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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 Apr 2013 - 06 Dec 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern study carried out under GLP and in accordance with the appropriate guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
A number of minor deviations from the protocol are noted in the report, however none of the deviations were considered to have affected the outcome or integrity of the study.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Solid
Details on test material:
- Name of test material (as cited in study report): BEPD
- Substance type:
- Physical state: White, neutral, solid/melt product
- Analytical purity: 99.63%
- Lot/batch No.: B184/1339
- Expiration date of the lot/batch: 01 Dec 2013
- Storage condition of test material: Ambient, Dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 126-150 g for males and 100-125 g for females
- Fasting period before study: None
- Housing: 2 or 3 per cage by sex, suspended polycarbonate cages with stainless steel grid tops, solid bottoms. Sterilised white wood shavings were used as bedding
- Diet (e.g. ad libitum): Rat and Mouse (modified) No. 1 Diet SQC Expanded, ad libitum
- Water (e.g. ad libitum): ad libitum from the public water supply
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-26°C
- Humidity (%): 35-73%
- Air changes (per hr): minimum of 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From: 16 Apr 2013 To: 17 Jul 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Hydroxypropyl Methylcellulose (aqueous)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly, based on a method established at the Test Facility, at appropriate concentrations to meet dosage level requirements. The formulations were stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing and continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): None given
- Concentration in vehicle: 1.5, 15, 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis from all groups at Day 1, Week 6 and Week 12. They were tested for concentration and homogeneity. Analyses were performed by Gas Chromatography with Flame ionisation Detection using a validated analytical procedure.
Duration of treatment / exposure:
90 days
Frequency of treatment:
single daily dose
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 150, 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were agreed with the Sponsor following examination of a previous toxicity study (Huntingdon Life Sciences Study No. NTO 17/951955) in which animals received 15, 150 or 1000 BEPD/kg/day for up to 28 days. In that study a NOAEL was considered to be 1000 mg/kg/day with a NOEL considered to be 15 mg/kg/day based on macroscopic and microscopic findings in the kidneys and livers of male rats, and clinical observation of unsteady gait in the high dose during Week 4 of treatment.
- Rationale for animal assignment: random
Positive control:
Not relevant for this study type.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for Mortality/Moribundity, at frequent intervals throughout the day for signs of ill health or reaction to treatment, staring immediately post dose
- Cage side observations: Mortality/Moribundity, ill health or reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week, starting during pretrial period
- detailed clinical examination including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: once during the pretrial period and daily from the first day of dosing (Day 1) until the end of the observation period

FOOD CONSUMPTION : Yes
- The quantity of food consumed by each cage of animals was recorded once during pretrial, and weekly during the dosing 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 : Yes
- Time schedule for examinations: Regular basis throughout the study
- Water consumption was monitored by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the pretrial period and during Week 13 of treatment
- Dose groups that were examined: all animals during the pretrial period. All animals in the control and high dose group were examined during Week 13 of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Days 92 – 93 and before unscheduled euthanasia where applicable
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Red blood cell count (haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin concentration, mean cell haemoglobin, reticulcytes, reticulocyte count (absolute), red blood cell distribution width), platelets, white blood cell count (neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells). Coagulation parameters (activated partial thromboplastin time, fibrinogen, prothrombin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Days 92 – 93 and before unscheduled euthanasia where applicable
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Urea, glucose, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatise, creatine phosphokinase, lactate hydrogenase, sodium, potassium, chloride, total protein, albumin, globulin, albumin/globulin ratio, cholesterol, creatinine, total bilirubin, calcium, inorganic phosphate.

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine: Days 87 - 88
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, over a period of 16-18 hours where the animals had access to water only
- Parameters checked: Microscopic evaluation of spun deposit, colour, turbidity, specific gravity, volume, ph, protein, glucose, bilirubin, ketones, leukocytes, blood pigments, urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during pretrial and once during the treatment period (Week 12/13)
- Dose groups that were examined: all animals
- Battery of functions tested:
1- Cageside Observations:
Posture/condition on first approach (animal undisturbed), checked for: (prostration, lethargy, writhing, circling, breathing abnormalities, gait abnormalities, tremor, fasciculation, convulsions, biting (of cage components or self mutilating), vocalisations, piloerection.) Ease of removal from the cage. Body temperature. Condition of the eyes, checking for: (pupillary function, miosis, mydriasis, exophthalmos, encrustation, lacrimation.) Condition of the coat. Presence of salivation. Overall ease of handling.
2- Observations in a Standardised Arena (2 Min Observation Period):
Latency (time to first locomotory movement), Level of mobility, Rearing, Grooming, Urination/defecation, Arousal (level of alertness), Posture, tremor/convulsions, vocalisation, piloerection (as done for cageside observations), Palpebral closure, Gait abnormalities, Stereotypy (excessive repetition of behaviours) and/or unusual behaviours
3- Functional Tests
Reaction to sudden sound (click above the head in the arena), Reaction to touch on the rump with a blunt probe (in the arena), Grip strength, Pain perception, Landing foot splay, Motor activity.
4- Any other abnormality not already recorded.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- A necropsy was conducted for animals that died on study, and specified tissues were saved.
- All animals 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 following organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanised in poor condition or in extremis. Paired organs were weighed separately and reported together. Organs were weighed before fixation unless otherwise noted. Organ to body weight ratio/percentage (using the terminal body weight) and organ to brain weight ratios were calculated.
Organs weighed at necropsy: brain, epididymis, adrenal gland, pituitary gland, prostate gland, thyroid gland, heart, kidney, liver, lung, ovary, spleen, testis, thymus, uterus.

HISTOPATHOLOGY: Yes
- Representative samples of the following tissues were collected from all animals, preserved in 10% neutral buffered formalin, with the exception of testes which were fixed in Modified Davidson’s solution for up to 72 hours, then trimmed and processed to wax impregnation. All other tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin. Bone marrow smears were stained with May-Grunwald-Giemsa.
- Tissue Collected and Preserved: animal identification, aorta artery, bone marrow smear, bone marrow from femur, bone marrow from sternum, femur bone, sternum bone, brain, cervix, epididymis, eye, adrenal gland, harderian gland, lacrimal gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (caecum, colon, rectum), larynx, liver, lung, mandibular lymph node, mesenteric lymph node, skeletal muscle, nasal cavity, optic nerve, sciatic nerve, oesophagus, ovary, oviduct, pancreas, pharynx, skin, small intestine (duodenum, ileum, jejunum), spinal cord, spleen, stomach, testis, thymus, tongue, trachea, ureter, urinary bladder, uterus, vagina
Other examinations:
None
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately. Pairwise comparisons were only performed against the control group.
Body weight, food consumption, selected functional observational battery and motor activity data, haematology, coagulation, clinical chemistry and selected urinalysis data were 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; i.e. 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 non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student’s t test).
In circumstances were it was not possible to perform the F-Max test due to zero standard deviation in at least one group, the non-parametric ANOVA results were reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above.
In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data were subjected to a rank transformation prior to analysis. Where it was not possible to perform the F-Max test due to the small sample size (i.e. less than 3 animals in any group), the untransformed parametric ANCOVA results are reported.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Three males at 1000 mg/kg bw.d and one at 150 mg/kg bw/d were found dead. Signs of toxicity were also noted in this group. An additional mortality in the 150 mg/kg bw/d is not considered to be treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
Three males at 1000 mg/kg bw.d and one at 150 mg/kg bw/d were found dead. Signs of toxicity were also noted in this group. An additional mortality in the 150 mg/kg bw/d is not considered to be treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Minor changes in blood chemistry parameters were also noted where urea levels were higher in males that received 150 or 1000 mg/kg/day and total protein and globulin levels were also higher in males that received 1000 mg/kg bw/d
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
specific gravity was higher in males that received 1000 mg/kg bw/d
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Iincidences of reduced activity/subdued behavior and abnormal respiration and/or locomotion were observed in the majority of males and females that received 1000 mg/kg bw/d. Excessive salivation was also noted in all rats in this group
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased kidney weights were seen in males at ≥ 150 mg/kg bw/d and higher liver weights were observed in animals at 1000 mg/kg bw/d.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Findings in the kidneys of males included tubular dilatation at ≥150 mg/kg bw/d and nephropathy at ≥ 15 mg/kg bw/d.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Five animals were found dead or killed prematurely before the end of the dosing period. The deaths of three males at 1000 mg/kg bw/d and one male at 150 mg/kg bw/d are considered to be related to treatment. The death of one female at 150 mg/kg bw/d is not considered to be treatment-related.

- Adverse clinical observations, including reduced/subdued activity and abnormal locomotion and respiration, were observed in the majority of animals that received BEPD at 1000 mg/kg bw/d. A review of the data indicated that these signs were transient, and were observed from approximately 15 minutes post-dose to approximately 1-2 hours post-dose with the animals returning to normal (i.e. no dosing abnormalities recorded). Additionally, other signs such as piloerection, hunched body posture and or eyes partially closed were also observed in a small number of animals at this dose. Furthermore excess salivation was also noted, however, it was not considered to be an adverse reaction to treatment, rather possibly represents a behavioural response to an unpleasant-tasting test material.

BODY WEIGHT AND WEIGHT GAIN
- There were no treatment-related differences in group mean body weight or body weight gain over the duration of the treatment period following administration of BEPD at dose levels up to 1000 mg/kg bw/d.

FOOD CONSUMPTION
- There were no treatment-related differences in food consumption following administration of BEPD at dose levels up to 1000 mg/kg bw/d.

WATER CONSUMPTION
- Visual inspection of the water bottles indicated no observable differences in water consumption between groups throughout the treatment period.

OPHTHALMOSCOPIC EXAMINATION
- There were no findings during ophthalmoscopic examinations that could be attributed to BEPD.

HAEMATOLOGY
- There were no notable inter-group differences in haematological parameters following administration of BEPD at dose levels up to 1000 mg/kg bw/d.
- Minor changes were observed in several parameters, where higher platelet counts, prolonged prothrombin and activated partial thromboplastin times and higher fibrinogen levels were observed in males that received 1000 mg/kg bw/d of BEPD, achieving statistical significance. A review of the data also suggested a possible dose-related trend at the lower dose levels in these parameters, although these did not achieve statistical significance (with the exception of prolonged activated partial thromboplastin times at 150 mg/kg bw/d). A similar pattern was observed in females, however, the magnitude of the changes was to a lesser degree with statistical significance generally not observed. As the changes were minor, with no correlating histopathological changes (such as an indication of inflammatory response or stress), the findings were considered to be toxicologically insignificant.

CLINICAL CHEMISTRY
- Urea levels were statistically significantly higher in males that received 150 or 1000 mg/kg bw/d when compared with controls. Additionally, minor increases in total protein and globulin levels, achieving statistical significance, were observed in males that received 1000 mg/kg bw/d.

URINALYSIS
- Urinary pH was statistically significantly lower in both males and females that received 1000 mg/kg bw/d while a statistically significant higher specific gravity was observed in males at this dose.

NEUROBEHAVIOUR
- A difference in the general behaviour of the animals in the arena was observed in females receiving 1000 mg/kg bw/d where 3/10 animals displayed signs of piloerection and hunched or high posture. Although these observations were not observed on approach to the home cage, as these adverse clinical observations were noted in-life the minor differences were considered to be treatment-related.
- There were no treatment-related differences in the quantitative functional observation parameters following administration of BEPD at dose levels up to 1000 mg/kg bw/d.
- There were no notable inter-group differences in motor activity following administration of BEPD at dose levels up to 1000 mg/kg bw/d.
The number of overall movements of females that received 150 or 1000 mg/kg bw/d was statistically significantly lower, when compared with controls during Week 13 evaluation. This was emphasised throughout the observation period in which movements were generally lower during all time periods, attaining statistical significance on a few occasions.
Although statistical significance was achieved at 150 or 1000 mg/kg bw/d in females, a review of the pre-treatment motor activity indicated that the number of control movements was relatively high during Week 13 in comparison to their pre-treatment values. Additionally, the number of movements for treated animals was generally consistent with pre-treatment values. As there was no correlation with other indices measuring movement and the fact that the data recorded for the BEPD treated animals were consistent with pre-treatment values, it is considered that there is no evidence of a treatment related effect.

ORGAN WEIGHTS
- Higher absolute and/or relative kidney weights were observed at ≥150 mg/kg bw/d in males and correlated with microscopic findings in the kidneys of tubular dilatation and nephropathy in animals that received ≥ 150 mg/kg bw/d. Although no microscopic findings were observed in the livers of animals that received 1000 mg/kg bw/d, the higher absolute and relative liver weight suggests a relation to treatment which was considered to be an adaptive response and of no toxicological significance.
- No other test item-related organ weight changes were noted.

GROSS PATHOLOGY
No test item-related gross findings were noted.

HISTOPATHOLOGY: NON-NEOPLASTIC
BEPD-related findings in the kidneys of males included tubular dilatation at ≥ 150 mg/kg bw/d and nephropathy at ≥ 15 mg/kg bw/d. The tubular dilatation was predominately at the corticomedullary junction. The epithelium lining the effected tubules was attenuated and the lumen contained an accumulation of eosinophilic cellular debris. The nephropathy, affecting cortical tubules, was characterized by multifocal tubules lined by basophilic epithelium with thickened basement membranes and variable infiltrates of mononuclear inflammatory cells.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Microscopic findings of tubular dilatation and nephropathy in the kidneys with correlating organ weight changes at 150 mg/kg bw/d and above in males.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Adverse clinical observations noted in females at 1000 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

 

M

F

0

15

150

1000

0

15

150

1000

Mortality (#)

-

-

1

3

-

-

1#

-

Clinical signs

-

-

-

ü

-

-

-

ü

Urea (mM)

5.4

5.6

6.0*

6.1**

5.6

5.9

5.9

5.2

Total protein (g/L)

62

61

64

66**

65

65

65

66

Globulin (g/L)

22

21

23

25**

19

19

20

21*

Calcium (mM)

2.69

2.64

2.70

2.79***

2.67

2.72

2.69

2.76**

Urine SG

1.011

1.017

1.015

1.028***

1.020

1.020

1.018

1.029

Urine pH

72

7.2

6.9

5.9***

6.6

6.4

6.3

5.9***

Kidney weight (g)

2.47

2.48

2.58

2.92**

1.57

1.63

1.62

1.67

Kidney weight (relative)

0.608

0.616

0.676*

0.701**

0.682

0.665

0.686

0.710

Liver weight (g)

14.28

13.73

13.96

19.11***

7.78

8.64*

8.20

10.19***

Liver weight (relative)

3.512

3.391

3.615

4.566***

3.374

3.520

3.466

4.340***

Nephropathy (minimal)

0

3

6

7

0

-

-

0

Tubular dilation minimal

0

0

2

1

0

-

-

0

Tubular dilation mild

0

0

5

4

0

-

-

0

*signficiantly different to controls (p<0.05); **p<0.01; ***p<0.001

#not related to treatment

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the NOAEL was considered to be 15 mg/kg bw/y for males and 150 mg/kg bw/d for females. This conclusion was based on the adverse clinical observations noted in both males and females at 1000 mg/kg/bwd and also the microscopic findings of tubular dilatation and nephropathy in the kidneys with correlating organ weight changes at 150 mg/kg bw/d and above in males.
Executive summary:

A study was conducted to determine the potential toxicity of BEPD when given orally by gavage for 90 days to rats. Ten rats per sex per dose were administered BEPD at dose levels of 0, 15, 150, or 1000 mg/kg bw/d.

The following were assessed at predetermined intervals from pre-trial until study completion for all animals: viability, clinical observations, body weights, food consumption, a visual assessment of water consumption and ophthalmoscopy examinations. All animals also received a detailed functional observation battery (including motor activity) during pre-trial and Week 12/13 of treatment. Blood samples for haematology, coagulation and blood chemistry investigations and urine samples were collected from all animals during Week 13 of treatment. All surviving animals were terminated after completion of at least 91 days of treatment and underwent a detailed necropsy examination with selected organs weighed. Tissues from all control and high dose animals were subjected to a comprehensive histological examination, with gross lesions (where appropriate) examined from low and intermediate dose animals. Additionally, the kidneys were evaluated from low and intermediate dose males.

There were five unscheduled deaths: a 150 mg/kg bw/d male was found dead, a 150 mg/kg bw/d female was euthanized moribund due to not properly using the right hind limb, and three 1000 mg/kg bw/d males were found dead. No test item-related gross findings were noted. In the four males found dead, kidney findings of minimal nephropathy and mild tubular dilatation was observed. Adverse clinical observations including incidences of reduced activity/subdued behaviour and abnormal respiration and/or locomotion were observed in the majority of males and females that received 1000 mg/kg bw/d. Excessive salivation was also noted in all animals that received 1000 mg/kg bw/d.

There was no effect on body weight gains or food consumption profiles during the treatment period. There were no ophthalmoscopy findings noted.

Minor changes in blood chemistry parameters were also noted where urea levels were higher in males that received 150 or 1000 mg/kg bw/d and total protein and globulin levels were also higher in males that received 1000 mg/kg bw/d. For urinalysis parameters, specific gravity was higher in males that received 1000 mg/kg bw/d.

There were no BEPD-related macroscopic observations at terminal euthanasia. BEPD-related increased kidney weights were seen in males that received ≥ 150 mg/kg bw/d and higher liver weights were observed in animals that received 1000 mg/kg bw/d. Following histological evaluation, BEPD-related findings in the kidneys of males included tubular dilatation at ≥ 150 mg/kg bw/d and nephropathy at ≥ 15 mg/kg bw/d.

The NOAEL was considered to be 15 mg/kg bw/d for males and 150 mg/kg bw/d for females. This conclusion was based on the adverse clinical observations noted in both males and females at 1000 mg/kg bw/d and also the microscopic findings of tubular dilatation and nephropathy in the kidneys with correlating organ weight changes at 150 mg/kg bw/d and above in males and the unscheduled deaths of males at 150 mg/kg bw/d and above.