Ammonium bromide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium bromide

EC Number:

235-183-8

EC Name:

Ammonium bromide

Cas Number:

12124-97-9

Molecular formula:

BrH4N

IUPAC Name:

Bromide activated chloramine (BAC) generated from ammonium bromide and sodium hypochlorite

Details on test material:

- Name of test material (as cited in study report): Ammonium bromide
- Physical state: White crystalline solid
- Analytical purity: 99.91%
- Lot/batch No.: 980060
- Stability under test conditions: Not determined; considered to be stable under the storage conditions
- Storage condition of test material: The material was stored in the dark at ambient temperature

Test animals

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
- 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

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

IN-LIFE DATES: From: 1999-12-10 To: 2000-09-08

Administration / exposure

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). 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.

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.

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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
225 mg/kg bw/day
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/kg bw/day (males)
Basis:
nominal in diet

Doses / concentrations 4

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

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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: Time points: during Week 12, and additionally for control and high dose rats during Week 17 (end of recovery)
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes / No / No data
- How many animals: All animals
- Parameters examined:

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: Yes / No / No data
- How many animals: All animals
- Parameters examined:

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);
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes
- Parameters examined:

NEUROBEHAVIOURAL EXAMINATION: Yes
Detailed functional observations were performed during pretrial and in Weeks 4, 8, 13 and 17. 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), 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.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

On completion of 13 weeks of treatment 10 males and 10 females per group were killed and underwent detailed necropsy. All control and high dose animals also underwent a full histological examination. A further 5 males and 5 females per group were killed by perfusion fixation (injection of 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) for neuropathological examination, with nervous tissues and organs from all perfused animals being examined histologically.
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

Other examinations:

None

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.

Results and discussion

Results of examinations

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.

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.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

haematology

urinalysis

2

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.

3

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

See Result Tables in attached document.

Applicant's summary and conclusion

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.