changes in exploratory behavior, ordinary behavior (e.g., changes in grooming, headshaking, gyration), abnormal behavior (e.g., autophagia, backward motion, abnormal vocalization) and aggression.

Body Weights
Body weights were recorded twice during the acclimation period, daily during the dose period and once weekly during the recovery period. A terminal
weight was collected prior to euthanasia. A fasted weight was recorded on the day prior to necropsy for rats assigned to urine collection.

Food Consumption
Food consumption values were recorded at least weekly throughout the dosing and recovery periods and the day prior to scheduled euthanasia.

Water Consumption
Water consumption values were recorded daily on DSs 1 through 8 and DSs 64 through 71 for all male and female rats .

Ophthalmic Examinations
Ophthalmological examinations were performed by a veterinary ophthalmologist prior to dose administration and on the week prior to scheduled euthanasia. An indirect ophthalmoscope in conjunction with a hand-held lens (diopter documented in the raw data) was used to examine ocular structures (lens and fundus oculi).

Estrous Cycle Evaluations
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Samples were collected for 21 consecutive days prior to scheduled euthanasia (including the day of scheduled euthanasia) for all main study female rats not selected for the recovery portion of the study.

Functional Observational Battery (Including Detailed Clinical Observations)
FOB was conducted during the fourth week of dosing (prior to dose administration), during the ninth week of dosing (between 1-3 hours postdose), during
the thirteenth week of dosing (between 1-3 hours postdose) for 10 male and 10 female rats per group, when possible, and during the week of scheduled euthanasia (recovery study only), for all male and female rats.
The functional observational battery (FOB) including detailed clinical observations, were conducted by an observer unaware of the group designation of the
rat. The Observer examined the rat in its home cage, while handling the rat, and/or in an open field to assess parameters including, but not limited to the following:
•lacrimation, salivation, palpebral closure, prominence of the eye, pupillary reaction to light, piloerection, respiration, and urination and defecation (autonomic functions)
• sensorimotor responses to visual, acoustic, tactile and painful stimuli (reactivity and sensitivity)
• reactions to handling and behavior in the open field (excitability)
• gait pattern in the open field, severity of gait abnormalities, air righting reaction, visual placing response and landing foot splay (gait and sensorimotor coordination)
• forelimb and hindlimb grip strength
• Abnormal clinical observations not sufficiently specified in the measurement scales for the parameters specified above. These observations included, but not limited to: general appearance (e.g., changes in skin, fur, and mucous membranes; presence or absence of discharge; emaciation; dehydration), body position and posture (e.g., hunchback posture), neuromuscular system (e.g., tremor, convulsion, muscular contractions, hypotonia, hypertonia), and behavior (e.g., headshaking, gyration, autophagia, backward motion, abnormal vocalization).
• Body temperature was measured at the completion of the FOB evaluation using a rectal probe (Physitemp Instruments Model RET-2) and digital thermometer (Physitemp Instruments Model Bat-10 R LOP).
Evidence of the ability of this battery to detect the effects of positive control substances was provided (Testing Facility Positive Control Data).

Motor Activity
Motor activity tests were conducted after completion of the FOB evaluations (the same 10 male and 10 female rats as FOB evaluation), at approximately the
same time of day (when possible).

Clinical Pathology:
Haematology
Coagulation
Clinical Chemistry
Urinalysis
Thyroid hormone analysis

Necropsy:
Organ weights
Histology
Histopatholgy

Sacrifice and pathology:

Method of Euthanasia: Male and female rats were euthanized by an intravenous injection of sodium pentobarbital into the inferior vena cava, following blood sample collection under isoflurane/oxygen anesthesia.

Scheduled Euthanasia: On DS 91 or 148, surviving rats were euthanized, and blood samples were collected and necropsy performed.

Necropsy: Male and female rats were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in rat anatomy and gross pathology. A veterinary pathologist was available for consultation during all scheduled necropsies. Images were generated for illustration of or consultation on gross observations. Generation of such images was documented. Images and associated documentation were retained and archived.

Statistics:

Statistical Evaluation - Means, standard deviations, percentages, proportions and/or incidences were calculated, as appropriate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males
Vehicle Control:
Clinical observations in control animals during the treatment period were limited to 2 males: 1558 showed decreased motor activity, rales, mild to moderate dehydration, ataxia, chromodacryorrhea, hunched posture, scant feces, chromorhinorrhea, thin body condition and no feces between DSs 82 to 87. As histopathological examination indicated minimal macrophage aggregation in the lung; these findings were considered secondary to administration of a small amount of the control article into the lung.
Male 1559 had misaligned incisors on DSs 47 to 60. During the recovery period, clinical signs were limited to sporadic observations of ungroomed coat (1566) and rales (1562).

Sodium Chloride Comparator Group:
Treatment with sodium chloride was not associated with adverse clinical signs. Those observations which did occur were considered not related to sodium chloride because the sign did not persist past the first day of dosing, there was no clear pattern of effect and/or the sign only occurred during the post-dose period. Four males (1511, 1513, 1514, 1521) had decreased motor activity following dosing on DS 1. This observation did not occur on any other day of dosing and was probably a reaction to the initial dose of sodium chloride.
Other observations were sporadic, common findings, observed in up to 3 males - chromorhinorrhea (1512), scabbing (1510, 1515, and 1519) soft or liquid feces (1505, 1517) on DSs 8, 63 and 81.

Sodium Bromide
60 mg/kg/day
Clinical observations in the 60 mg/kg/day dose group were considered not treatment-related because most were secondary to an injury which occurred in one animal and the other observations demonstrated no clear pattern of effect and/or the sign did not persist. Male 1521 had a series of adverse clinical observations between DSs 85 and 91 relating to an injured left hind limb: impaired pinch reflex, lost grip reflex, no/limited use of the limb, purple color, swollen and reddening. Mild dehydration, hunched posture, irregular gait and reduced feces were also observed in this animal. Other observations were limited to transient observations in single animals of chromodacryorrhea and misaligned/broken incisors (1523) or mild dehydration (1526).

175 mg/kg/day
The incidence of adverse observations in males in this dose group was increased compared with controls. Decreased motor activity was observed in males treated at 175 mg/kg/day but the frequency and incidence was not so severe as the high dose group: 6/10 males (p≤ 0.05) were affected for at least one day between DSs 12 to 15 and some of these animals also had transient observations of chromodacryorrhea (1573), mild dehydration (1575) or scabbing (1580). Other observations were limited to a swollen right ear and hunched posture (1578), chromodacryorrhea (1573), scab (1580) and eye changes including chromodacryorrhea, ptosis, periorbital swelling, mass and scab (1579).

500 mg/kg/day
Adverse effects of treatment at 500 mg/kg/day included decreased motor activity, dehydration (mild and moderate), ataxia, ungroomed coat, urine-stained abdominal fur, hunched posture, chromodacryorrhea, ptosis, low carriage and limited use of limb(s)/paw(s) which affected an increased incidence of males (p≤ 0.01) compared to the control males:
Decreased motor activity was first observed from 2 hours after the first dose on DS 1, in 5 males, with the incidence of affected animals increasing to 9 during the first week of treatment and all males by week 3 of treatment. Ataxia/prostrate was first observed in 9 males after three weeks of dosing, generally apparent between 1 to 2 hours after dosing and persisting until the end of the normal working day, and all males were affected by week 11. Impaired righting reflex was first observed on DS 22 in one male and then on or about DS 50 in 2 males and was observed at an incidence of 3/20 until the end of treatment. After approximately 6 weeks of dosing, one or more of these observations persisted throughout the working day, and was evident prior to dosing the next day for at least 2 males in this group throughout the rest of the dosing period.
Mild to moderate dehydration (as assessed by skin turgor) was observed in all animals in the group, generally beginning within the first 3 or 4 weeks of treatment and with recurring episodes throughout the treatment period.
During the recovery period, all 8 males (p≤ 0.01) in the 500 mg/kg/day dose group continued to exhibit decreased motor activity, ungroomed coat, urine-stained abdominal fur and dehydration, for up to 30 days after the cessation of treatment. The severity and incidence of these signs generally decreased with time off-dose but 1 rat still showed signs of urine-stained abdominal fur until DS 120. Other clinical observations occurred in a male that was euthanized or in only a single male and were considered incidental.
Although not statistically significant, the number of males in poor clinical condition, characterised by the number of observations of limbs/paws swollen, reduced (scant) feces, mass (eye, limbs, paws or inguinal area), and red or purple limb(s)/paw(s), chromorhinorrhea, thin body condition, impaired righting reflex, scab (mouth, head, neck, eye, nose, inguinal area, fore or hindlimb), head tilt to the left, lost grip reflex, abrasion (forepaw or inguinal area), piloerection, and hypernea, was also increased compared to the control group males. Each of these observations were considered related to the 500 mg/day dose of sodium bromide as many persisted throughout the dose period, became less evident in the recovery period, and had also occurred in the rats that were euthanized before scheduled termination.

Females
Vehicle Control
Minimal and sporadic common clinical observations occurred in this group.
Four females had sparse hair coat on the limbs, underside and/or head. Other observations were limited to one or 2 females and included scab on head (1659), misaligned incisors (1662) or mild dehydration (1669, 1670). During the recovery period, only one or 2 females showed each clinical sign. One female (1662) had a mass on the hindlimb from DSs 99 to 148 and another (1663) had limited use of the hindlimb and irregular gait only on DS 147. Other observations were limited to sparse hair coat on the limbs(1664, 1667), ungroomed coat (1670) or a swollen ear (1668).

Sodium Chloride Comparator Group
There was no effect of sodium chloride on clinical condition. During the dosing period, observations were limited to sparse hair coat on the limbs and underside (1613) or a scab on the mouth (1612, 1613). During the recovery period, ptosis was observed in one female (1616) on two separate days and other signs were limited to sparse hair coat on the limbs and/or underside (1613, 1618) or a swollen ear (1615, 1619).

60 mg/kg/day
Clinical observations were minimal and sporadic, and as most occurred in a single rat (secondary to an injury) or demonstrated no clear pattern of effect and/or did not persist, were considered unrelated to treatment with sodium bromide.
Three females had chromodacryorrhea (1621, 1622, and 1625), one had a mass around the eye (1625) and 2 had sparse hair coat on the head and/or limbs (1622, 1626). Female 1622 also had an ulceration (0.8 cm) on the head on the first day of dosing which healed and scabbed by DS 14. Female 1626 also had a scab on the neck.

175 mg/kg/day
Decreased motor activity was observed in females treated at 175 mg/kg/day but was transient, only apparent in the second week of treatment, and did not persist. Eight of ten females (vs 0/10 in controls, p≤ 0.01) had decreased motor activity DSs 11 to 13. These observations were only noted postdose and were no longer apparent the following morning, with the exception of two females (1671 and 1672) that had decreased motor activity, prior to dosing the next day. Other clinical signs were minimal and transient, and included bent tail (1671), a mass on the head (1678) or scabbing (1680).

500 mg/kg/day
The number of females with decreased motor activity, dehydration, ataxia, ungroomed coat, urine-stained abdominal fur, hunched posture, chromodacryorrhea, ptosis, and low carriage was significantly increased (p≤ 0.01) compared to control group females.
Decreased motor activity was first observed from 1 hour after the tenth dose (DS 10), in 5 females, with the incidence of affected females increasing to 16 during the second week of treatment and generally affecting all females by week 3 of treatment. Ataxia/prostrate was first observed in 9 females after two weeks of dosing, generally apparent between 1 to 2 hours after dosing and persisting until the end of the normal working day, and increasing to 18 affected by week 8. Impaired righting reflex was first observed on DS 49 in 1 female and continued until DS 50. Mild to moderate dehydration (as assessed by skin turgor) was observed in 17/20 females. Whilst the incidence was far higher than control females (2/20), the duration and/or frequency of these episodes was lower than that observed in males treated at 500 mg/kg/day, and in many cases the onset was later in the treatment period.
During the recovery period, 3 females in the 500 mg/kg/day dose group continued to exhibit ungroomed coat, urine-stained abdominal fur, and dehydration, for up to 9 days after the cessation of treatment.
Although not statistically significant, the number of females in poor clinical condition, characterized by the number of observations/animal with head tilt to the right, a mass (eye, limbs, or inguinal area), and/or thin body condition, choromorhinorrhea, pale ears, impaired righting reflex, cold to touch, pale extremities, and bradypnea was also increased compared to the control group. These observations were considered related to the 500 mg/day dose of sodium bromide as many persisted throughout the dose period, became less evident in the recovery period, and also occurred in the rats that were euthanized before scheduled termination.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 60 mg/kg/day, female 1628 was found dead on Day 28 of study (DS 28) with no previous adverse clinical observations. Body weight and food consumption values were unremarkable. Necropsy revealed a red gelatinous material in the right axillary subcutis indicating that the cause of death was secondary to the jugular bleed to collect blood for hormone analysis and therefore incidental to treatment.

All animals in the group treated at 175 mg/kg/day survived to scheduled termination.

Four male rats from the 500 mg/kg/day group were killed on study days 52, 56, 86 or 107 owing to adverse clinical signs that were similar to, but more severe than, others in the dose group. These 4 animals had generally gained weight for the first six weeks of dosing and then showed reduced gain/weight loss as their clinical signs increased in severity.

All animals in the sodium chloride treated group survived to scheduled termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no significant effect of treatment at 60 or 175 mg/kg/day on body weight or body weight gain in males and females, although values at 175 mg/kg/day were very slightly lower than controls. At the end of the treatment period mean body weights of males were 97.2% and 93.0% of controls and for females 108.7% and 95.2%, in the 60 and 175 mg/kg/day groups, respectively. Body weight gain was similar, with values for males of 97.3% and 90% of controls and for females 118.1% and 92.1% of controls, in the 60 and 175 mg/kg/day groups, respectively.

At 500 mg/kg/day there was a significant reduction in body weight gain in males for most weekly intervals after week 2, and at the end of the dosing period body weight and body weight gain were significantly lower than control (81.2 % and 68.8% of control, respectively, p≤ 0.01). Body weight remained significantly lower than controls for the first two weeks post-dosing but increased to 90.4% of control values at the end of the recovery period, owing to weight gain markedly higher than control (134.7%). Female body weight and weight gain were unaffected during the treatment period but gain at the end of the recovery period was substantially lower (66.5% of control values).

Figures of body weight vs time are attached.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake generally paralleled changes in body weight. There was no adverse effect in males or females at 60 mg/kg/day and at 175 mg/kg/day significant
reductions over the treatment period were limited to males with absolute intake of 91.4% of controls (p≤ 0.05).

At 500 mg/kg/day, average and relative food consumption in males were significantly reduced (p≤ 0.01) over the dosing period (DS 1 to 90) and for each weekly interval after week 1. During the recovery period, average absolute food consumption values were significantly reduced (p≤ 0.01) in first 2 weeks, remained
lower than controls for the next 2 weeks but were similar thereafter. Relative food consumption values in males were significantly lower (p≤ 0.05) at the start of the recovery period (Days 90 to 92), comparable to controls for the next two weeks and significantly increased (p≤ 0.01) for each weekly interval for the
remainder of the recovery period, resulting in an overall significant increase (DS 90 to 147, p≤ 0.01).

In females treated at 500 mg/kg/day, within the dosing period there were transient decreases in absolute (DS 43 to 50, p≤ 0.01) and in relative food consumption (DS 8 to 15, p≤ 0.01 and DS 43 to 50, p≤ 0.05) values for the entire period (DS 1-90) were comparable to controls. Over the entire recovery period (DS 90-147) average absolute and relative food consumption were similar to control values as reductions (p≤ 0.01) observed in the first 2 weeks were followed by increases (p≤ 0.05 to p≤ 0.01) in the last 2/3 weeks.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There was no adverse effect on water intake in any group during the first period measured (DS 1 7). In the second period (DS 64-70), significant reductions
were observed in both sexes in all groups treated with sodium bromide. A dosage-related trend was apparent for males, with values of 87.4%, 81.7% and 78.8% in the low, intermediate and high dose groups, respectively, but not for females (82.5%, 77.3% and 83.9% of controls, respectively). However, in the low
dose group significant reductions in water intake were confined to DS 66 to 67 in males (when a similar reduction was seen the sodium chloride treatment group males) and DS 66 to 67 and 67 to 68 in females.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The 284 mg/kg/day dose of sodium chloride and doses of 60, 175 or 500 mg/kg/day of sodium bromide did not result in any treatment-related ophthalmic changes
# Prior to Dose Administration - Prior to assignment to the study, there were no ophthalmic findings present for any male or female.
# Evaluation Days 80 to 84 of Study (Main Study) - retinal degeneration was noted in the right eye in 1 male in the 175 mg/kg/day sodium bromide group. Focal
retinal degeneration was noted in either left or right eye in 1 male and 1 female in the 175 mg/kg/day sodium bromide group and one control female. These findingswere considered incidental, of the nature commonly observed in this strain and age of rats, and were therefore considered unrelated to administration of sodium bromide.
# Evaluation Days 140 to 143 of Study (Recovery Study) - There were no ophthalmic findings present for the male and female rats during the recovery study.

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology including Coagulation:
Hematology parameters values evaluated were not affected by treatment with sodium chloride. There was no effect in males or females at 60 mg/kg/day or in males at 175 mg/kg/day. Any statistically significant differences that did occur in these groups were considered unrelated to either test substance because the difference reflected lower body weight, food or water intake, was not dosage-related, did not persist into the recovery period and/or the value was within the historical control range for the Testing Facility.
The only toxicologically significant findings were the increased number of segmented neutrophils (a normal response to infection) in males on DS 91 at 500 mg/kg/day, which was no longer apparent at the end of the recovery period (DS 148).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There was no treatment- related effect on coagulation or clinical chemistry value in male or female rats from any treated group when compared to concurrent and/or historical control values.
Sodium Chloride: No significant differences from control were observed in males and females treated with sodium chloride with the exception of a reduction in cholesterol (p≤ 0.05) at the end of the recovery period (DS 147) in males. This difference was considered unlikely to be toxicologically significant as the value was within the historical control range.

Sodium Bromide: At the end of the dosing period (DS 91), chloride levels were significantly increased (p≤ 0.05) in males and females in the 175 and 500 mg/kg/day dose groups. These values probably reflected blood levels of sodium bromide which, under the applied method of analysis of chloride levels, would not be distinguished from chloride.
All other clinical chemistry parameters evaluated were not affected by treatment with sodium bromide at up to 500 mg/kg/day. Statistically significant differences that did occur were considered not related to sodium bromide because the difference reflected lower body weight, food and/or water consumption in the dose group, the difference was not dose-dependent, the change did not persist into the recovery period, the change only occurred in the recovery period and/or the values were within the historical control range for this Testing Facility.

#Males
At the end of the dosing period (DS91), urea nitrogen was reduced (p≤ 0.05) in the 60 mg/kg/day and higher dose groups, but no dose response was observed and the values were within the historical control range for this Testing Facility. Albumin was reduced (p≤ 0.05) in the 175 mg/kg/day dose group and total bilirubin, triglycerides and creatine kinase were reduced (p≤ 0.05) in the 175 and 500 mg/kg/day males. All these values were within the historical control range and therefore any changes were considered unrelated to treatment.
At the end of the recovery period, DS 147, only phosphorus was increased compared to the control group (p≤ 0.05) in the 500 mg/kg/day males, but the value was well within the historical control range.

#Female Rats
On DS 91, creatinine was increased (p≤ 0.05) in the 60 and 500 mg/kg/day dose group. Total bilirubin was reduced (p≤ 0.05) in the 175 and 500 mg/kg/day females and cholesterol was reduced (p≤ 0.05) at 500 mg/kg/day: these values were within the historical control range. Total protein, albumin, globulin and calcium levels were increased in females at 500 mg/kg/day, compared to concurrent controls (p≤ 0.05), but these values were within the historical control range.
At the end of the recovery period (DS 147), only alkaline phosphatase was increased (p≤ 0.05) in the 500 mg/kg/day females but this value within the historical control range.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Effects on urine were limited to the 500 mg/kg/day group.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional Observational Battery- Animals were assessed prior to dosing in week 4, post-dosing in weeks 9 and 13 and at the end of the recovery period for groups 1, 2
and 5. Results for males and females are presented in Text Table 14 and Text Table 15 (attached). Effects were limited to 500mg/kg group.

Sodium Chloride Comparator Group:
No effects of sodium chloride at 284mg/kg/day were noted in the functional observational battery when evaluated in males or females during study weeks 4, 9 or 13 or at the end of the recovery period. A significant increase (p≤ 0.05) in the mean auditory reaction for female rats during week 13 was not considered toxicologically important as the difference slightly higher than the control group value but the change was small and comparable to Sodium Bromide groups. A significant decrease (p≤ 0.05) in the maximum hindlimb grip strength in male rats at the end of the recovery period was not considered toxicologically important as the difference it occurred in only one sex and no effect on this parameter was observed during the dose period.

NaBr Males:
#60 mg/kg/day
There was no effect of sodium bromide at 60 mg/kg/day in the functional observational battery when evaluated during study weeks 4, 9 or 13.

#175 mg/kg/day
There was no effect of sodium bromide at 175 mg/kg/day in the functional observational battery when evaluated during study weeks 4 or 9.
At week 13, 3/10 males had difficulty in air righting (landed on side) which was considered associated with treatment as similar effects were observed in 8/10 males in the 500 mg/kg/day group in the same week of treatment (see below). The occurrence of this finding only after 12 weeks of treatment may be indicative of an increase in toxicity over time.

# 500 mg/kg/day
No statistically significant differences from control occurred for any parameter evaluated when assessed prior to dosing in week 4. There was, however,
someindication of a difference as this group had the lowest mean number of rears in the home field (3.1 vs 6.3 in controls) and 2/10 rats with ataxia and slight
abnormality of gait.
During week 9, when assessed post dosing, all males showed ataxia and slight gait abnormality (p≤ 0.01), 4/10 were unkempt and 6/10 had urine and/or fecal
staining. At week 13, 7/10 males showed ataxia (p≤ 0.01) and slight /moderate abnormalities of gait, 5/10 had unusual posture (p≤ 0.01), 3/10 were unkempt and 7/10 had urine and/or fecal staining. There was also a higher mean difference (p≤ 0.05) for urination and increased incidences of urination in the open field
(9/10) and landing on side in the air-righting test (8/10).
During the last week of the recovery period, there were significant increases in the incidence of urination in open field (7/10 vs 4/10 in controls) and in average and maximum mean values for forelimb grip strength (p≤ 0.01). The number of males with no normal movement (sleeping) in the home cage was reduced in this group compared to the control group, but no movement (sleeping) in the home cage is an expected finding when nocturnal animals are observed during the day (lights on).

NaBr Females:
#60 mg/kg/day
No effects of sodium bromide at 60 mg/kg/day were noted in females in the functional observational battery when evaluated during study weeks 4, 9, 13 or at the end of recovery period.

#175 mg/kg/day
When assessed post dosing in week 13, the number of females sleeping in the home cage prior to testing was significantly increased (p≤ 0.01).

#500 mg/kg/day
When assessed prior to dosing in week 4, the only parameter affected was air righting: the number of females that landed on their side was increased and the mean score was significantly increased (p≤ 0.05).
During week 9, when assessed post dosing, ataxia (6/10, p≤ 0.01), unusual posture (5/10, p≤ 0.01), abnormal gait (6/10), urine and/or fecal staining (3/10) were all observed only in this dose group and unkempt appearance was observed in 5/10 females compared to 1/10 in the other dose groups and 2/10 control females. Hindlimb maximum and average grip strength was also significantly reduced (p≤ 0.05) in the 500 mg/kg/day dose group.
During week 13, when assessed post dosing, the number of females sleeping in the home cage (prior to testing) was significantly increased (8/10 vs 4/10 in controls, p≤ 0.01). The number with ataxia (8/10), unusual posture (6/10) and abnormal gait (8/10) had increased from week 9 and these signs were still not seen in control females.
Full recovery from these changes was demonstrated during testing in the last week of the recovery period, as no statistically significant differences from control occurred in females treated at 500 mg/kg/day for any parameter evaluated.
No other statistically significant differences occurred in any group for any parameter evaluated in weeks 4, 9 or 13 and at the end of the recovery period.

Selected functional observation parameters are presented in Table text 14 and 15 (attached).

Motor Activity
Sodium Chloride
No effects of sodium chloride were noted in the motor activity testing of males or females when evaluated during study weeks 4, 9, 13 or at the end of recovery.

Sodium Bromide - Males
#At 60 mg/kg/day no effects of sodium bromide were noted in the motor activity testing of males when evaluated during study weeks 4, 9, 13 or at the end of the recovery.
period.
#At 175 mg/kg/day, when tested prior to dosing in week 4, the number of ambulations in the first 10 minute interval was significantly increased (p≤ 0.05)
compared to the control group males. No effects were observed in this group at weeks 9 and 13 or at the end of recovery.

#At 500 mg/kg/day, when tested prior to dosing in week 4, the number of ambulations in the first 10 minute interval was also significantly increased (p≤ 0.05) and continued for the next two 10 minute intervals. The number of fine movements for all intervals and the overall mean for the hour tested was significantly
increased (p≤ 0.05) compared to control males. During week 9, when tested post dosing, the number of ambulations in the first 10 minute interval was again
significantly increased (p≤ 0.05) compared to controls. During week 13, when tested post dosing, there was no effect on the number of ambulations but the
number of fine movements was significantly decreased (p≤ 0.05) compared to controls. This difference was no longer apparent during the last week of the
recovery period as no statistically significant differences from control occurred in males treated at 500 mg/kg/day for any parameter tested.
No other statistically significant differences for any parameter evaluated occurred in males from any group in weeks 4, 9 or 13.

Sodium Bromide - Females
#No effects of sodium bromide at 60 or 175 mg/kg/day were noted in the motor activity testing when evaluated during study weeks 4, 9 or 13 or at the end of
recovery.

#At 500 mg/kg/day, prior to dosing in week 4, the number of ambulations was significantly increased (p≤ 0.05) for all intervals evaluated, compared to the
control group.
During week 9, post dosing, only the number of ambulations in the first 10 minute interval was higher than controls (p≤ 0.05) and no statistically significant differences occurred at week 13 or in the last week of the recovery period.
No other statistically significant differences occurred among the groups for any parameter evaluated at week 4, 9 or 13.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in absolute thyroid weight and increases relative to body weight were only significant (p≤ 0.05) in females treated at 500 mg/kg/day. This did not, however, generally correlate with hormone levels or the microscopic finding of colloid depletion in individual animals. No other significant organ weight changes were observed.

Sodium Chloride: There was no effect on organ weights in males treated with sodium chloride. In females, there was a significant increase (p≤ 0.01) in absolute thyroid/parathyroid weight and in values expressed as a percentage of brain and body weight. These changes occurred in the absence of any change at histopathology but hormone analysis indicated slightly (although not significantly) higher values for T3 and T4.

Sodium Bromide:
# Males: Absolute brain weight was decreased in males treated at 60 (p≤ 0.05) and 175 mg/kg/day (p≤ 0.01) but was considered not adverse as there was no effect relative to body weight.
There was also an increase in lung weight as a percentage of body weight (p≤ 0.01) at 175 mg/kg/day, which was considered to be associated with the slightly lower terminal mean body weight (93% of control value at DS91).
At 500 mg/kg/day, the significant reduction in terminal body weight (81.2% of control value at DS91) was considered consistent with the higher lung weight as a percent of body weight (not statistically significant) and the increased relative brain weight but there was also a significant decrease in absolute brain weight.

#Females: In females, a trend for a dose-related decrease in terminal body weight was not significant. Absolute thyroid/parathyroid weights in females at 500 mg/kg/day were slightly higher and the difference from control was significant when weights were expressed as a percentage of terminal body weight and brain weight. However, as this did not correlate with hormone levels or the microscopic finding of colloid depletion in individual animals and as there was one control female (1659) with extremely low thyroid/parathyroid weight, this difference was considered equivocal.
Any other organ weight differences observed in females were considered incidental.

#Recovery Euthanasia Rats (Day 148): The organ weight changes considered associated with sodium bromide treatment at DS 91 were not observed in animals examined at the end of the recovery period (Day 148). Some organ weight values were statistically different from control at Day 148 but as there were no patterns, trends or correlating data to suggest these changes were toxicologically relevant they were considered incidental and unrelated to treatment.

Summary of selected organ weight data are presented in Text Table 18


There was no adverse effect on reproductive organ weights in males treated at 60, 175 or 500 mg/kg/day.
Some statistically significant differences in absolute weights were considered not biologically significant, but a consequence of the lower body weights, as values relative to body weight were comparable to controls or significantly higher.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One male in each of the 60 and 175 mg/kg/day groups and 1 male and 2 females in the 500 mg/kg/day group were found to have a mass on the limbs at gross necropsy. Histopathology confirmed these findings as abscesses associated with bacterial infection and not directly related to treatment.

Masses in the subcutaneous tissues of the skin were observed in 1 animal from each of the 60 and 175 mg/kg/day groups and 3 in the 500 mg/kg/day group and correlated with the microscopic finding of abscess in skin sections.
Other gross findings observed were considered incidental. Gross findings in animals killed at the end of the recovery period (Day 148) were considered incidental. There was no evidence of the subcutaneous masses which had been observed in 5 animals killed at the end of the treatment period.

Summary of gross pathology findings are presented in Text Table 17 (attached).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mild/moderate depletion of colloid (noted when the majority of follicles in the thyroid contained no identifiable colloid) was observed in 2 males and 2 females in the groups treated at 175 and 500 mg/kg/day. It is, however, noteworthy that there was no correlation between these findings and decreased T3 and T4
levels in individual animals. These changes were not apparent at 60 mg/kg/day.

Microvesicular vacuolation in the periportal hepatocytes was observed in 2, 5, 4 males and 2, 3, 4 females in the 60, 175 and 500 mg/kg/day groups,
respectively. This is a common incidental finding in rats, but can also result from nutritional disturbances. At 175 and 500 mg/kg/day these findings tended to
occur in animals also showing lung changes of accumulation of macrophages (within alveoli) and mixed cell infiltrates scattered in the pulmonary parenchyma

Tension lipidosis, often related to increased body weights, was observed at a higher incidence in females at 500 mg/kg/day, in which terminal body weights
were lower than control females. This is a common incidental finding in rodents. At terminal euthanasia 1/19, 1/20 and 3/19 animals in the 60, 175 and 500 mg/kg/day groups multiple abscesses in the skin/subcutis containing bacteria with associated formation of Splendore-Hoppeli material which usually forms secondary to local antigen-antibody reaction.
Skin abscess with evidence of bacteria and neutrophilic infiltrates were also contributory to the moribund condition of 2 males in the 500 mg/k/day group killed
before scheduled termination. As these findings rarely occur spontaneously in animals at this facility they were considered treatment-related. As the skin
abscesses were all located on the limbs/paws, however, these infections may be secondary to damage sustained during periods of test-substance related immobility or abnormal movement and/or poor clinical condition and lack of grooming.
Other microscopic findings observed at the end of the treatment period were considered incidental.

At the end of the recovery period, there was no evidence of previous signs other than macrophage aggregates in the lung. This is a common incidental finding in rats. As this was observed in 3/17 animals treated at 500 mg/kg/day sodium bromide/day, a comparable incidence to the control and sodium chloride treated groups (2/20 and 3/20 animals, respectively), it was considered not to indicate any irreversible effect of treatment.

Female reproductive organ histopathology and ovarian follicle counts:
Histopathology revealed no effects on the uterus, oviduct, vagina or mammary gland at any dose level.
Depletion of corpora lutea was observed in 3 females in the 500 mg/kg/day group, which were all in estrus at termination. Further sections taken from these
females, examined in the Ovarian Follicle Quantification investigation, also had no corpora lutea present, although no effect on follicle count was apparent.
At recovery euthanasia, all females in the 500 mg/kg/day group had corpora lutea but one control female was also found to have no corpora lutea.
As sectioning of the ovaries for follicle counts involved some deviations from protocol in positioning of the sections within the ovary, allocation of left and right
ovaries and some incomplete sections (including the females with no corpora lutea) and as there were no other effects on female reproductive organ weight or histopathology, it is considered that no conclusions can be drawn from the above observations and further investigation would be necessary to investigate any potential effect on female reproductive function.
The absence of corpora lutea did not correlate with depletion of colloid in the thyroid or with thyroid hormone levels in individual animals.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The pathology summary indicates:
• There were no adverse histopathology findings for males or females treated with sodium chloride.

• The minimal/mild depletion of colloid in the thyroid observed in 2 males and 2 females from each of the 175 mg/kg/day and 500 mg/kg/day groups was recorded when the majority of follicles contained no identifiable colloid.

• The minimal/mild vacuolation observed in the periportal hepatocytes of 2-5 males and 2 4 females in the sodium bromide-treated groups was microvesicular, and tended to occur in animals which also showed lung changes, e.g. accumulation of macrophages in the lung (within alveoli) and mixed cell infiltrates (scattered in the pulmonary parenchyma). These were likely adaptive changes.

• Tension lipidosis is sometimes related to increased body weight, but this was not the case for the 5 females treated at 500 mg/kg/day as the mean terminal body weight (DS90) for this group was marginally lower (1%) than the control mean and weights of affected animals were not the highest in the group.

• At terminal euthanasia 1/19 animals in Group 3, 1/20 animals in Group 4 and 3/19 in Group 5 had gross findings that correlated to microscopic findings of abscesses in the skin. Two of 5 males in Group 5 which were killed before scheduled termination also had evidence of bacteria or abscess in the skin. Most of the abscesses contained colonies of bacteria with associated formation of Splendore-Hoppeli material which usually forms secondary to local antigen-antibody reaction. The incidence of animals with bacteria present (either in the abscess or in other tissues) in both early deaths and scheduled euthanasia was considered unusually high (as this is an uncommon finding in a study where the test substance is given by gavage) and was therefore considered likely to be associated with treatment with sodium bromide, probably secondary to poor clinical condition and lack of grooming.

• Depletion of corpora lutea was observed in 3 females (1633, 1634 and 1637) in the 500 mg/kg/day group, which were all in estrus at termination. Further sections taken from these females, examined in the Ovarian Follicle Quantification investigation, also had no corpora lutea present, although no apparent effect on follicle count was observed. There was however, one control female killed at the end of the recovery period (DS148) which also had no corpora lutea. As sectioning of the ovaries for follicle counts involved some deviations from protocol in positioning of the sections within the ovary, allocation of left and right ovaries and some incomplete sections (including those for the females with no corpora lutea) and as there were no other effects on female reproductive organ weight or histopathology, it is considered that no conclusions can be drawn from the above observations and further investigation would be necessary to investigate a potential effect on female reproductive function.

• A peer review of the testes and epididymides was conducted . Sodium bromide -related findings were found in 2/10 in the 175 and in 9/9 500 mg/kg terminal euthanasia animals. All 4 early deaths (two of which were recovery group animals that died either during the dosing period or shortly thereafter) in the NaBr 500 mg/kg dose groups had NaBr-related findings. NaBr-related findings consisted of minimal to moderate retained spermatids at the lumen of the seminiferous tubule epithelium-primarily at Stages X-XII and minimal to moderate retained spermatid heads in the Sertoli cell near the basement membrane-primarily at Stages XI-XII. A secondary change in the epididymis, originating from the corresponding testis, was increased cellular debris in the lumen.
Any other microscopic findings observed in males or females were considered incidental.
There were no adverse findings at histopathology for the male reproductive organs at any dose of sodium bromide.

Recovery Euthanasia (Day 148)
Most findings noted at the terminal euthanasia were not observed at the end of the recovery period (Day 148). Microscopic findings noted at the terminal euthanasia which were also observed at the end of the recovery period (Day 148)
At the end of the recovery period, there was no evidence of the microscopic findings observed at the end of the treatment period i.e. depletion of colloid in the thyroid, or periportal vacuolation and tension lipidosis in the liver or the mixed cell infiltrates in the lung. Additionally, none of the females previously treated with sodium bromide had depletion of corpora lutea in the ovary, although there was one control female (1664) with moderate depletion of corpora lutea. Recovery of all microscopic findings was therefore apparent, with the exception of macrophage aggregates in the lung, observed in 3/17 animals treated at 500 mg/kg/day sodium bromide/day. As this finding was also observed at a comparable incidence in the control and sodium chloride treated groups (2/20 and 3/20 animals, respectively) at this time, it was considered not to be associated with previous treatment.
Other microscopic findings observed were considered incidental.

Summary of toxicolofical significant Microscopic findings at terminal Euthanasia are presented in Text Table 19.
Summary of Microscopic findings in animals after the recovery are presented in Text Table 20

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone analysis in serum was conducted on a single occasion in Week 4.
Interpretation of thyroid hormone data was constrained by the limited amount of historical control data (2 studies), the consequent variation of some control
values from this study in comparison to these values and the high number of values below the LLOQ.

There was however, an apparent reduction (p≤ 0.01) in T3 (males only) and in T4 (males and females) at 500 mg/kg/day. At 175 mg/kg/day, differences from
control in T3 (p≤ 0.05, males) and T4 (p≤ 0.01, males and females) were less marked and values were comparable to historical control values. Single animals in these groups also showed depletion of colloid in the thyroid at histopathology (2 males and 2 females in each group) but there was generally no correlation between this finding, hormone levels or thyroid weight in individuals.

In the absence of effects on thyroid weight and histopathology at 60 mg/kg/day, the slight decreases in T3 and T4, to values which were within the range of
historical control data, are considered to be of questionable toxicological significance and may fall into the normal adaptive response range of the thyroid.

There was no effect on TSH in males or females at 60 mg/kg/day. Although not statistically significant (probably due to wide variation in individual results)
mean TSH levels were 36% and 74% higher than controls in the 175 and 500 mg/kg/day male groups, respectively. All mean values, including controls were, however, markedly higher (~2 to 6 fold) than the historical control range. There was no significant effect on TSH in females.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

mortality

water consumption and compound intake

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

respiratory system: upper respiratory tract

Organ:

liver

lungs

skin

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

SUMMARY
The objectives of this study were to provide information on general toxicity and possible adverse effects in male and female Crl:CD(SD) rats resulting from repeated exposure to sodium bromide over an extended period of time covering growth into adulthood; with emphasis on estrous cycles, sperm evaluation and histopathology.

Conclusion General Toxicity
Administration of 500 mg/kg/day of sodium bromide for 90 days produced severe toxicity, characterized by adverse clinical observations and reductions in body weight gain, food intake and water consumption, with effects generally more severe in males than females. Histopathology indicated adverse effects in the liver, lungs and skin, with indicators of an increased susceptibility for bacterial infection, especially in the limbs, probably associated with the treatment-related decreased mobility and lack of grooming.
Administration of 175 mg/kg/day of sodium bromide produced similar but less severe toxicity including adverse clinical observations and reductions in food and water consumption, occurring more frequently in males than females. Effects on thyroid hormones, brain and lung weights and thyroid, liver, lung and skin histopathology were observed in occasional males only and cannot unequivocally be attributed to the test substance.
Effects observed in the 60 mg/kg group were comparable to the sodium chloride or historical controls and/or were findings common in this strain of rat, which cannot be unequivocally related to treatment and are regarded as not adverse. This dose level is therefore considered a NOAEL.

NaCl Conclusion
Administration of 284 mg/kg/day of sodium chloride at an equivalent osmololarity as 500 mg/kg/day of sodium bromide produced no toxicity, indicating that the toxicity observed in the 500 mg/kg/day dose group was due to exposure to bromide. The 500 mg/kg/day dose of sodium bromide was also demonstrated to be too toxic for use as a high dose in any evaluation of the effect of bromide on reproduction or other functional parameters as the general toxicity would preclude an appropriate evaluation of these functions.

Reproductive Toxicity
In the presence of severe toxicity in males treated at 500 mg/kg/day sodium bromide, there were effects on sperm motility, morphology and sperm count but no adverse effects on reproductive organ weights, or testicular spermatid counts. Retained spermatids at the luminal surface or in basal Sertoli cell cytoplasm occurred in 2/10 in the 175 and in 9/9 500 mg/kg terminal euthanasia animals . Three of 10 females in the 500 mg/kg/day dose group had no corpora lutea noted in the ovary, but overall follicle counts were not affected and there were no other adverse effects on reproductive organ weight, histopathology or estrous cycles.
The 500 mg/kg/day dose of sodium bromide was demonstrated to be too toxic for use as a high dose in any further evaluation of the effect of bromide on reproduction or other functional parameters as the general toxicity would preclude an appropriate evaluation of these functions.
There were minimal toxic effects on reproductive parameters at 175 mg/kg/day and no significant adverse effects on the reproductive parameters evaluated at 60 mg/kg/day sodium bromide. The NOAEL for reproductive effects was thereforbe established as 60 mg/kg/day.

Executive summary:

SUMMARY

The objectives of this study were to provide information on general toxicity and possible adverse effects in male and female Crl:CD(SD) rats resulting from repeated exposure to sodium bromide over an extended period of time covering growth into adulthood; with emphasis on estrous cycles, sperm evaluation and histopathology.  

The study design was as :

Text Table 1

Experimental Design

Group No.       Test Material       Dose Level (mg/kg/day)       Dose Volume (mL/kg)       Dose Concentration (mg/mL)       No. of Rats

                                  Males       Females

1       R.O. deionized water       0       5       0       10a + 10b       10a + 10b

2       Sodium Chloride       284       5       56.8       10a + 10b       10a + 10b

3       Sodium Bromide       60       5       12       10a       10a

4       Sodium Bromide       175       5       35       10a       10a

5       Sodium Bromide       500       5       100       10a + 10b       10a + 10b

a.       The first 10 rats were euthanized following completion of the dosing period (Day 91 of study).

b.       The remaining 10 rats were euthanized following completion of the 8 week recovery period (Day 148 of study).

The test or control substances were administered to the appropriate rats by once daily oral gavage from Days 1 to 90 of Study (DSs 1 to 90).  The sodium chloride

group was included to determine the effect of a sodium dose of equivalent osmolarity to that of the high dose sodium bromide group, and hence to distinguish effects

of the sodium and bromide components of the test material.  

All formulations were considered appropriate for use. The sodium chloride formulation was confirmed to be of equivalent osmolarity to the high dose sodium bromide

formulation.

Sodium Chloride Comparator Group

Administration of 284 mg/kg/day sodium chloride did not result in mortality, adverse clinical observations, or changes in body weight, food or water consumption.  There was no effect on ophthalmic observations, hematology (including coagulation values), urine or clinical chemistry values.  T3, T4 and TSH hormone levels were not affected although mean thyroid weights were higher than in any other group. There was no effect on organ weights, histopathology, or reproductive parameters (including estrous cycles, ovarian follicle counts or sperm count, motility and morphology).

Sodium Bromide

General Toxicity:

Mortality, Clinical Condition (routine and detailed clinical observations)

There were no deaths in the control group but one male (1558) showed adverse clinical signs and lung changes at histopathology which were considered likely due to aspiration of control substance into the lungs.  Other control males and females showed common, transient signs (e.g. scab, misaligned incisors, swollen ear, ungroomed coat) during the treatment and recovery periods in one or two animals/sex only.

All males and females treated at 175 mg/kg/day survived to scheduled euthanasia.  In males, treatment related signs of decreased motor activity postdose were observed in 6/10 males in week 2 but all animals recovered before the end of the working day.  In females, these signs were only observed on DS 11-13, in 8/10 animals.  Other clinical signs, including chromodacryorrhea, mild dehydration, swollen ear and/or periorbital area and hunched posture were generally infrequent and transient in both sexes.

At 500 mg/kg/day, multiple clinical signs, including numerous episodes of ataxia and decreased motor activity, prostration or breathing abnormalities (tachnypnea/dyspnea /hyperpnea), limb abnormalities (limited or no use and/or swollen/lacerated/purple color, no grip reflex in fore or hindlimbs) and signs indicative of poor condition (dehydration, ungroomed coat, chromodacryorrhea, chromorhinorrhea, fur staining), were considered evidence of marked toxicity in all males.  In 4 of these animals, these signs were so severe as to require euthanasia on DS 52, 55, 86 or 107: all of these males had shown marked reductions in body weight, and 2/4 had decreased food intake.  At necropsy, 2/4 had tan, firm lung lobes with masses: one also had tan material in the trachea and the other adhesions to the pericardium.  Histopathology confirmed the presence of bacterial infections in these animals, possibly related to mis-intubation or aspiration of the dosing solution in animals which may have still been affected by the dose from the previous day.

In surviving males clinical signs, consistent with the known sedative effects of sodium bromide, were apparent from 2 hours after dosing on the first day of treatment

in 5 males and increased in incidence, duration and severity over the treatment period, with all animals showing decreased motor activity by week 3 and all males

showing ataxia/prostration which persisted beyond the end of the working day by week 11.  Significant increases (p≤ 0.01, compared to controls) were observed in

the incidence of decreased motor activity, dehydration (mild and moderate), ataxia, ungroomed coat, urine-stained abdominal fur, hunched posture, chromodacryorrhea, ptosis, low carriage and limited use of limb(s)/paw(s).  Impaired righting reflex, first observed in week 8 in 2 males, affected 3 males by the end of the treatment period.  After approximately 6 weeks of dosing, one or more of these observations persisted throughout the working day, and was still evident at predose the next day in 2 or more males for the remainder of the dosing period.  The number of males in poor clinical condition (defined by limbs/paws red or purple and/or swollen, scabbed, chromorhinorrhea, reduced feces, thin, piloerection, head tilt, hypernea, lost grip reflex, abrasion and/or mass present in the eye, limbs, paws or inguinal area) was also increased compared to the control group and, although not statistically significant, was considered treatment-related as many effects persisted throughout the dose period, then became less evident in the recovery period.

After treatment ceased, 8 males (p≤ 0.01) continued to exhibit decreased motor activity, ungroomed coat, urine stained abdominal fur and dehydration for up to 30 days of the recovery period.  The severity and incidence of these signs generally decreased with time off-dose but one male still showed signs of lack of grooming (urine-stained abdominal fur) until DS 120.

Females showed similar clinical signs, with significant increases (p≤ 0.01) in ataxia, decreased motor activity, hunched posture, ptosis, low carriage and  limited use of limb(s)/paw(s), chromodacryorrhea, ungroomed coat, urine-stained abdominal fur and dehydration (mild and moderate) but they appeared later in the treatment period, did not persist until the following day and recovery was faster, suggesting a higher tolerance than males.  Decreased motor activity was first observed in 5 females from 1 hour after dosing on DS 10, increasing to 16 females during the second week of treatment and generally affecting all females by week 3.  Ataxia/prostration began after 2 weeks of dosing, generally between 1 to 2 hours after dosing and persisting until the end of the normal working day, initially in 9 females and increasing to 18 during week 8.  Impaired righting reflex was also observed in week 8.  During the recovery period, ataxia/decreased motor activity was not apparent from the first day off-dose but 3 females continued to exhibit ungroomed coat, urine-stained abdominal fur and dehydration, for up to 9 days after treatment ceased.

Detailed clinical observations recorded once weekly generally confirmed the daily clinical observations reported above.

Functional Observation Battery & Motor Activity

Functional observations and motor activity generally reflected the clinical observations.

There was no effect of treatment at 60 mg/kg/day on functional observations or motor activity at any of the scheduled evaluations.Functional observations were not

affected by treatment at 175 mg/kg/day.  Effects on motor activity were limited to significantly increased (p≤ 0.05) ambulations in week 4 (prior to dosing), only at the

first 10 minute interval and only in males.

At 500 mg/kg/day, when assessed predose during week 4 of treatment, males showed a decreased incidence of rears, and two animals exhibited ataxia and

abnormal gait.  These signs were not apparent in females but there was evidence of lack of righting reflex in 5 females (4 landing on their side, one on the back) and a reduction in hindlimb grip strength.  Increased motor activity (p≤ 0.05) was observed in males (ambulation and fine movements) and females (ambulations only).  

When assessed postdose in week 9, ataxia and abnormal gait were observed during the Functional Observation Battery in all males and in 6/10 females, respectively.  Five females also showed unusual posture.  Increased ambulations (p≤ 0.05) were recorded for both sexes at the first 10 minute interval of motor activity monitoring only, and the incidence of unkempt appearance/stained fur was 4-6 in males and 3-5 in females.

By week 13 (post-dosing), although the number of males showing ataxia was slightly lower (7), 5 animals showed unusual posture and 8 showed lack of air righting response (landing on their side).  Signs indicating lack of grooming (unkempt, urine/fecal staining) were more frequent and 9/10 males urinated in the open field.  In females, ataxia was observed in 8 females which was a slightly higher incidence than week 9 (6 females), abnormal gait and unusual posture had increased slightly and 8/10 females were sleeping in the home cage prior to examination.  Decreased fine movement was observed in males but there was no effect on motor activity in females.

By the end of the recovery period, functional effects were limited to the males.  There was an increased incidence of urination in the open field and there was an increase in forelimb grip strength (mean and maximum).

Body weight

There was no significant effect of treatment at 60 or 175 mg/kg/day on body weight or body weight gain in males and females, although values at 175 mg/kg/day were very slightly lower than controls.  At the end of the treatment period mean body weights of males were 97.2% and 93.0% of controls and for females 108.7% and 95.2%, in the 60 and 175 mg/kg/day groups, respectively.  Body weight gain was similar, with values for males of 97.3% and 90% of controls and for females 118.1% and 92.1% of controls, in the 60 and 175 mg/kg/day groups, respectively.

At 500 mg/kg/day there was a significant reduction in body weight gain in males for most weekly intervals after week 2, and at the end of the dosing period body weight and body weight gain were significantly lower than control (81.2 % and 68.8% of control, respectively, p≤ 0.01).  Body weight remained significantly lower than controls for the first two weeks post-dosing but increased to 90.4% of control values at the end of the recovery period, owing to weight gain markedly higher than control (134.7%).  Female body weight and weight gain were unaffected during the treatment period but gain at the end of the recovery period was substantially lower (66.5% of control values).

Food intake

Food intake generally paralleled changes in body weight.  There was no adverse effect in males or females at 60 mg/kg/day and at 175 mg/kg/day significant reductions over the treatment period were limited to males with absolute intake of 91.4% of controls (p≤ 0.05).

At 500 mg/kg/day, average and relative food consumption in males were significantly reduced (p≤ 0.01) over the dosing period (DS 1 to 90) and for each weekly interval after week 1.  During the recovery period, average absolute food consumption values were significantly reduced (p≤ 0.01) in first 2 weeks, remained lower than controls for the next 2 weeks but were similar thereafter.  Relative food consumption values in males were significantly lower (p≤ 0.05) at the start of the recovery period (Days 90 to 92), comparable to controls for the next two weeks and significantly increased (p≤ 0.01) for each weekly interval for the remainder of the recovery period, resulting in an overall significant increase (DS 90 to 147, p≤ 0.01).  

In females treated at 500 mg/kg/day, within the dosing period there were transient decreases in absolute (DS 43 to 50, p≤ 0.01) and in relative food consumption (DS 8 to 15, p≤ 0.01 and DS 43 to 50, p≤ 0.05) values for the entire period (DS 1-90) were comparable to controls.  Over the entire recovery period (DS 90-147) average absolute and relative food consumption were similar to control values as reductions (p≤ 0.01) observed in the first 2 weeks were followed by increases (p≤ 0.05 to p≤ 0.01) in the last 2/3 weeks.

Water intake

There was no adverse effect on water intake in any group during the first period measured (DS 1 7).  In the second period (DS 64-70), significant reductions were observed in both sexes in all groups treated with sodium bromide.  A dosage-related trend was apparent for males, with values of 87.4%, 81.7% and 78.8% in the low, intermediate and high dose groups, respectively, but not for females (82.5%, 77.3% and 83.9% of controls, respectively).  However, in the low dose group significant reductions in water intake were confined to DS 66 to 67 in males (when a similar reduction was seen the sodium chloride treatment group males) and DS 66 to 67 and 67 to 68 in females.

Clinical chemistry and hematology

There was no treatment- related effect on any hematology, coagulation or clinical chemistry value in male or female rats from any treated group when compared to concurrent and/or historical control values.

Thyroid Hormone Analysis

Thyroid hormone analysis in serum was conducted on a single occasion in Week 4.

Interpretation of thyroid hormone data was constrained by the limited amount of historical control data (2 studies), the consequent variation of some control values from this study in comparison to these values and the high number of values below the LLOQ.

There was however, an apparent reduction (p≤ 0.01) in T3 (males only) and in T4 (males and females) at 500 mg/kg/day.  At 175 mg/kg/day, differences from control in T3 (p≤ 0.05, males) and T4 (p≤ 0.01, males and females) were less marked and values were comparable to historical control values.  Single animals in these groups also showed depletion of colloid in the thyroid at histopathology (2 males and 2 females in each group) but there was generally no correlation between this finding, hormone levels or thyroid weight in individuals.

In the absence of effects on thyroid weight and histopathology at 60 mg/kg/day, the slight decreases in T3 and T4, to values which were within the range of historical control data, are considered to be of questionable toxicological significance and may fall into the normal adaptive response range of the thyroid.  

There was no effect on TSH in males or females at 60 mg/kg/day.  Although not statistically significant (probably due to wide variation in individual results) mean TSH levels were 36% and 74% higher than controls in the 175 and 500 mg/kg/day male groups, respectively.  All mean values, including controls were, however, markedly higher (~2 to 6 fold) than the historical control range.  There was no significant effect on TSH in females.  

Urinalysis

Effects on urine were limited to the 500 mg/kg/day group.  Males and females showed increases in leucocytes and males also showed increased incidences of yellow coloration and hemolysed blood in the urine, possibly indicating infection.

Ophthalmology

Ophthalmology revealed no significant changes in any treated group.

Gross Pathology

One male in each of the 60 and 175 mg/kg/day groups and 1 male and 2 females in the 500 mg/kg/day group were found to have a mass on the limbs at gross necropsy.  Histopathology confirmed these findings as abscesses associated with bacterial infection and not directly related to treatment.

Organ Weights

There were no statistically significant differences in absolute thyroid weight and increases relative to body weight were only significant (p≤ 0.05) in females treated at 500 mg/kg/day.  This did not, however, generally correlate with hormone levels or the microscopic finding of colloid depletion in individual animals.  No other significant organ weight changes were observed.  The effects on the reproductive organs are discussed in the Reproductive Toxicology section below.

Histopathology

Mild/moderate depletion of colloid (noted when the majority of follicles in the thyroid contained no identifiable colloid) was observed in 2 males and 2 females in the groups treated at 175 and 500 mg/kg/day.  It is, however, noteworthy that there was no correlation between these findings and decreased T3 and T4 levels in individual animals.  These changes were not apparent at 60 mg/kg/day.

Microvesicular vacuolation in the periportal hepatocytes was observed in 2, 5, 4 males and 2, 3, 4 females in the 60, 175 and 500 mg/kg/day groups, respectively.  This is a common incidental finding in rats, but can also result from nutritional disturbances.   At 175 and 500 mg/kg/day these findings tended to occur in animals also showing lung changes of accumulation of macrophages (within alveoli) and mixed cell infiltrates scattered in the pulmonary parenchyma.

Tension lipidosis, often related to increased body weights, was observed at a higher incidence in females at 500 mg/kg/day, in which terminal body weights were lower than control females.  This is a common incidental finding in rodents.   At terminal euthanasia 1/19, 1/20 and 3/19 animals in the 60, 175 and 500 mg/kg/day groups multiple abscesses in the skin/subcutis containing bacteria with associated formation of Splendore-Hoppeli material which usually forms secondary to local antigen-antibody reaction.  Skin abscess with evidence of bacteria and neutrophilic infiltrates were also contributory to the moribund condition of 2 males in the 500 mg/k/day group killed before scheduled termination.  As these findings rarely occur spontaneously in animals at this facility they were considered treatment-related.  As the skin abscesses were all located on the limbs/paws, however, these infections may be secondary to damage sustained during periods of test-substance related immobility or abnormal movement and/or poor clinical condition and lack of grooming.  

Other microscopic findings observed at the end of the treatment period were considered incidental.  At the end of the recovery period, there was no evidence of previous signs other than macrophage aggregates in the lung.  This is a common incidental finding in rats.   As this was observed in 3/17 animals treated at 500 mg/kg/day sodium bromide/day, a comparable incidence to the control and sodium chloride treated groups (2/20 and 3/20 animals, respectively), it was considered not to indicate any irreversible effect of treatment.

Reproductive Toxicity:

Females

Estrous cycles

There was no adverse effect of treatment with sodium chloride or sodium bromide on estrous cycles.

Ovary weight

There were no statistically significant differences in ovary weight (absolute or relative to brain or body weight) in any treatment groups compared with controls.

Female reproductive organ histopathology and ovarian follicle counts

Histopathology revealed no effects on the uterus, oviduct, vagina or mammary gland at any dose level.

Depletion of corpora lutea was observed in 3 females in the 500 mg/kg/day group, which were all in estrus at termination.  Further sections taken from these females, examined in the Ovarian Follicle Quantification investigation, also had no corpora lutea present, although no effect on follicle count was apparent.  At recovery euthanasia, all females in the 500 mg/kg/day group had corpora lutea but one control female was also found to have no corpora lutea.

As sectioning of the ovaries for follicle counts involved some deviations from protocol in positioning of the sections within the ovary, allocation of left and right ovaries and some incomplete sections (including the females with no corpora lutea) and as there were no other effects on female reproductive organ weight or histopathology, it is considered that no conclusions can be drawn from the above observations and further investigation would be necessary to investigate any potential effect on female reproductive function.

The absence of corpora lutea did not correlate with depletion of colloid in the thyroid or with thyroid hormone levels in individual animals.

Males

Organ weights

There was no adverse effect on reproductive organ weights in males treated at 60, 175 or 500 mg/kg/day.

Some statistically significant differences in absolute weights were considered not biologically significant, but a consequence of the lower body weights, as values relative to body weight were comparable to controls or significantly higher.

Histopathology

Microscopic examination of testes, prostate gland, epididymides, cauda epididymis, seminal vesicles and coagulating glands indicated no effect of treatment at any dose level with the exception of  sodium bromide -related findings  that were found in 2/10 in the 175 and in 9/9 500 mg/kg terminal euthanasia animals. All 4 early deaths (two of which were recovery group animals that died either during the dosing period or shortly thereafter) in the NaBr 500 mg/kg dose groups had NaBr-related findings.  NaBr-related findings consisted of minimal to moderate retained spermatids at the lumen of the seminiferous tubule epithelium-primarily at Stages X-XII and minimal to moderate retained spermatid heads in the Sertoli cell near the basement membrane-primarily at Stages XI-XII. A secondary change in the epididymis, originating from the corresponding testis, was increased cellular debris in the lumen.

Seminology

There was no effect on sperm count, motility or morphology at 60 mg/kg/day or 175 mg/kg/day.  At 175 mg/kg/day, the number of sperm with detached/no head was higher than the concurrent control but considered unlikely to have been an effect of treatment as it was closer to the expected (historical control mean) value than was the unusually low control value.

At 500 mg/kg/day, there was a reduction (88.6% of control, p≤ 0.01) in the number of normal sperm, a reduction in the percent motile sperm from the vas deferens (75.3% of control, p≤ 0.05) and increases in mean non-motile sperm (110.4% of control, p≤ 0.01), percent abnormal sperm (11.9%, p≤ 0.01) and mean number of sperm with detached head (20.6%, p≤ 0.01) or no head (3.2%, p≤ 0.01) compared to the control group values.  Mean epididymal sperm counts were also below the historical control range, although not significantly different (68.5%) from the concurrent control.

Testicular sperm counts (heads of homogenized sperm) were, however, comparable among the groups.  The lack of effect on this parameter, or on testis weight or histopathology suggests that the changes in epididymal sperm count and vas deferens sperm parameters, may be indicative of an effect of sodium bromide on the sperm occurring only after they have left the testes.  The changes observed may therefore have been a direct effect on the sperm or may have been mediated by a change in the osmolarity of the fluids in the epididymal lumen or vas deferens, possibly associated with altered electrolyte balance and/or the hydration state of the male.

Conclusion  

Administration of 284 mg/kg/day of sodium chloride at an equivalent osmololarity as 500 mg/kg/day of sodium bromide produced no toxicity, indicating that the toxicity observed in the 500 mg/kg/day dose group was due to exposure to bromide.  The 500 mg/kg/day dose of sodium bromide was also demonstrated to be too toxic for use as a high dose in any evaluation of the effect of bromide on reproduction or other functional parameters as the general toxicity would preclude an appropriate evaluation of these functions.

General Toxicity

In conclusion, administration of 500 mg/kg/day of sodium bromide for 90 days produced severe toxicity, characterized by adverse clinical observations and reductions in body weight gain, food intake and water consumption, with effects generally more severe in males than females.  Histopathology indicated adverse effects in the liver, lungs and skin, with indicators of an increased susceptibility for bacterial infection, especially in the limbs, probably associated with the treatment-related decreased mobility and lack of grooming.

Administration of 175 mg/kg/day of sodium bromide produced similar but less severe toxicity including adverse clinical observations and reductions in food and water consumption, occurring more frequently in males than females.  Effects on thyroid hormones, brain and lung weights and thyroid, liver, lung and skin histopathology were observed in occasional males only and cannot unequivocally be attributed to the test substance.

Effects observed in the 60 mg/kg group were comparable to the sodium chloride or historical controls and/or were findings common in this strain of rat, which cannot be unequivocally related to treatment and are regarded as not adverse.  This dose level is therefore considered a NOAEL.  

Conclusion  

Administration of 284 mg/kg/day of sodium chloride at an equivalent osmololarity as 500 mg/kg/day of sodium bromide produced no toxicity, indicating that the toxicity observed in the 500 mg/kg/day dose group was due to exposure to bromide.  The 500 mg/kg/day dose of sodium bromide was also demonstrated to be too toxic for use as a high dose in any evaluation of the effect of bromide on reproduction or other functional parameters as the general toxicity would preclude an appropriate evaluation of these functions.

General Toxicity

In conclusion, administration of 500 mg/kg/day of sodium bromide for 90 days produced severe toxicity, characterized by adverse clinical observations and reductions in body weight gain, food intake and water consumption, with effects generally more severe in males than females.  Histopathology indicated adverse effects in the liver, lungs and skin, with indicators of an increased susceptibility for bacterial infection, especially in the limbs, probably associated with the treatment-related decreased mobility and lack of grooming.

Administration of 175 mg/kg/day of sodium bromide produced similar but less severe toxicity including adverse clinical observations and reductions in food and water consumption, occurring more frequently in males than females.  Effects on thyroid hormones, brain and lung weights and thyroid, liver, lung and skin histopathology were observed in occasional males only and cannot unequivocally be attributed to the test substance.

Effects observed in the 60 mg/kg group were comparable to the sodium chloride or historical controls and/or were findings common in this strain of rat, which cannot be unequivocally related to treatment and are regarded as not adverse.  This dose level is therefore considered a NOAEL.  

Reproductive Toxicity

In the presence of severe toxicity in males treated at 500 mg/kg/day sodium bromide, there were effects on sperm motility, morphology and sperm count but no adverse effects on reproductive organ weights, or testicular spermatid counts.  Retained spermatids at the luminal surface or in basal Sertoli cell cytoplasm occurred in 2/10 in the 175 and in 9/9 500 mg/kg terminal euthanasia animals .  Three of 10 females in the 500 mg/kg/day dose group had no corpora lutea noted in the ovary, but overall follicle counts were not affected and there were no other adverse effects on reproductive organ weight, histopathology or estrous cycles.

The 500 mg/kg/day dose of sodium bromide was demonstrated to be too toxic for use as a high dose in any further evaluation of the effect of bromide on reproduction or other functional parameters as the general toxicity would preclude an appropriate evaluation of these functions.

There were minimal toxic effects on reproductive parameters at 175 mg/kg/day and no significant adverse effects on the reproductive parameters evaluated at 60 mg/kg/day sodium bromide. The NOAEL for reproductive effects was thereforbe established as 60 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1 according to appropriate OECD testing guidleine and GLP

System:

respiratory system: upper respiratory tract

Organ:

liver

lungs

skin

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

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.

The short term oral toxicity of ammonium bromide was investigated in rats (90-day toxicity study with neurotoxicity screening battery; 4-week dose range finding study). In addition, short term oral toxicity of sodium bromide was investigated in rats and dogs. The nervous system and the endocrine system were the main target organs in rats. In dogs, the nervous system was the main target organ. In both species, effects on bodyweight growth were noted (reduced bodyweight/bodyweight gain). Gastrointestinal toxicity (bloody stool, vomiting, diarrhea) and skin lesions were other effects noted in dogs.

 

No repeated dose toxicity studies performed with sodium bromide in animals by the dermal and inhalation routes are available. A subchronic dermal toxicity study conducted on sodium bromide is not considered necessary since the potential dermal absorption rate of sodium bromide is expected to be low and the systemic dose is not expected to be higher than the doses used in the available oral 90 day rat study. Nor is a subchronic inhalation toxicity study conducted on sodium bromide considered necessary since sodium bromide is not a volatile substance with a particle size that precludes inhalation.

Ammonium bromide

A 4-week dose-range finding study was performed in rats with ammonium bromide concentrations of 100, 500 and 1000 mg/kg bw/day mixed with the diet. In this study clinical signs were noted in male and female rats at a dose level of 500 mg/kg bw/day and above. The observed clinical signs consisted of agitated, nervous and hyperactive behaviour, subdued behaviour, rolling gait, hunched posture, piloerection and eyes partially closed. To a lesser extent unkempt coat and irregular breathing were noted. Statistically significant reduced body weight gains (males:-49%; females:-31%), reduced food consumption (males: 29%) and increased rel. liver weight (females) were noted in addition in animals of the high dose group (1000 mg/kg bw/day). The NOEL for male and female rats was determined at 100 mg/kg bw/day (corresponding to 82 mg bromide/kg bw/day). The NOAEL for male and female rats was determined at 100 mg/kg bw/day based on clinical signs indicating neurotoxicity and subdued behaviour (both sexes at ≥500 mg/kg bw/day), reduced body weight gain (both sexes at 1000 mg/kg bw/day) and increased liver weight (females at 1000 mg/kg bw/day) (Willerton and Robins, 1999).

 

A 90-day feeding study which included also a neurotoxicity screening battery was performed with ammonium bromide in rats following administration of 100 and 225 mg/kg bw/day in both sexes and500 mg/kg bw/day (males) as well as 750 mg/kg bw/day (females), respectively. Main study and neuropathology animals were treated continuously for at least 13 consecutive weeks. Recovery animals were fed treated diet for 13 weeks, and were then fed untreated diet for a period of at least 4 weeks. Clinical signs of subdued behaviour and neurotoxic effects (abnormalities of gait) were noted during the routine daily clinical examination (in males at ≥225 mg/kg bw/day; in females at 750 mg/kg bw/day). Additional findings included hunched posture, unkempt coat and claws that were longer than normal. The signs generally became apparent after approximately 8 weeks of treatment, and persisted until necropsy (main study animals) or at least the third week of the recovery period. Clinical signs of neurotoxicity were also noted during the detailed neurotoxicity examination (in males at ≥100 mg/kg bw/day; in females at ≥225 mg/kg bw/day). The findings noted during the detailed neurotoxicity examination (included viability, clinical signs, detailed functional observations) consisted of increased limpness, decreased alertness, increases in landing foot splay and decreases in fore and hind limb grip strength. At the low dose (100 mg/kg bw/day) the findings were limited to slight limpness in 3 males; of these, only one showed the finding on more than one occasion. Functional alterations were noted in both sexes at ≥225 mg/kg bw/day. All of these effects had reversed following the 4 week recovery period, except for hind limb grip strength in females at 750 mg/kg bw/day. In the absence of histopathological findings it was considered that all neurotoxicological effects were probably reversible. Other effects noted in the study consisted of: reduced bodyweight gain (males at ≥225 mg/kg bw/day; females at 750 mg/kg bw/day), reduced food consumption (males at 500 mg/kg bw/day), minor changes in haematology parameters (males at 500 mg/kg bw/day; females at 750 mg/kg bw/day), minor changes in biochemical parameters (males at ≥225 mg/kg bw/day; females at ≥100 mg/kg bw/day), decreased urine pH (females at 750 mg/kg bw/day) and reduced epididymides weight (males at 500 mg/kg bw/day). No treatment related necropsy or histopathological findings were observed. No NOEL was determined for male and female rats. The NOAEL for female rats was determined at 100 mg/kg bw/day (corresponding to 82 mg bromide/kg bw/day) based on clinical signs of neurotoxicity (noted at ≥225 mg/kg bw/day), clinical signs of subdued behaviour (noted at 750 mg/kg bw/day), reduced bodyweight gain (noted at 750 mg/kg bw/day), changes in haematological parameters (noted at ≥225 mg/kg bw/day), changes in biochemical parameters (noted at 750 mg/kg bw/dag) and decreased pH urine (noted at 750 mg/kg bw/day). No NOAEL was determined for male rats but the NOAEL was considered to be close to the dose level of 100 mg/kg bw/day (Bartonet al., 2000).

 

Sodium bromide supporting data

Observations in a 4-week oral study in female rats (Van Logten M.J.et al., 1973) and a 90-day oral study in male and female rats (Van Logten M.J.et al., 1974) demonstrated that sodium bromide caused behavioural changes, growth reduction, increased thyroid and adrenals weights, and a dose-related disturbance of the endocrine system. The NO(A)EL for rats was 15 mg (Br^-)/kg bw/day from the 90-day oral study. The results of an additional 90-day repeat dose study with sodium bromide (Van Logten M.J.et al., 1976) and a 90-day study with a similar salt, ammonium bromide (Barton S.J.et al., Inveresk Research, Report No. 18612) did not show any evidence of cellular change, even in potential target tissues such as the endocrine (thyroid) or neural systems, that could be considered preneoplastic change. Repeat dose studies in dogs were performed according to non-standard tests in which animals received 78 rising to 312 mg (Br^-)/kg bw/day for 400 days (Rosenblum I., 1958). Signs of toxicity noted were stated as being comparable with signs noted in human after suffering bromide intoxication. Although no NO(A)EL was determined, the study author states that dogs receiving 78 mg (Br^-)/kg/day showed no mortalities and only minimal signs of toxicity.

Conclusion

The NO(A)EL from sub-chronic toxicity studies is 95 mg/kg bw/day (modified for sodium bromide) from the 90-day oral study with ammonium bromide in rats.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The ion of concern for systemic toxicity is bromide. The study by Barton et al on ammonium bromide gives the lowest reliable NOAEL for bromide salts = 100 mg/kg bw/day. The NOAEL has been extrapolated to NaBr giving a final NOAEL for inorganic bromide salts of 95 mg/kg bw/day.

Justification for classification or non-classification

The substance should not be classified based on NOAEL 60 mg/kgbw/day in 90 day of oral repeated dose exposure.