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Administrative data

Description of key information

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

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1999-12-10 to 2000-09-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- 84 males & 84 females (80 animals of each sex were assigned to the study, spare animals were not considered to be part of the study)
- Females were nulliparous and non-pregnant
- Age at study initiation: approximately 4 weeks
- Weight at study initiation: 77-81 g (males) and 52-58 g (females).
- Fasting period before study:
- Housing: one per cage, solid bottomed polycarbonate cages with stainless steel grid tops containing bedding material
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days
- Animals allocated to treatment group by a measured allocation based on their body weight to ensure a mean body weight equivalent between the group
- Identified individually with electronic subcutaneous chip

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 21°C
- Humidity (%): Humidity levels were noted to be generally lower than the target range of 50 % ± 15 % respectively; actual range was 22-47 %.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour light /dark cycle
- Cage and hoopers changed at least once every two weeks. Bottles were changed once each week. Entire room washed with disinfectant solution once each study week

IN-LIFE DATES: From: 1999-12-10 To: 2000-09-08
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
Formulated diets were prepared from premix (Groups 3 and 4) or from the Group 4 diet (group 2).

The premix was prepared by manually mixing a sieved quantity of test material with the same weight of sieved diet, prior to addition to the blank diet to form a premix. The premix was then mixed for ca 2h in a Hobart mixer before being transferred to an appropriately labelled premix container.Treated diets were formulated weekly throughout the treatment period. The exact concentration (ppm) of test material in the diet varied over the treatment period to allow for the varying bodyweight and food consumption data produced by the animals. This enabled as near a constant dose level (mg/kg/day) as possible to be achieved.

Analysis of formulated diets were undertaken with regards to stability, concentration and homogeneity.
Triplicate samples were taken from each formulated diet( including control) immediately after preparation on Day 1 and during weeks 7 and 13 of treatment. The samples were assayed using methodology previously supplied by the sponsor and validated.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulated diets was undertaken with regard to stability, concentration and homogeneity. Triplicate samples were taken from each formulated diet (including control) immediately after preparation on Day 1 and during Weeks 7 and 13 of treatment.
Analysis of formulated diets indicated that actual dose levels administered were within ± 10 % of the nominal concentration, indicating acceptable accuracy of formulation. The coefficients of variation were all less than 10 %, indicating acceptable homogeneity.
The concentration of the test material in the diet was adjusted at weekly intervals to achieve (as far as practical) a constant dose level of test material per kg animal's body weight per day.
Duration of treatment / exposure:
13 weeks (90-days)
Post-exposure period: 4 weeks for the control and high dose group (10 animals/sex/group);
no postexposure period for the low and intermediate dose group and the remaining animals of the control and high dose group
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
225 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
500 mg/kg bw/day (males)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
750 mg/kg bw/day (females)
Basis:
nominal in diet
No. of animals per sex per dose:
25/sex/group (control and high doses), 15/sex/group (low and intermediate dose)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
4 week dose range-finding study.
Positive control:
Not applicable
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked early each morning and as late as possible each day for viability.All animals were examined for reaction to treatment each day. The onset, intensity and duration of any signs were recorded.
In addition, each week, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.
Mortality: Each animal was checked twice per day for viability

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once weekly during the pretrial period, up until the end of the recovery period. Animals showing weight loss or deterioration in condition were weighed more frequently as necessary.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
The quantity of food consumed by each animal was measured and recorded once during the week prior to the start of treatment and then weekly up until the end of the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored by visual inspection on a weekly basis throughout the study, commencing during the pretrial period.

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmoscopic examination was carried out on all animals before treatment commenced and on control and high dose animals during Week 12 of treatment. The eyes were examined using an indirect ophthalmoscope after the application of 1 % Tropicamide (Mydriacyl). Anterior, lenticular and fundig areas were evaluated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken via the orbital sinus under isoflurane anaesthesia. Time points: during Week 12, and additionally for control and high dose rats during Week 17 (end of recovery).
- From this blood sample, ca 0.5 ml of whole blood was transferred into EDTA for haematology assessment, 0.45 ml was transferred into trisodium citrate tubes for assessment of coagulation parameters. The reminder was taken for clinical chemistry investigations.
- Anaesthetic used for blood collection: Yes
- Animals fasted: No data
- How many animals: All animals
- Parameters examined: Haemoglobin, red blood cell count, Haematocrit, mean cell volume, mean cell haemogloboin, mean cell haemoglobin concentration, platelets, white blood cell count, differential (Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, unclassified cells)/ Moreover, Prothrombin time is regarded.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Time points: during Week 12, and additionally for control and high dose rats during Week 17 (end of recovery)
- Animals fasted: No data
- How many animals: All animals
- Parameters examined: Urea, Glucose, Aspartate Amino Transferase, Alanine Amino transferase, Total, protein, Albumin, Albumin, Globulin ratio, Creatinine, total bilirubin, sodium, potassium, Chloride, Calcium, Phosphate, Alkaline Phosphatatse, Total Cholesterrol, Gamma Glutamyltransferasen Sorbitol Dehydrogenase, Globulin.

URINALYSIS: Yes
- Time schedule for collection of urine: Time points: during Week 12, and additionally for control and high dose rats during Week 17 (end of recovery); By oversight, the urine samples in week 17 were taken over a period of 5h; this deviation was considered not to have affected the integrity of the study.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: pH, Specific Gravity, Volume, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood pigments, Microscopy of the Spun Deposit

NEUROBEHAVIOURAL EXAMINATION: Yes
Detailed functional observations were performed during pretrial and in Weeks 4, 8 and 13 of dosing together with week 17 for recovery animals, a detailed examination was performed for all designated animals. Observations included, but were not limited to: posture/condition, ease of removal from the cage, body temperature, condition of eyes and coat, presence of salivation, overall ease of handling, latency, level of mobility, rearing, grooming, urination/defecation, arousal (level of alertness), posture, tremor/convulsions, vocalisation, piloerection, palpebral closure, gait abnormalities, stereotypy and/or unusual behaviours, reaction to sudden sound, reaction to touch on the rump, grip strength, pain perception, landing foot splay, motor activity and any other physical/functinal abnormalities.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

On completion of 13 weeks of treatment main and neuropathological stud animals were killed and underwent detailed necropsy (After 17 weeks for recovery study animals). All control and high dose animals also underwent a full histological examination. The main and recovery study animals were killed by exposure to carbone dioxide asphyxiation follwed by exsanguination.
The control and high dose animals that were retained for 4 weeks after treatment received a detailed necropsy, but no histological examination was conducted.

Organ weights: adrenal, brain, epididymis, heart, kidney, liver, lung, ovary, pituitary, prostate, spleen, submaxillary salivary gland, testis, thymus, thyroid, parathyroid, uterus

HISTOPATHOLOGY: Yes
adrenal, aortic arch, brain, epididymis, eye, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, heart, kidney, larynx, liver, lung, mesenteric lymph node, nasal cavity, oesophagus, optic nerve, ovary, pancreas, pharynx, pituitary, prostate, sciatic nerve, seminal vesicles, skin and mammary gland, spinal cord, sleen, sternum, submandibular lymph node, submaxillary salivary gland, testis, tigh muscle, thymus, thyroid, parathyroid, tongue, trachea, urinary bladder, uterus, vagina
The tissues designated for examination were processed and examined histologically for main study and neuropathology control and high dose animlals. To follow up an apparent effect on thyroid weights, histological examination of the thyroids was extended to main study low and intermediate dose animals, and to recovery study control and high dose animals.
Other examinations:
Neuropathological examination, with nervous tissues and organs from all perfused animals being examined histologically. The animals were aneaesthesied using a sublethal dose of sodium pentobarbitone. They were then injected via tail vein 1 % sodium nitrite and 1 % heparin via the tail vain, perfusion with phosphate buffered saline to remove most of the blood, fixation by perfusion with paraformaldehyde/glutaraldehyde-mixture. All neuropathology study animals were dissected with brain, spinal cord, tibial nerve, sacral nerve and sciatic nerve preserved. All peripheral nerve tissues were to be processed to methacrylate resin blocks
Statistics:
Bodyweight, food consumption, haematology, clinical chemistry and urinalysis data were subjected to analysis of variance or the Kruskal-Wallis non-parametric analysis, as appropriate.
Organ weight data were subjected to analysis of variance and of covariance using the terminal bodyweight as the single covariate.
Histological incidence data were analysed using Fisher´s Exact Probability test where considered appropriate.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See details below
Mortality:
mortality observed, treatment-related
Description (incidence):
See details below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See details below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See details below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See details below
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
See details below
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
See details below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See details below
Gross pathological findings:
no effects observed
Description (incidence and severity):
See details below
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details below
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details below
Details on results:
CLINICAL SIGNS AND MORTALITY
The principal signs were rolling gait, intermittent staggering, subdued behaviour, nasal bleeding, unkempt coat, hunched posture, discharge from the eyes and splayed hind limbs. Some or all of these signs were observed for all animals at 500 mg/kg/day (males) and 750 mg/kg/day (females); among males 225 mg/kg/day, 11/15 showed subdued behaviour, sometimes accompanied by other signs. These signs generally became apparent after approximately 8 weeks of treatment, and persisted until necropsy (main study animals) or until at least the third week of the recovery period.
Additionally, the claws of many male rats at 225 and 500 mg/kg/day and females at 750 mg/kg/day were longer than normal. The presence of occasional females at 100 and 225 mg/kg/day with longer claws could not be positively associated with treatment.
Irregular respiration was noted for 7 males at 500 mg/kg/day, although 2 animals did not show this finding until the recovery period.

There were 3 premature descendents, all among males at 500 mg/kg/day. One animal was killed in the 6th week of treatment, with signs including laboured respiration; necropsy and histology indicated a widespread purulent pneumonia. Another animal was killed in the 13th week of treatment, with limping/swelling affecting the right hind foot; necropsy and histology indicated marked pododermatitis. The third animal was killed in the 9th week of treatment due to limping. None of the reasons for the premature terminations appeared to be associated with treatment.

BODY WEIGHT AND WEIGHT GAIN
Males:
At 500 mg/kg/day there was a slight increase in bodyweight gain to Day 6; from Day 6-13, gain was similar to control, but by Day 28 mean weights at this level were lower than Control, with differences from Day 35 attaining statistical significance (-23% on Day 91 of treatment). During the 4th week recovery period, there was an increase in weight gain, compared with control, although absolute weights remained significantly lower. At 225 mg/kg/day, there was a slight increase in bodyweight gain to Day 13, with the difference at Day 13 attaining statistical significance. From Day 13, gain was slightly lower than control, although absolute weights were slightly greater to Day 28. By Day 49, however, weights at this level were lower than control, attaining statistical significance from Day 63 (-10% on Day 91 of treatment). At 100 mg/kg/day, bodyweight performance was similar to control.
Females:
At 750 mg/kg/day, there was a slight increase in bodyweight gain to Day 6, with the difference at Day 6 attaining statistical significance, but by Day 21 mean weights at this level were lower than control, with the differences from Day 35 attaining statistical significance (-22% on Day 91 of treatment). During the 4th week recovery period, there was a slight increase in weight gain, compared with control, although absolute weights were significantly lower in most weeks. At 225 mg/kg/day, mean weight gain and absolute weights were greater than Control in the early part of the dosing period, with many differences being statistically significant. During the latter part of the dosing period, bodyweight gain was slightly lower than control, with absolute weights at this level being similar to control. At 100 mg/kg/day, bodyweight performance was similar to control.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Because the animals were receiving powdered diet and were housed in solid bottomed cages containing bedding material, it was difficult to measure scattered diet, and this impacted upon the accuracy of the food consumption calculations. However, it was considered that the inherent errors in assessment of scatter would apply equally to animals in all groups of the study, and therefore the validity of the study was not affected.
Males: At 500 mg/kg/day, mean food consumption during the first 3 weeks of treatment was significantly greater than control. During Weeks 4-13, consumption was lower than control, with most differences statistically significant. During the first week of recovery, consumption was slightly greater than control, in the other weeks of recovery, consumption was similar to control. At 225 mg/kg/day, mean food consumption during the first 3 weeks of treatment was also significantly greater than control, but thereafter consumption was slightly lower; some differences were statistically significant. At 100 mg/kg/day, there was no consistent effect on food consumption. Slight differences from the food consumption of the controls were recorded, a small number of which achieved statistical significance, but these were considered to be of no toxicological significance.
Females: At 750 mg/kg/day, food consumption in the first and fourth week of treatment was significantly greater than control; in later weeks, consumption was generally lower than control, with occasional differences attaining statistical significance. During the first week of recovery, consumption was significantly greater than control; in the other weeks of recovery, consumption was similar to control. At 225 mg/kg/day, mean consumption was generally greater than control, with some differences attaining statistical significance. At 100 mg/kg/day, there was a tendency towards slightly increased food consumption, although sporadic values were lower than the corresponding control values.
The achieved intakes of test material, expressed as mg Ammonium Bromide/kg/day were close to the target dose levels.

HAEMATOLOGY
Week 12:
For males treated at 500 mg/kg/day, mean values for haemoglobin and haematocrit were higher than those of the concurrent controls. Although the statistical significance of the increase may have been exaggerated by the concurrent controls being at the lower end of the background control range, both of the values for treated males were above the background control range. Females treated at 750 mg/kg/day also showed minimal increases in haemoglobin and haematocrit, but the differences from the concurrent control values did not achieve statistical significance. For males at 100 and 225 mg/kg/day mean haemoglobin level was slightly increased compared with the concurrent controls, the difference achieving statistical significance, but no dose-relationship was apparent and there was no corresponding increase in haematocrit. The increase in these groups was considered to be a consequence of the low concurrent control value, as the haemoglobin level of both treated groups was similar to the background control mean, and, therefore, was of no toxicological significance. Values for females in the low and intermediate treatment groups were similar to those of the controls.
For males and females in the high dose group (500 and 750 mg/kg/day respectively), the mean total white blood cell count was slightly increased, although the differences from the controls were not statistically significant. In the case of males, the increase was largely due to two animals. For both sexes the overall increase reflected increases in the majority of white cell types, with the values for neutrophils and monocytes in females achieving statistical significance. At 225 mg/kg/day for females, minimal increases were seen in the same parameters but males were unaffected, as were both sexes at 100 mg/kg/day.
There were no obvious treatment related differences in platelet count and prothrombin time of males and females at any treatment level.

Week 17:
At the end of the 4-week recovery period, mean haematocrit and haemoglobin values of males and females previously treated with 500 and 750 mg/kg/day respectively were similar to control values. For males, mean cellular haemoglobin (MCH) was statistically significantly higher than that of the concurrent controls. However, the increase was considered to be of doubtful toxicological significance, as the concurrent control value was lower than the Week 12 control value and was at the lower end of the Week 12 background control range, whereas the value for the previously treated males was similar to both the Week 12 control value and background control mean value. MCH for previously treated females was similar to that of the controls.
In contrast to Week 12 findings, mean values for total and differential white cell counts of previously treated males and females were generally lower than those of the corresponding concurrent controls, with the differences for total white cell count and lymphocytes (both sexes), monocytes (males) and large unclassified cells (females) achieving statistical significance.
For previously treated females, although mean prothrombin time appeared to be significantly increased, the magnitude of the increase was minimal and it was considered not to represent any toxicological response to treatment.

CLINICAL CHEMISTRY
Week 12:
In both sexes, mean assayed chloride levels were apparently greater in all groups, with values for males at 225 and 500 mg/kg/day and females at all levels attaining statistical significance. However, to some extent these increases may have reflected increased plasma bromide levels, because the bromide ion has been found to interfere with the chloride ion in the assay.
Mean cholesterol levels in males at 225 and 500 mg/kg/day and females at all levels were significantly lower than control.
The mean total bilirubin level in females at 750 mg/kg/day was significantly reduced.
Mean phosphate levels of males at 225 and 500 mg/kg/day were significantly higher than control; a similar effect was not observed for females or for males in the low treatment group. The apparent increase in calcium levels among females at 100 and 225 mg/kg/day was influenced by a control group in which only 2 animals had the calcium level determined.
The mean glucose level among males at 100 mg/kg/day was significantly lower than control; this observation was not seen at higher levels.

Week 17:
After 4 weeks of recovery, mean assayed chloride levels of both sexes of treated animals were very slightly, but significantly greater than control, although the levels were slightly lower than the control values at Week 12.
Mean calcium levels of both sexes were significantly lower than control, but the actual differences were small and considered to be of doubtful toxicological significance.
Mean cholesterol among females at 750 mg/kg/day was significantly lower than control.
Among females at 750 mg/kg/day, mean glucose level was significantly greater than control, but this observation was not supported by a similar finding in males. Alkaline phosphatise at this level was significantly increased, but this increase was influenced by a low control value. Albumin at this dose level was decreased, but was within the background range; the control value was at the upper and of that range: the slight differences in globulin and A-G ratio were considered to be secondary observations. The differences in glucose, alkaline phosphatise and albumin were considered not to be of toxicological significance.

URINALYSIS
At Week 12, the urine pH of both sexes at the high dose levels was lower than control, with the value for females attaining statistical significance; this finding was not seen at Week 17.
At Week 17, slight differences in specific gravity were considered to be incidental, despite their statistical significance, in view of the low actual differences in the values and because males showed a decrease whilst females showed an increase. The increased urine volume of males at Week 17 was also considered to be incidental.

NEUROBEHAVIOUR
Males: At 500 mg/kg/day many males were limp when handled (Weeks 4, 8 and 13) and/or showed stumble/rolling gait (Weeks 8 and 13). These signs were observed for occasional animals at 225 mg/kg/day, and limpness was also recorded for a few animals at 100 mg/kg/day of ammonium bromide. Occasional males had reduced alertness (225 and 500 mg/kg/day) and unkempt coat (500 mg/kg/day). All of the signs at 500 mg/kg/day had disappeared by the end of the recovery period.
Females: At 750 mg/kg/day, many females were limp in the hand (Weeks 8 and 13) and/or showed stumble/rolling gait (Weeks 4, 8 and 13). Limpness was also recorded for 4 animals at 225 mg/kg/day in Week 8. All of these signs at 750 mg/kg/day had disappeared by the end of the recovery period. Occasional females at 750 mg/kg/day had unkempt coat, which persisted into the recovery period.
Some inter-group variations in motor activity parameters were recorded for males and females at each of the test intervals, a small number of which achieved statistical significance compared with the concurrent controls. There were, however, no consistent findings that were indicative of a dose or treatment related response to treatment regarding motor activity.

Detailed functional observations:
Males: Among males, landing foot splay at 225 and 500 mg/kg/day was increased during Week 4, with differences for trial 2 attaining statistical significance. At other times, values for foot splay showed no statistically significant inter-group differences. At 500 mg/kg/day, hind limb grip strength was significantly lower at Weeks 4, 8 and 13; fore limb grip strength was significantly lower at Weeks 8 and 13. By the end of the 4th week recovery period, there were no significant differences in these parameters. At 225 mg/kg/day, hind limb strength at Week 4 was significantly lower than control, although the fore limb grip strength was slightly increased. At Weeks 8 and 13, hind limb grip strength values were also lower than concurrent control values although statistical significance was not attained; forelimb grip strength was unaffected. At 500 mg/kg/day the tail flick response was slightly slower than control, with the difference for the trial 3 value at Week 13 attaining statistical significance. However, the control value for trial 2 was low compared with the trial 1 value, which tended to exaggerate the group 4 result and by the end of the 4th week recovery period, there was no difference in response time. At 225 mg/kg/day, values for tail flick were similar to Control. At 500 mg/kg/day, the number of sectors traversed was significantly increased in Week 8; this observation was not repeated at other times. At 100 mg/kg/day, there were no obvious differences in the detailed functional observations for male rats.
Females: Among females, landing foot splay at 225 and 750 mg/kg/day was increased during Week 4, with differences for trial 1 at 750 mg/kg/day attaining statistical significance. At other times, foot splay values were similar to control. At 750 mg/kg/day fore and hind limb grip strength were reduced at Weeks 4, 8 and 13, with the differences at Weeks 8 and 13 attaining statistical significance. At the end of the 4th week recovery period, hind limb strength remained lower than control, although fore limb strength was similar to control. There were no other obvious differences among females in the detailed functional observations.

ORGAN WEIGHTS
Week 13:
Among males, most absolute organ weights at 500 mg/kg/day, together with weights for brain, epididymides, heart, liver and testes at 225 mg/kg/day, were significantly lower than control, reflecting the lower mean bodyweights at these levels. Following covariance analysis to adjust for bodyweight, none of the differences were statistically significant, although the validity of this statistical method was questionable because of the marked treatment-related effect on bodyweight. At 100 mg/kg/day, brain and epididymides weights were significantly lower than control, although adjusted weights were not significantly decreased.
In females at 750 mg/kg/day, absolute brain, lung and thymus weights were significantly lower than control, although the differences were no longer statistically significant after covariance adjustment. Absolute salivary gland and uterus weights at this level were significantly reduced; for these organs in this study, the covariance adjustment indicated decreasing organ weight with increasing body weight, and therefore, the assessment was based on the absolute weights.
The thyroid weights of males at 100 and 225 mg/kg/day, both absolute and adjusted, were significantly lower than control, although weights at the high dose (500 mg/kg/day) were not significantly different. A similar but slighter effect was noted for females, although none of the differences were statistically significant. Thyroid weights were taken by two technicians. The weights from control group animals were separated between the two technicians and recorded weights from one technician were slightly lower than from the other. Because of this difference and because of the small actual weights and the absence of an effect at the high dose, the apparent decreases at 100 and 225 mg/kg/day were not attributed to treatment.
Among females at 100 and 225 mg/kg/day, there were no significant differences for any organ.

Week 17:
At 500 mg/kg/day, following the 4-week recovery period, epididymides weights (absolute and adjusted) were significantly lower than control. Absolute prostate and testes weights were significantly reduced, although the statistical significance had disappeared after covariance adjustment. Adjusted spleen weights were increased, although absolute weights were similar to control; this apparent increase appeared to have been due to an over-correction by the analysis of covariance.
At the end of the 4-week recover period, the thyroid weights (absolute and adjusted) of high dose males were significantly greater than control, although the high dose value was very similar to the control value at Week 13. A slight, but not significant increase was noted for thyroid weights of high dose females. The apparent slight increases were attributed to 4 animals with noticeably higher weights rather than to an increase across all animals, and the differences considered to be of no toxicological significance.
At 750 mg/kg/day, following the recovery period, absolute adrenal gland, brain and lung weights were significantly lower than control, although differences were no longer statistically significant after covariance adjustment.

GROSS PATHOLOGY
There were no necropsy findings attributed to administration of ammonium bromide. One female rat treated at 750 mg/kg bw/dayhad a subcutaneous mass on the head. The left testis and epididymis of another animal of the 500 mg/kg bw/day group were small and flaccid. All other findings recorded reflect the usual range of congenital or spontaneously arising background findings in rats of this strain, at this age, on this type of study at the performing laboratory.

HISTOPATHOLOGY:
Main study animals:
The subcutaneous mass on the head of one female rat was found to be a granuloma, containing areas of acute inflammation, bacterial colonies, and pigmented macrophages. The male animal with the abnormal finding had widespread left testicular tubular atrophy. Due to the isolated occurrence in individual animals only and since no dose-response relationship was evident, neither of these findings was attributed to the test material. All other findings recorded reflect the usual range of congenital or spontaneously arising background findings in rats of this strain, at this age, on this type of study at the performing laboratory.

Neuropathology study animals:
Ventricular dilatation was seen in animals from all groups. Occasional ballooned axons and digestion chambers, extending one internode length, were noted in animals from all groups. An epidermal cyst was seen in the white matter of the lumbar cord of one female at 225 mg/kg/day. Epidermal cysts are congenital lesions of the spinal cord. They are attributed to improper closure of the neural tube in the later stage of embryonal development. The finding of ventricular dilatation was considered to be artefactual, as perfusion fixation is known to cause minimal to mild ventricular distension. Occasional ballooned axons and digestion chambers were seen. Such small segmental demyelination lesions are recognised spontaneous lesions of peripheral nerves.

Recovery study animals: The thyroids from the control and high dose recovery study animals showed no changes that were attributed to treatment.

Ophthalmoscopy finding: No treatment related findings were noted.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: One animal had slight limpness recorded one more than one occasion, and this recording was restricted to the detailed neurotoxcicological examination.
Dose descriptor:
LOAEL
Effect level:
225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Changes in haematological parameters and clinical signs of neurotoxicity noted at 225 mg/kg bw/day
Critical effects observed:
not specified

See Result Tables in attached document.

Conclusions:
LOAEL males: 100 mg/kg bw/day. The observed effect of slight limpness noted in males at 100 mg/kg bw/day during the detailed neurotoxicological observations is an expected effect and should be regarded as an adverse effect, although the sign had disappeared by the end of the recovery period. LOAEL: females: 225 mg/kg bw/day.
Clinical signs of subdued behaviour and neurotoxic effects 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. 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. It was considered that all neurotoxicological effects were probably reversible. Other effects noted in the study consisted of: reduced bodyweight gain (in males at ≥225 mg/kg bw/day; in females at 750 mg/kg bw/day), reduced food consumption (in males at 500 mg/kg bw/day), minor changes in haematology parameters (in males at 500 mg/kg bw/day; in females at 750 mg/kg bw/day), minor changes in clinical parameters (in males at ≥225 mg/kg bw/day; in females at ≥100 mg/kg bw/day), decreased urine pH (in females at 750 mg/kg bw/day) and reduced epididymides weight (in 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 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, changes in haematological parameters noted at ≥225 mg/kg bw/day, changes in biochemical parameters noted at 750 mg/kg bw/day and decreased urine pH 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.
Executive summary:

Materials and method

The objective of this study was to assess the toxicity (including neurotoxicity) of the test material in the rat after oral administration with the diet for 13 weeks including as recovery period of 4 weeks for control and high dose animals. As part of the assessment of toxicity, it was also intended to identify the no observed effect level (NOEL) and any toxic effects.

The main study and neuropathology animals were treated continuously by the diet for at least 13 consecutive weeks. The recovery animals were fed treated diet for 13 weeks, and were then fed untreated diet for a period of at least 4 weeks. The concentration of the test material in the diet was adjusted at weekly intervals to achieve a constant dose level of test material per kg of bodyweight per day.

All animals were observed for viability and examined for reaction to treatment. In addition, each week, all animals received a detailed clinical examination. Detailed functional observations were performed during pretrial and at several time points throughout the study. Once per week bodyweight and food and water consumption were recorded. Ophthalmoscopic examination was performed during the pretrial and for control and high dose animals again during Week 12 of treatment.

At the end of the treatment and recovery periods organ weights were taken; several organs, including nerve tissue, were fixed and stained with haematoxylin and eosin for histological examination.

Results and discussion

Clinical signs of reaction at the highest treatment levels of ammonium bromide tested in this study (500 mg/kg/day for males and 750 mg/kg/day for females) included subdued behaviour and abnormalities of gait. Additional findings included hunched posture, unkempt coat and claws that were longer than normal. The detailed neurotoxicological findings included increased limpness, decreased alertness, increases in landing foot splay, and decreases in fore and hind limb grip strength, with hind limb strength remaining lower in females after the 4 week recovery period.

At the intermediate treatment level (225 mg/kg/day) subdued behaviour, abnormalities of gait, hunched posture and claws longer than normal were observed for males. Neurological findings at this level were limpness and increased landing foot splay in both sexes, and decreased hind limb grip strength in males. At the low level (100 mg/kg/day) the findings were limited to slight limpness in 3 males; of these, only one showed the finding on more than one occasion. Slight limpness was only noted during the detailed neurotoxicity examination, but was not recorded during routine daily clinical examination.

Bromide is known to have a sedative effect and the decreased alertness and reduced muscle tone (as indicated by reduced grip strength, and increased limpness and landing foot splay) can be regarded as expected effects. The increased claw lengths were probably related to the decreased activity.

The pattern of effects on bodyweight was an initial increased gain, compared with control, followed by reduced gain, which was observed for males at 225 and 500 mg/kg/day and for females at 750 mg/kg/day. A lesser effect, manifest as a weight gain followed by a return to control values was observed for females at 225 mg/kg/day, During the recovery period, there was an increase in weight gain, although absolute weights remained lower than control. Food consumption essentially mirrored the body weight performance.

At the high treatment level, for both sexes after 12 weeks, there were increases in haemoglobin, haematocrit and total white cell count, the latter reflecting increases in the majority of white cell types. At the intermediate treatment level, in females only, minimal increases were recorded in total and differential white cell counts. After the 4-week recovery period, haemoglobin and haematocrit values of animals in the high treatment group were similar to those of the controls. In contrast, total and differential white cell counts of previously treated animals were significantly lower than those of the controls. Other inter-group variations in haematological parameters were considered to be of no toxicological significance.

Mean assayed chloride levels were greater in all test groups. To some extent these increases may have reflected increased bromide levels, because bromide ion interfered with the chloride ion assay. Only a slight increase was still present after the 4-week recovery period, and the values were similar to control values at Week 12. Because of the interference of the bromide ion it was not possible to comment on the actual chloride levels.

Mean cholesterol levels in males at 225 and 500 mg/kg/day and females at all levels were lower than control. For the females, the control value was slightly larger than the expected value and therefore the effect, particularly at 100 mg/kg/day may have been incidental.

Mean total bilirubin level in females at 750 mg/kg/day was reduced. Mean phosphate levels of males at 225 and 500 mg/kg/day were significantly increased. Urine pH was decreased at the high dose levels of both sexes.

Assessment of the effect of ammonium bromide on organ weights was complicated by the marked effect on bodyweight. Many weights were reduced, especially for males at 500 mg/kg/day, but it was not clear whether the reductions were true effects on the organ or were merely a consequence of the lower bodyweight. Taking all the data into consideration, it was considered that epididymis weight was reduced at the high dose group and that this reduction persisted into the recovery period. A lesser effect, never significant by analysis of covariance, was seen on the testes. Neither of these organs had any histological finding after Week 13 necropsy.

Apparent slight differences in thyroid weights of main study animals were attributed to variations between the recordings of two technicians, rather than to an effect of treatment. This opinion is reinforced by the absence of any treatment related finding at histopathological examination. Equally, the apparent slight increase in thyroid weights among recovery study animals were also considered to be of no toxicological significance in the absence of histopathological findings.

In assessing neurotoxicity, it was noted that effects (decreased alertness and grip strength, and increased limpness and landing foot splay) were seen in males at 225 and 500 mg/kg/day and in females at 750 mg/kg/day, together with slight limpness in 3 males at 100 mg/kg/day. All of the effects had reversed following the 4-week recovery period except for hind limb grip strength in females at the highest dosage group. Hind limb grip strength was the more sensitive marker in this study in that decreased hind limb strength occurred without the fore limb being affected, but decreased fore limb strength only occurred when the hind limb strength was also reduced. There was no indication of any histological effect on the nervous system following 13 weeks of treatment. It was considered that all neurotoxicological effects were probably reversible.

During the pretrial period (observation of animals one week prior to study initiation) one animal had an unacceptable ophthalmoscopic finding and another one showed poor performance during detailed functional observations. These animals were replaced by spare animals prior to study initiation.

Analysis of formulated diets indicated that all were within ± 10 % of the nominal concentration, indicating acceptable accuracy of formulation. The achieved intakes of the test material, expressed as mg ammonium bromide/kg/day, were close to the target dose levels.

LOAEL males: 100 mg/kg bw/day. The observed effect of slight limpness noted in males at 100 mg/kg bw/day during the detailed neurotoxicological observations is an expected effect and should be regarded as an adverse effect, although the sign had disappeared by the end of the recovery period. LOAEL: females: 225 mg/kg bw/day.

Clinical signs of subdued behaviour and neurotoxic effects 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. 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. It was considered that all neurotoxicological effects were probably reversible. Other effects noted in the study consisted of: reduced bodyweight gain (in males at ≥225 mg/kg bw/day; in females at 750 mg/kg bw/day), reduced food consumption (in males at 500 mg/kg bw/day), minor changes in haematology parameters (in males at 500 mg/kg bw/day; in females at 750 mg/kg bw/day), minor changes in clinical parameters (in males at ≥225 mg/kg bw/day; in females at ≥100 mg/kg bw/day), decreased urine pH (in females at 750 mg/kg bw/day) and reduced epididymides weight (in 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 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, changes in haematological parameters noted at ≥225 mg/kg bw/day, changes in biochemical parameters noted at 750 mg/kg bw/day and decreased urine pH 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
95 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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 95 mg/kgbw/day in 90 day of oral repeated dose exposure.