Ammonium perrhenate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Jul 2012 to 12 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A good quality guideline study, carried out to GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Only minor deviations, for example in temperature and humidity which were not determined to have any impact on the integrity of the study or results.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium perrhenate

EC Number:

237-075-6

EC Name:

Ammonium perrhenate

Cas Number:

13598-65-7

Molecular formula:

H4N.O4Re

IUPAC Name:

Ammonium oxido(trioxo)rhenium

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Ammonium perrhenate (also known as Ammonium tetraoxorhenate (VII))
- Substance type: Technical product
- Physical state: White solid
- Analytical purity: 99.95%
- Purity test date: 5 Apr 2012
- Lot/batch No.: 11411
- Expiration date of the lot/batch: 31 Oct 2012
- Storage condition of test material: Ambient / Dark
- Other: Supplied by Heraeus Precious Metals GmbH & Co.KG

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 Limited, Margate, Kent, UK
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 305-381 g (males); 224-282 g (females)
- Housing: Polycarbonate cages with stainless steel grid tops and solid bottoms, approximately 61 x 43.5 x 24 cm. White paper tissue was supplied to females as nesting material from Day 20 of gestation
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded), supplied by Special Diets Services Limited, Witham, Essex, UK provided ad libitum
- Water (e.g. ad libitum): Public water supply (Scottish Water, Edinburgh, UK) was available ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 (daily monitoring indicated that temperature went above the target range on 1 occasion. Temp reached a max of 24 deg C for about 1.5 hr. This was not considered to have affected the outcome or integrity of the study).
- Humidity (%): 40-70 (humidity was above target ranges on several occasions - actual range 48-77%. Daily target average humidity was within the target range and deviations were short in duration. They were considered not to have had any impact on the outcome or integrity of the study).
- Air changes (per hr): minimum 10
- Photoperiod (hrs dark / hrs light): 12 hours light / dark

IN-LIFE DATES: Dosing was initiated on 09 Jul 2012. In-life phase of the study was completed on 24 Aug 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 40% PEG400 in Milli-Q water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4 deg C, and dispensed daily. They were prepared by adding an appropriate amount of vehicle to the required amount of test item and were mixed by magnetic stirring until a visible clear solution was obtained. The dosing formulations were removed from the refrgerator and stirred for at least 30 mins prior to dosing and continuously during dosing.

Homogeneity and stability were previously established by Charles River (Study No. 428793).

All formulations were used within an established 8 day stability period.

VEHICLE
- Concentration in vehicle: 0, 11, 33, 100 mg/ml (0, 110, 330, 1000 mg/kg bw/day dose levels, respectively)
- Amount of vehicle (if gavage): Dose volume 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate dosing formulations were sampled for analysis at 1 and 4 weeks. Analyses were performed by ICP-Optical Emmission Spectroscopy using a validated analytical procedure. Results of the sample concentrations were considered acceptable as they were all within +/-10% of the theoretical concentrations.

Duration of treatment / exposure:

Males were dosed for 4 weeks, starting 2 weeks prior to mating. Females were dosed 2 weeks prior to mating, throughout mating and through to at least day 4 of lactation.

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
110, 330, 1000 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The study was designed to provide information to allow the test item to be used safely as well as to assist in the classification for STOT-RE (Specific Target Organ Toxicity - Repeated Exposure) under CLP (Classification, Labelling and Packaging).

Dose levels were agreed after review of existing relevant toxicological data including a 7 day dose range finding study (Charles River Study No. 495676) where dose levels up to 1000 mg/kg bw/day produced no adverse reaction to treatment.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once during the pre-treatment period and weekly thereafter.
- Cage side observations included: posture/condition (including e.g. prostration, lethargy, writhing, circling, breathing abnormalities, gait abnormalities, tremor, fasciculation, convulsions, biting of cage components or self mutilating, vocalisations, piloerection); ease of removal from cage; body temperature; condition of eyes and coat; presence of salivation, overall ease of handling.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded one week prior to the start of treatment. From the start of treatment, individual body weights were recorded daily.

FOOD CONSUMPTION:
- Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating. After pairing, the female food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-4 of lactation. Male food consumption did not recommence after pairing for mating.

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretrial (Week -1) - all animals; Week 4 - all males; Shortly prior to sacrifice - all females

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 for males; on or after Day 4 of lactation for females
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: first 5 animals/group (males); first 5 animals in each group to rear their litter to Day 3 of lactation
- Parameters examined: red blood cell count; haemoglobin concentration; haematocrit; mean corpuscular volume; red blood cell distribution width; mean corpuscular haemoglobin concentration; mean corpuscular haemoglobin; reticulocyte count (absolute); platelet count; white blood cell count; neutrophil count (absolute); lymphocyte count (absolute); monocyte count (absolute); eosinophil count (absolute); basophil count (absolute); large unclassified cells.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 for males; on or after Day 4 of lactation for females
- Animals fasted: No
- How many animals: first 5 animals/group (males); first 5 animals in each group to rear their litter to Day 3 of lactation
- Parameters examined: urea; glucose; aspartate aminotransferase; alanine aminotransferase; alkaline phosphatase; creatine phosphokinase; lactate dehydrogenase; sodium; potassium; chloride; total protein; albumin; globulin; albumin/globulin ratio; cholesterol; creatinine; total bilirubin; calcium; phosphate.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 5 males per group during Week 4; 5 females per group during lactation
- Battery of functions tested: sensory activity / grip strength / motor activity / other: landing foot splay

Sacrifice and pathology:

The males were killed when mating was completed and the animals had been dosed for at least 4 weeks. The females and litters were killed between Day 5 and 7 of lactation.

Premature decedents were necropsied with a view to diagnosis of the cause of the animal’s condition or cause of death. An external examination was followed by inspection of the cranial, thoracic and abdominal contents. Representative samples of abnormal tissues, together with any other tissues considered appropriate, were fixed in neutral buffered 10% formalin. The reproductive tracts of females were examined for signs of implantation and the number of implantation sites recorded.

All adult animals surviving to scheduled euthanasia were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system: all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded and the numbers of corpora lutea graviditatis on the ovaries were counted.

The following organs were weighed at necropsy for all adult animals: brain; epididymis; adrenal gland; pituitary gland; prostate gland; thyroid gland; heart; kidney; liver; lung; ovary; spleen; testis; thymus; uterus.

Histopathological evaluation of all tissues indicated in Table 1, below, was undertaken for 5 males and 5 females in the Control and High dose groups (the same animals that were used for laboratory investigations).

Statistics:

Unless otherwise stated, statistical tests were two-sided and performed at the 5% significance level. Pairwise comparisons were only performed against the control group. Body weight, food consumption, haematology, coagulation, clinical chemistry and selected FOB and motor activity data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis nonparametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test). Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.

Incidence data was analysed as proportions in a Kruskal-Wallis analysis, or by categorical methods using contingency tables with the Fisher’s Exact Probability test or the Chi-squared test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two animals were prematurely killed in the top dose group of 1000 mg/kg bw/day and considered to be treatment-related. At 1000 mg/kg bw/day there was an increase in the incidence and severity of clinical observations in both males and females, when compared to controls. At 330 mg/kg bw/day there was a slight increase in ploughing in both males and females and excess salivation in males only. No treatment-related clinical observations were noted at 110 mg/kg bw/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two animals were prematurely killed in the top dose group of 1000 mg/kg bw/day and considered to be treatment-related. At 1000 mg/kg bw/day there was an increase in the incidence and severity of clinical observations in both males and females, when compared to controls. At 330 mg/kg bw/day there was a slight increase in ploughing in both males and females and excess salivation in males only. No treatment-related clinical observations were noted at 110 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males at 1000 mg/kg bw/day, had a statistically significant decrease in mean body weight gain from week 1. At 330 mg/kg bw/day, a slight reduction could not be positively associated with treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced food consumption at 1000 mg/kg bw/day in males and females. At 330 mg/kg bw/day, food consumption was similar to controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Effect on circulating red blood cell parameters in males and females at 1000 mg/kg bw/day. Also at this does, slight differences in some white blood cell parameters in males, compared to controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Some clinical chemistry changes in males and females at 1000 mg/kg bw/day. Slight changes were also seen in 2/5 males at 330 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, there was an increase in incidence of several neurotoxicity clinical observations. Detailed functional observations and motor activity were within normal parameters, however.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 330 mg/kg bw/day and above, increased thyroid weight in males and females. Several additional organ weight changes were observed at 1000 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged thyroid glands and mandibular lymph nodes at 1000 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment at 1000 mg/kg bw/day in both sexes was associated with effects on the thyroid, bone marrow and spleen. Males at this dose also had effects on the pituitary gland.

Details on results:

CLINICAL SIGNS AND MORTALITY
Two animals were prematurely killed in the top dose group of 1000 mg/kg bw/day and considered to be treatment-related, with cause of death in both due to widespread haemorrhage and congestion.

At 1000 mg/kg bw/day there was an increase in the incidence and severity of clinical observations in both males and females, when compared to controls. Dose related signs included ploughing, body hunched, piloerection, rolling gait, walking on tip toes, redness of the ears/extremities, swelling of the ears/feet/muzzle/ventral neck and abdomen, excess salivation, pale ears/extremities, excessive chewing of cage parts/shavings (females only),
eyes partially closed (males only) and irregular respiration. The observed redness of the ears/extremities and the swelling of the ears/feet/muzzle/ventral neck and abdomen, was evident in most high-dose animals from ca Study Day 10/11 onwards. The redness of the ears/extremities became more transient as dosing continued with most animals ceasing to show these signs beyond Study Day 19. A similar pattern of recoverability was apparent with the signs of swelling in the females; however the signs of swelling remained evident in the majority of males until necropsy. At 330 mg/kg bw/day there was a slight increase in ploughing in both males and females and excess salivation in males only. No treatment-related clinical observations were noted at 110 mg/kg bw/day.

BODY WEIGHT AND WEIGHT GAIN
In males at 1000 mg/kg bw/day, a statistically significant decrease in mean body weight gain was observed from Week 1 onwards, when compared to Controls. At 330 mg/kg bw/day in males there was a slight reduction in mean body weight gain, when compared to Controls. The group mean body weight gain at 330 mg/kg bw/day was 122g over Days 0-28 compared to 130g in Controls, which is a 6% reduction in body weight gain compared to Controls. This did not achieve statistical significance and the group mean value has been slightly skewed by the weights of 2 outlier males. Both these animals started to show a reduced body weight gain from ca Week 1/2 of the study onwards and when these two animals are removed from the group mean body weight gain, the group mean gain from Day 0-28 is 129g compared to 130g in Controls. As the reduction in body weight gain of these two animals is slight and due to all other males at this level having weight gains comparable to Control, this reduced body weight gain could not be positively associated with treatment. At 110 mg/kg bw/day the group mean body weight and the group mean body weight gain in males were similar to Controls.

In all treated groups, group mean body weight gain of females prior to mating was similar to Control. In females at 1000 mg/kg bw/day, a reduction in group mean body weight gain was observed over Gestation Days 0-20 and with increased severity during Lactation Days 0-4, when compared to Controls. At 1000 mg/kg bw/day group mean body weight gain over Day 0-20 of gestation was 13% lower than Controls and over Days 1-4 of lactation was 59% lower than Controls.

FOOD CONSUMPTION
During the first two weeks of dosing, males dosed at 1000 mg/kg bw/day had a statistically significant reduction in group mean food consumption, when compared to Controls. Prior to mating, females at 1000 mg/kg bw/day had a very slight reduction in group mean food consumption compared to Controls. During the 2 weeks prior to mating, high-dose females ate an average of 5% less food than Control animals. This did not achieve statistical significance and the change was considered minor. At 1000 mg/kg bw/day in females during gestation and lactation, there was a notable reduction in group mean food consumption when compared to Controls. Over Day 0-20 of gestation high-dose females ate an average of 15% less food than Controls, rising to 19% reduction in food consumption over Day 0-4 of lactation, when compared to Controls. At levels up to 330 mg/kg bw/day food consumption performance in both sexes was similar to Control.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopy findings which were considered to be related to treatment with Ammonium Perrhenate. All findings were considered to be typical of rats of the age and strain used.

HAEMATOLOGY
At 1000 mg/kg bw/day there was an effect on circulating red blood cell parameters in males and females. Decreases were observed in haemoglobin, red blood cell count and haematocrit along with increases in mean cell haemoglobin and mean cell volume. These effects were particularly evident in one male and two female animals. For these three animals, an increase in red blood cell distribution width was also recorded; however, the reticulocyte count was increased for the male but decreased in the two females. One female also had an increase in red blood cell distribution width when compared to Controls; however the reticulocyte count in this animal was similar to Controls. At 1000 mg/kg bw/day there were also some slight differences in some white blood cell parameters in males, when compared to Controls. Increases were noted in large unclassified cells along with decreases in eosinophils. The differences from Control were slight and all other white blood cell parameters were similar to Controls. These differences were considered incidental and too small to be positively related to treatment. In both sexes at 1000 mg/kg bw/day there was a slight increase in prothrombin time. However due to the increase being so slight and a lack of any changes in all other coagulation parameters this difference cannot be positively attributed to treatment with Ammonium Perrhenate. All other haematology and coagulation parameters tested were similar to Controls in all dose groups for both males and females.

CLINICAL CHEMISTRY
At 1000 mg/kg bw/day in both sexes an increase in aspartate aminotransferase, glucose and a slight increase in total protein were observed in a proportion of animals, when compared to Controls. A decrease in lactate dehydrogenase and creatine phosphokinase was also evident in one male and two females, when compared to Controls. Treatment at 1000 mg/kg bw/day was also associated with a slight increase in urea in the males and a slight decrease in urea in the females. Although the female values were slightly lower than Controls, they are still within the normal range of this species and strain. In females the difference did not achieve statistical significance and so this apparent reduction was considered to be due to normal variation and considered not to be related to treatment. The increase in the males did achieve statistical significance and a dose related increase was also observed at 330 mg/kg bw/day indicating that these differences in Urea in males were most likely associated with treatment. Treatment in males at 330 mg/kg bw/day was associated with a slight increase in aspartate aminotransferase, urea and glucose, when compared to Controls. These increases were only evident in 2/5 males for each parameter, the remaining 3/5 males had values similar to Controls. All other clinical chemistry parameters tested were comparable to controls in all dose groups for both males and females and any minor variations were considered too small to be related to treatment.

NEUROBEHAVIOUR
At 1000 mg/kg bw/day an increase in incidence of several neurotoxicity clinical observations was observed in a number of animals in both sexes from Week 2 onwards. Abnormal findings with an increased incidence include abnormalities in posture/gait (high/rolling/hunched/splayed hind limbs), partial palpebral closure, irregular respiration and piloerection. These increases in abnormal findings were only evident during the arena observations and are consistent with the clinical observations. During Week 5 there was an increase in females with piloerection, in all dose groups including Controls. This was the week prior to the females giving birth so is most likely due to the animals nearing their day of parturition. For both sexes, the incidence of all other neurotoxicity clinical observations remained comparable to controls in all dose groups for the duration of the study.

In all dose groups, all the detailed functional observations in males and females were comparable to Controls for the duration of the study. Any slight intergroup differences were considered too small to be attributed to treatment.

Slight inter-group differences in motor activity were intermittent and considered incidental, therefore could not be associated with treatment with Ammonium Perrhenate.

ORGAN WEIGHTS
At 330 mg/kg bw/day and above, a dose related increase in thyroid weight was observed for males, when compared to Controls. Group mean thyroid weight increased by 47% at 330 mg/kg bw/day and 86% at 1000 mg/kg bw/day, compared to the control group mean thyroid weight. At 110 mg/kg bw/day there was an apparent slight increase in group mean thyroid weight when compared to Control. However when looking at the individual data only one male has a thyroid weight above the weight range observed in the Control animals. As all other males in this group had a thyroid weight similar to Control, the slight increase in thyroid weight in this one animal could not be positively attributed to treatment. At 330 mg/kg bw/day and above, a dose related increase in thyroid weight was observed for females, when compared to Controls. Group mean thyroid weight increased by 58% at 330 mg/kg bw/day and 88% at 1000 mg/kg bw/day, compared to the control group mean thyroid weight. Administration of Ammonium Perrhenate at 1000 mg/kg bw/day was associated with the following additional organ weight differences in males: thymus (decrease), lung (decrease), heart (decrease), kidney (decrease) and spleen (increase). No other test item-related organ weight changes were noted. There were other isolated organ weight values that were different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, other organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of Ammonium Perrhenate.

HISTOPATHOLOGY
Treatment at 1000 mg/kg bw/day in both sexes was associated with follicular cell hypertrophy in the thyroid, decreased cellularity and congestion/haemorrhaging of the bone marrow (femur, sternum and rib) and increased cellularity and macrophages in the red pulp of the spleen. Treatment at 1000 mg/kg bw/day was also associated with pituicyte hypertrophy of the pituitary in males only. The follicular cell hypertrophy in the thyroid was considered related to higher thyroid weights in high-dose animals. The increase in macrophages in the red pulp was considered to be related to the higher spleen weights observed in males. There were no histology findings that could be related to the lower weights observed in the thymus, heart, lungs and kidneys of the high-dose males. There were two females at 1000 mg/kg bw/day with atrophy of the thymus compared to 1 in Controls. The stresses endured during parturition can cause effects on the thymus and the lack of supporting organ weight effects suggest that this slight increase in incidence of thymus atrophy cannot be positively related to treatment. Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of Ammonium Perrhenate.

Effect levels

Dose descriptor:

NOAEL

Effect level:

110 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

A no-observed-adverse-effect level (NOAEL) of 110 mg/kg bw/day was found in a repeated dose toxicity study combined with a reproduction/developmental toxicity screening test in male and female rats given ammonium perrhenate by oral gavage. Males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (approximately 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least day 4 of lactation (approximately 6 weeks of treatment).

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD TG422) was conducted with ammonium perrhenate by oral gavage in rats.

Sprague-Dawley rats were randomised into 3 test groups (given 110, 330 or 1000 mg/kg bw/day) and one Control group, each containing 10 males and 10 females. The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca 6 weeks of treatment). The following parameters and end points were evaluated: clinical signs, body weights, body weight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathological examinations, mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights).

Treatment with Ammonium Perrhenate at 1000 mg/kg bw/day was associated with an increase in the incidence and severity of clinical observations along with a decrease in group mean body weight gain and food consumption performance (during gestation and lactation only for females). Treatment at this level also resulted in the premature death of 2 animals and effects on several clinical chemistry parameters and circulating red blood cell parameters. At necropsy an increase in the incidence of enlarged thyroid glands and mandibular lymph nodes was noted along with several organ weight effects; thyroid (increase), thymus (decrease in males), heart (decrease in males), lung (decrease in males) kidney (decrease in males) and spleen (increase in males). Microscopic findings were noted in the thyroid (follicular cell hypertrophy), pituitary gland of males (pituicyte hypertrophy), bone marrow (decreased cellularity and congestion/haemorrhage) and the spleen (increased cellularity, macrophages in the red pulp).

Treatment at 330 mg/kg bw/day was associated with an increase in thyroid weight in both sexes. A slight increased incidence of ploughing and excess salivation (males only) was also noted at this dose level.

Treatment at 110 mg/kg bw/day revealed no significant changes in any of the parameters assessed that were considered to be indicative of a reaction to treatment. Overall, under the conditions of this study, a No-Observed-Adverse-Effect Level (NOAEL) was considered to be 110 mg/kg bw/day for both males and females.