Registration Dossier

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 2018 to TBC

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar HanTM:RccHanTM:WIST strain

Details on species / strain selection:

Based on guideline requirement

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS - Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes 
- Date of birth: Not specified 
- Age at study initiation: male 11 weeks and female 12 weeks 
- Weight at study initiation: Male 284 to 354g and female 193 to 225g
- If immature animals, whether or not supplied with dam or foster dam and date of weaning: N/A 
- Fasting period before study: No 
- Housing: Groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Free access to food, a pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used.
- Water (e.g. ad libitum): Free access to mains drinking water supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 20 days

  DETAILS OF FOOD AND WATER QUALITY  
ENVIRONMENTAL CONDITIONS 
- Temperature (°C):22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness
 IN-LIFE DATES: From: To: Not indicated

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. The volume of test and control item administered to each animal were based on the most recent scheduled body weight and adjusted accordingly.

Four dose groups (control, low, intermediate and high) each comprising 24 animals (12 male and 12 female) for toxicology evaluation and one positive control group comprising of 3 male animals for comet assay evaluation were allocated to test groups

Groups 1 to 4: Dose levels of 0 (Control), 100, 350 and 750 mg/kg bw/day were selected, in collaboration with the sponsor, based on available toxicity data, including preliminary results obtained from the dose range finder study (Envigo Study number VS54SJ). In the preliminary study, a dosage of 1000 mg/kg bw/day was demonstrated to be too high for long term investigation of toxicity, but a dosage of 750 mg/kg bw/day was considered likely to be suitable. At higher dosages in the preliminary study, less toxicity appeared to be observed when the dose volume was increased from the standard 4 mL/kg to 6mL/kg, probably due to the physical nature of the dosing formulation. A dose volume of to 6mL/kg was therefore employed on this study.

Group 5: Positive control animals were untreated until Day 44 of the study when they were dosed with N-Nitroso-N-methylurea on Days 44 and 45. The volume of reference item administered to each animal was based on the scheduled body weight on Day 44.

Control animals will be treated with the vehicle (Polyethylene glycol 400) alone over the same treatment period and at the same dose volume as that of test animals.

Vehicle:

other: Polyethylene glycol 400 (PEG 400)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:  The test item was prepared at the appropriate concentrations in Polyethylene glycol 400 (PEG 400). Formulations were therefore prepared in batches of up to two weeks and made up to three days in advance of first use and stored at approximately 4°C in the dark.
  VEHICLE   - Justification for use and choice of vehicle (if other than water): The substance was miscible in Polyethylene glycol 400 grade
- Concentration in vehicle: 99-104% of the nominal concentration
- Amount of vehicle (if gavage): Dose Volume of 6mL/kg
- Lot/batch no. (if required): 1729820 
- Purity: Not stated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.

The homogeneity and stability was confirmed for the test item in PEG 400 formulations at nominal concentration of 3.75 mg/mL and 250 mg/mL when stored refrigerated for 18 days.

The mean concentrations of the test item formulations analysed for the study were within +/- 10% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

6 - 8 weeks

Frequency of treatment:

Daily

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

other: Positive control

Details on study design:

i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
iii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iv. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
v. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
vi. On completion of the pairing phase, five selected males per dose group were evaluated for functional/sensory responses to various stimuli during Week 6.
vii. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance, visible nipple counts (male offspring) and clinical signs were also recorded during this period.
viii. On Day 4 post partum, where possible, blood sampling was performed on two randomly allocated offspring from each litter in order to obtain serum samples.
ix. At Day 12 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli
x. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 43. The male dose groups were killed and examined macroscopically on Day 44 or 45.
xi. On Day 13 post partum, where possible, blood sampling to produce serum samples for assessment of thyroid hormones was performed on two randomly selected offspring (one male and one female) per litter. Where possible, a further two randomly selected offspring (one male and one female) per litter were sampled to produce surther serum samples as a contingency against the need to measure TSH. Thyroid/parathyroid samples were also retained from one male and one female from each litter where litter sizes allowed. All surviving offspring were killed and examined externally; where external observations were detected an internal necropsy was performed.
xii.Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 13 post partum. All surviving females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. In addition, blood samples to produce both serum and plasma were taken from all adult animals at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
xiii.Selected tissues were taken from five males from Groups 2 to 4 and all 3 positive control males (Group 5) for subsequent processed as part of the comet assay assessment.

Positive control:

Three males were dosed with N- Nitroso-N-methylurea (vehicle: distilled water) for two consecutive days to act as a positive control group for the comet assay investigation.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study - Cage side observations checked in table [No.?] were included. Yes
  DETAILED CLINICAL OBSERVATIONS: Yes
 - Time schedule: Group 1 - 4: 30 mins post dosing & an hour after dosing. Comet male, immediately before dosing and up to 30 mins post dosing.  Group 5: Daily but formal recording began on day 44 when observations were made immediately before dosing and up to 30 mins post dosing and day 45 only immediately before dosing and up to 30 mins post dosing.

BODY WEIGHT: Yes  - Time schedule for examinations:
Groups 1 to 4: Individual body weights were recorded for males on Day -5 (Day of allocation), Day 1 and then weekly until termination. Individual body weights were also recorded at terminal kill.
For females, individual body weights were recorded on Day -5 (Day of allocation), Day 1 and then weekly until pairing. During the pairing phase, females were weighed daily until mating was confirmed. Mated females were weighed on Day 0, 7, 14 and 20 post coitum and body weights for females which give birth were recorded on Days 1, 4, 7 and 14 post partum.
Group 5:
During the pre-treatment period, individual body weights will be recorded on a weekly basis to coincide with the scheduled weighing of Group 1 to 4 animals. Individual body weights were recorded on Day 44 (prior to start of dosing) and were also recorded on the day of termination (Day 45).
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes - Time schedule for examinations:
Group 1 to 4: During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females during lactation, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14.
Group 5: No formal measurement of food consumption was performed for these animals.

FOOD EFFICIENCY: Yes - Time schedule for examinations:
Group 1 to 4: Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

 WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes  - Time schedule for examinations: Daily  

OPHTHALMOSCOPIC EXAMINATION: No
 - Time schedule for examinations: 
- Dose groups that were examined:  

HAEMATOLOGY: Yes  
- Time schedule for collection of blood: Male day 43 & female Day 13 Post partum 
- Anaesthetic used for blood collection: No 
- Animals fasted: No - How many animals: Five male and five female from groups 1 -4 
- Parameters checked in table [No.?] were examined. Yes  

CLINICAL CHEMISTRY: Yes 
- Time schedule for collection of blood:Male day 43 & female Day 13 Post partum
- Animals fasted: No 
- How many animals:Five male and five female from groups 1 -4
- Parameters checked in table [No.?] were examined. Yes  

URINALYSIS: No 
- Time schedule for collection of urine:
 - Metabolism cages used for collection of urine:  
- Animals fasted:  
- Parameters checked in table [No.?] were examined.  

NEUROBEHAVIOURAL EXAMINATION: Yes  
- Time schedule for examinations: Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

- Dose groups that were examined:Groups 1 - 4
- Battery of functions tested: sensory activity / grip strength / motor activity /: All
other: Behaviourals ( Gait, Tremors, Twitches,Convulsions Bizarre/Abnormal/Stereotypic behavior, Salivation ,Pilo-erection ,Exophthalmia Lachrymation,
Hyper/Hypothermia Skin color Respiration Palpebral closure Urination Defecation, Transfer arousal & Tail elevation.

IMMUNOLOGY: No 
- Time schedule for examinations:
 - How many animals:
 - Dose groups that were examined:
 - Parameters checked in table [No.?] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
 HISTOPATHOLOGY: Yes

Other examinations:

Thyroid Hormone Analysis:
Groups 1 to 4
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60°C. Samples were taken as follows:
Where possible serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
Where possible, serum samples for T4 analysis were taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. Where possible, further serum samples as a contingency against any need to measure TSH were also taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.
Serum samples were taken from all adult males and females at termination.
All serum samples, excluding those taken as a contingency against the need to measure TSH, were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator (Salman Alam). A complete Thyroid Hormone Analysis report is presented in Annex 3. All serum samples taken as a contingency against the need to measure TSH, were retained at the Test Facility.

Statistics:

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module.
The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. None dose response with data showing non- homogeneity of means, a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data not analyzed by the Provantis data capture system, R Environment for Statistical Computing was used. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal- Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression. Where the data were unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p):
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one unscheduled adult death (Female 94)on day 50, occurring at a dosage of 750 mg/kg bw/day. The animal had shown hunched posture, pallor of the extremities, apparent hypothermia and piloerection on the previous day. This female had littered but had shown total litter on Day 1 post partum. Necropsy examination revealed enlarged liver, spleen and right adrenal, a pale area on the liver and thin appearance of the non-glandular region of the stomach and raised limiting ridge. Additionally, a pale mass was observed in the right ventricle of the heart during tissue processing. At histopathology examination, the main changes observed were abscessation in the lungs and marked inflammatory change in the heart with the presence of bacterial colonies. It is considered likely that sepsis, as a result of complications of pregnancy, was the cause of death. This isolated atypical death was, therefore, considered to be incidental and unrelated to treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on food consumption of males throughout the study or of females during the pre-pairing or gestation phases at 100, 350 or 750 mg/kg bw/day.
At all dosages, food consumption was lower than control from Day 4 of lactation and whilst group mean values showed no dosage relationship, the difference from control was most notable at 750 mg/kg bw/day during the second week of lactation.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For males at 750 mg/kg bw/day, mean values for mean corpuscular hemoglobin and mean corpuscular volume were statistically significantly lower than control. For mean corpuscular volume, 3/5 individual values for these treated animals were below the historical control range but all individual values for mean corpuscular hemoglobin were within the historical control range. These findings, in isolation, were considered most likely to be incidental and, in the absence of any supporting histopathology, were insufficient to be regarded as an adverse effect.
For males at 350 or 750 mg/kg bw/day, mean reticulocyte counts were statistically significantly higher than control but values showed no dosage relationship. All individual values at 350 mg/kg bw/day were within the historical control range but 2/5 values at 750 mg/kg bw/day were below this historical range. However, all individual values for control animals were also below the historical range and it is considered that the observed differences in mean values were due to atypical low values for the control group rather than any effect of treatment.
For males at 350 or 750 mg/kg bw/day, mean total leucocyte count were statistically significantly higher than control principally due to statistically significant higher mean number of lymphocytes. For total leucocyte count, 2/5 values at 350 mg/kg bw/day and 3/5 values at 750 mg/kg bw/day exceeded the historical control range and for lymphocytes count, 2/5 values at each dosage exceeded the historical control range. In the absence of any supporting histopathology, this finding was considered to be of little toxicological significance and insufficient to represent an adverse effect.
For females at 750 mg/kg bw/day, the mean value for mean corpuscular hemoglobin was statistically significantly lower than control. All individual values for these treated females were within the historical control range whist 2/5 control exceeded this historical range. This finding, in the absence of any supporting histopathology, was therefore considered to be incidental and unrelated to treatment.
For females at 350 or 750 mg/kg bw/day, mean total leucocyte count were statistically significantly higher than control principally due to statistically significant higher mean number of lymphocytes. All individual values for these treated females were within the historical control range and this finding, in the absence of any supporting histopathology, was considered to be of little toxicological significance and insufficient to represent an adverse effect.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The mean levels of thyroxine (T4) in adult males were statistically significant higher then control at 100, 350 and 750 mg/kg bw/day, although mean values showed no dosage-relationship at the lower dosage levels. These differences from control for T4 levels in males occurred in the absence of any effect on organ weight or evidence of histopathological change for the male thyroid and, therefore, this finding was considered to be incidental and unrelated to treatment.
For females at 350 or 750 mg/kg bw/day, mean total protein levels, albumin levels and albumin/globulin (A/G) ratio were statistically significantly lower than control. For total protein and albumin, all or the majority of individual values at these dosages were below the respective historical control range, however for albumin/globulin ratio only one individual value was below the respective historical control range at each dosage. Whilst these levels may indicate slight changes in liver metabolism, in the absence of any supporting histopathological change, they were considered to be of little toxicological significance and not to indicate any adverse effect.
For both sexes at 350 or 750 mg/kg bw/day, mean total cholesterol levels were statistically significantly higher than control, although mean values showed no dosage relationship for females. For males only 2/5 individual values at 750 mg/kg bw/day exceeded the historical control. For females all values at 350 or 750 mg/kg bw/day exceeded the historical control but so did 3/5 of the control values. Whilst these levels may indicate slight changes in liver metabolism, in the absence of any supporting histopathological change, they were considered to be of little toxicological significance and not to indicate any adverse effect.
For females at 350 or 750 mg/kg bw/day, mean creatinine levels were statistically significantly lower than control but, all individual values were within the historical control range. Whilst changes in mean creatinine levels may indicate slight changes in liver metabolism or kidney performance, in the absence of any supporting histopathological change, they were considered to be of little toxicological significance and not to indicate any adverse effect.
For both sexes at 350 or 750 mg/kg bw/day, mean total bilirubin levels were statistically significantly higher than control. One female value at 350 mg/kg bw/day and another female value at 750 mg/kg bw/day exceeded the historical control range, all other individual values for both sexes at these dosages were within the respective historical control range. There were no consistent effects on hematology parameters apparent for either sex that indicated any increase in erythrocyte turnover and, in the absence of any supporting histopathological change, this finding was considered to be of little toxicological significance and did not represent an adverse effect.
For males at all dosages, mean inorganic phosphorus levels were statistically significantly higher than control, but all individual values were within the historical control range. For females at 750 mg/kg bw/day, mean calcium levels were statistically significantly higher than control, but all individual values were within the historical control range. There were no other statistically differences observed for other blood electrolytes in either sex, and these isolated findings, in the absence of any histopathological change, were considered to be incidental and unrelated to treatment.
For males at 750 mg/kg bw/day, mean bile acids levels were statistically significantly higher than control, with 3/5 individual values exceeding the historical control range compared to only one for the control group. Whilst changes in bile acids levels may indicate slight changes in liver metabolism, in the absence of any supporting histopathological change, this finding was considered to be of little toxicological significance and not to indicate any adverse effect.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Isolated occasions of noisy respiration were observed for one male at 100 mg/kg bw/day, one female at 350 mg/kg bw/day and one male and one female at 750 mg/kg bw/day during these assessments. These findings were consistent with the clinical signs observed during routine pot-dosing observations throughout the study and were considered not to indicate any systemic effect of treatment.
At 350 mg/kg bw/day, one female showed piloerection during the behavioral assessment for the third week of gestation but this finding, in isolation, was considered to be incidental and of no toxicological significance.
Grip strength and motor activity assessments did not indicate any adverse effect of treatment for either sex at 100, 350 or 750 mg/kg bw/day.
For females at all dosages, mean hind limb grip strength was statistically significantly lower than control during trial two, however there was no dosage relationship and no other statistically significant differences were apparent during the other trial of grip strength. This finding was therefore considered to be incidental and unrelated to treatment.
For females at 750 mg/kg bw/day, lower motor activity, compared to control was apparent during at the last 20% of the testing period. Motor activity followed the expected pattern of behavior and there was no similar decrease apparent for males at this dosage. There were no histopathological findings apparent for the brain or nervous system and no other findings apparent during the study to indicate any neurological effect. In isolation, this finding was considered to be incidental and unrelated to treatment

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

For females at all dosages, absolute and body weight-relative ovary weights were statistically significantly higher than control, however, mean values showed no dosage relationship. At 100 mg/kg bw/day, 2/12 absolute and 3/12 relative weights were below the respective historical control range, at 350 mg/kg bw/day, 1/12 absolute values exceeded and 3/12 relative weights were below the respective historical control range and at 750 mg/kg bw/day, 2/11 absolute and 4/11 relative weights were below the respective historical control range. For the control group, however, 6/12 absolute and 11/12 relative weights were below the respective historical control range and it is considered that the observed differences in ovary weight represented atypically low values for the control groups and was unrelated to treatment. For females at all dosages, absolute and body weight-relative ovary weights were statistically significantly higher than control, however, mean values showed no dosage relationship. At 100 mg/kg bw/day, 2/12 absolute and 3/12 relative weights were below the respective historical control range, at 350 mg/kg bw/day, 1/12 absolute values exceeded and 3/12 relative weights were below the respective historical control range and at 750 mg/kg bw/day, 2/11 absolute and 4/11 relative weights were below the respective historical control range. For the control group, however, 6/12 absolute and 11/12 relative weights were below the respective historical control range and it is considered that the observed differences in ovary weight represented atypically low values for the control groups and was unrelated to treatment.
For males at 350 and 750 mg/kg bw/day, absolute and body weight-relative seminal vesicles weights were statistically significantly higher than control, although absolute mean values showed no dosage relationship. Except for one body weight relative value at 750 mg/kg bw/day, all individual seminal vesicle weights at these dosages were within the respective historical control range. In the absence of any supporting histopathological change, the observed differences in organ weights were considered to be incidental and of no toxicological significance. At 750 mg/kg bw/day, absolute and body weight-relative prostate weights were statistically significantly higher than control. In the absence of any supporting histopathological change, the observed differences in organ weights were considered to be of little no toxicological significance and did not represent an adverse effect. In particular, there were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological examinations did not indicate any adverse effect of treatment at 100, 350 and 750 mg/kg bw/day.
It was noted that follicular hypertrophy was present in the thyroid glands of 1/12, 9/12 and 7/11 females at 100, 350 and 750 mg/kg bw/day respectively, although no histopathological changes in the thyroid were apparent for males. The follicular hypertrophy for females appeared to correlate with increased thyroid weights compared to control, however neither the incidence of the microscopic finding nor the differences in mean thyroid weights showed any dosage relationship. Whilst this finding could possibly represent a direct stand-alone change in the thyroid gland, this is considered to be unlikely when occurring only in females. Thyroid activity is increased in females which are pregnant/lactating and, in view of the lack of a true dose-response, this findings was considered to reflect an uneven incidence of normal variability for these female animals within the study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Table 3. Summary Incidence of Daily Clinical Observations

Observation

In Male

Group 10(Control)

Group 2 100 mg/kg bw/day

Group 3

350 mg/kg bw/day

Group 4

750 mg/kg bw/day

Observation

In Female

Group 1

0(Control)

Group 2 100 mg/kg bw/day

Group 3

350 mg/kg bw/day

Group 4

750 mg/kg bw/day

Open wound #Number of Animals Days from - to

1

18 - 30

-

Found dead Number of Animals Days from - to

-

1

50 -50

Scab #Number of Animals Days from - to

1

20 -45

-

Staining around the snout@ Number of Animals Days from - to

-

1

42 - 42

-

Scheduled kill Number of Animals Days from - to

12

44 - 45

12

44 - 45

12

44 - 45

12

44 - 45

Scheduled kill Number of Animals Days from - to

12

51 -62

12

51 - 54

12

51 - 55

11

51 - 62

Increase salivation Number of Animals Days from - to

-

3

9 - 32

12

9 - 45

Increase salivation Number of Animals Days from - to

-

6

38 - 51

11

9 - 53

Noisy Respiration Number of Animals Days from - to

1

11 - 11

2

11 - 11

7

1-15

8

1 - 42

Noisy Respiration Number of Animals Days from - to

1

9 - 10

-

3

13-39

9

7 -51

Hunched posture Number of Animals Days from - to

-

1

31 -32

Hunched posture Number of Animals Days from - to

-

1

49 -49

Pilo-erection Number of Animals Days from - to

-

Pilo-erection Number of Animals Days from - to

-

1

49 -49

Hypothermia Number of Animals Days from - to

-

Hypothermia Number of Animals Days from - to

-

1

49 -49

Pallor of the extremities Number of Animals Days from - to

-

Pallor of the extremities Number of Animals Days from - to

-

1

42 -43

1

49 -49

Table 4. Summary Incidence of Behavioral Assessments (Observations)

Male
Group/ sex	Clinical Sign	Severity	Day Numbers Relative to Start Date
Group/ sex	Clinical Sign	Severity	5	7	14	21	28	35	39
1(M)	Animals alive		12	12	12	12	12	12	12
	Animals Normal		12	12	12	11	11	11	12
	Open wound	Present	.	.	1	1	1	.
	Total	.	.	1	1	1	.
2(M)	Animals alive		12	12	12	12	12	12	12
	Animals Normal		12	12	12	12	12	11	12
	Respiration Noisy	Present	.	.	.	.	1	.
	Total		.	.	1	.
3(M)	Animals alive		12	12	12	12	12	12	12
3(M)	Animals Normal		12	12	12	12	12	11	12
4(M)	Animals alive		12	12	12	12	12	12	12
	Animals Normal		12	11	12	12	12	11	12
	Respiration Noisy	Present	.	1	.	.	1
	Total		.	.	.	1
Female
Group/ sex	Clinical Sign	Severity	Day Numbers Relative to Start Date
Group/ sex	Clinical Sign	Severity	5	7	14	15	16	17	18	19
1(F)	Animals alive		12	12	12	12	12	12	12	12
1(F)	Animals Normal		12	12	12	12	12	11	12	12
2(F)	Animals alive		12	12	12	12	12	12	12	12
2(F)	Animals Normal		12	12	12	12	12	11	12	12
3(F)	Animals alive		12	12	12	12	12	12	12	12
	Animals Normal		12	11	12	12	12	11	12	12
	Respiration Noisy	Present		1
	Total	1
4(F)	Animals alive		12	12	12	12	12	12	12	12
	Animals Normal		12	12	12	12	12	11	12	12
	Respiration f Noisy	Present				1				1
	Total			1
	Autonomic Piloerection	Slight					1
	Total				1

Table 5. Summay Incidence of Behavioral Assessments (Scores)

Male

Group

Scores

Day 5 Relative to start date

Day 7 Relative to start date

Day 14 Relative to start date

Day 21 Relative to start date

Day 28 Relative to start date

Day 35 Relative to start date

Day 39 Relative to start date

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Group 1

0(Control)

Mean

S.D

N

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.2

0.4

12

0.0

12

0.0

12

Group 2

100 mg/kg bw/day

Mean

S.D

N

0.0

12

0.2

0.4

12

0.0

12

0.0

12

0.1

0.3

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.2

0.6

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

Group 3

350 mg/kg bw/day

Mean

S.D

N

0.0

12

0.1

0.3

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.3

0.5

12

0.3

0.8

12

0.0

12

Group 4

750 mg/kg

Mean

S.D

N

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.3

0.5

12

0.1

0.3

12

0.0

12

0.0

12

0.1

0.3

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

Group

Scorse

Day 5 Relative to start date

Day 7 Relative to start date

Day 14 Relative to start date

Day 15 Relative to start date

Day 16 Relative to start date

Day 17 Relative to start date

Day 19 Relative to start date

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Transfer arousal

Urination

Defecation

Tail elevation

Group 1

0(Control)

Mean

S.D

N

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.4

0.5

12

0.0

12

0.0

12

Group 2100 mg/kg bw/day

Mean

S.D

N

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.2

0.4

12

0.0

12

0.0

12

Group 3

350 mg/kg bw/day

Mean

S.D

N

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.3

0.7

12

0.0

12

0.0

12

Group 4

750 mg/kg

Mean

S.D

N

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.1

0.3

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.0

12

0.2

0.4

12

0.1

0.3

12

0.0

12

0.0

11

0.5

11

0.1

0.3

11

0.0

11

Table 6. Group Mean Functional Test Values

Group/ sex	No of animal /Group	Test 1 Forelimb	Test 1 Hindlimb	Test 2 Forelimb	Test 2 Hindlimb	Test 3 Forelimb	Test 3 Hindlimb	Overall activity	Overall Mobile	Last 20%activity	Last 20%mobile	Group/ sex	No of animal /Group	Test 1 Forelimb	Test 1 Hindlimb	Test 2 Forelimb	Test 2 Hindlimb	Test 3 Forelimb	Test 3 Hindlimb	Overallactivity	OverallMobile	Last 20%activity	Last 20%mobile
1(M)	5	1084.4	503.0	1118.8	336.2	1035.2	407.4	448.8	0.0	34.6	0.0	1(F)	5	975.8	434.2	933.0	423.0	963.2	285.0	401.0	0.0	50.8	0.0
2(M)	5	1349.2	306.2	1215.6	415.6	1096.2	273.0	396.2	0.0	43.8	0.0	2(F)	5	950.6	338.4*	964.2	303.4	894.0	290.0	412.6	0.0	57.0	0.0
3(M)	5	1156.0	360.4	1081.8	385.8	918.6	454.6	372.0	0.0	31.4	0.0	3(F)	5	794.8	212.6**	883.0	343.2	1018.0	413.8	351.4	0.0	32.0	0.0
4(M)	5	1247.2	430.4	1280.6	386.4	1141.0	287.0	447.8	0.0	39.4	0.0	4(F)	5	1012.6	340.4**	1012.8	321.2	979.0	282.8	301.6	0.0	11.6*	0.0

Table 7. Summary Incidence of Sensory Reactivity Assessments (Scores).

Group/ sex	No of animal /Group	Grasp response	Vocalisation	Toe pinch	Tail pinch	Finger approach	Touch escape	Pupil reflex	Blink reflex	Startle reflex	Group/ sex	No of animal /Group	Grasp response	Vocalisation	Toe pinch	Tail pinch	Finger approach	Touch escape	Pupil reflex	Blink reflex	Startle reflex
1(M)	5	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	1(F)	5	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
2(M)	5	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	2(F)	5	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
3(M)	5	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	3(F)	5	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
4(M)	5	0.0	0.0	0.2	0.0	0.0	0.0	0.0	0.0	0.0	4(F)	5	0.0	0.0	0.0	0.2	0.0	0.0	0.0	0.0	0.0

Table 8. Group Mean Body Weight Values

Body Weight (g)

Group/ sex

Day Numbers Relative to Start Date

Group/ sex

Day Numbers Relative to Start Date

Gestation

Lactation

1

8

15

22

29

36

43

1

8

15

0

7

14

20

1

4

7

14

1(M)

315.8

337.3

352.2

361.3

375.8

390.0

399.1

1(F)

211.5

217.8

224.7

227.4

254.5

280.2

339.0

257.8

270.8

276.0

307.8

2(M)

314.4

337.2

352.6

366.0

378.5

387.4

397.1

2(F)

211.5

217.2

227.5

226.9

251.0

273.8

325.8

251.6

267.5

277.7

303.1

3(M)

320.3

347.2

365.1

379.9

393.4

404.8

416.0

3(F)

206.6

214.3

223.5

220.9

249.8

272.4

324.7

251.0

265.8

277.0

297.9

4(M)

317.9

342.7

360.2

368.7

383.8

394.2

407.7

4(F)

211.5

215.0

222.3

220.3

241.8

265.8

315.8

249.5

261.9

268.5

290.0

Table 9. Group Mean Body Weight Gains

Increase in Body Weight (g)
Group/ sex	Day Numbers Relative to Start Date						Abs gain	% gain	Group/ sex	Day Numbers Relative to Start Date		Abs gain	% gain	Gestation Days			Cumulative Body Weight Change (g)Days		Lactation			Cumulative Body Weight Change (g)Days
Group/ sex	1 - 8	8 - 15	15 -22	22 - 29	29 – 36	36 - 43	1 - 43	1 - 43	Group/ sex	1 - 8	8 - 15	1 - 15	1 - 15	0 - 7	7-14	14-20	0 - 14	0 - 20	1 - 4	4 - 7	7-14	1 - 7	1 - 14
1(M)	21.6	14.8	9.1	14.5	14.3	9.1	83.3	26.3	1(F)	6.3	6.8	13.2	6.2	27.1	25.7	58.8	52.8	111.6	13.1	5.2	31.8	18.3	50.0
2(M)	22.8	15.4	13.4	12.5	8.9	9.7	82.7	26.1	2(F)	5.7	10.3	16.0	7.6	24.1	22.8	51.9	46.9	98.8	15.9	10.2	25.4	26.1	51.5
3(M)	26.8	17.9	14.8	13.5	11.3	11.3	95.7	29.6	3(F)	7.8	9.2	16.9	8.2	28.8	22.7	52.3	51.5	103.8	14.8	11.2	20.9	26.0	46.9
4(M)	24.8	17.5	8.5	15.1	10.4	13.5*	89.8	28.0	4(F)	3.5	7.3	10.8	5.1	21.5	23.9	50.1	45.4	95.5	12.4	6.5	21.5	18.9	40.5

Table 10. Group Mean Food Consumptions

Group/ sex	Day Numbers Relative to Start Date				Group/ sex	Day Numbers Relative to Start Date		Gestation			Lactation
Group/ sex	1 - 8	8 - 15	29 – 36	36 - 43	Group/ sex	1 - 8	8 - 15	0 - 7	7-14	14-20	1 - 4	4 - 7	7-14
1(M)	23.8	23.1	24.5	25.4	1(F)	16.1	16.6	21.6	24.5	25.4	31.9	47.7	54.0
2(M)	22.8	22.6	22.8	23.7	2(F)	17.5	16.3	19.7	22.9	24.8	30.0	41.8	49.4
3(M)	24.3	23.4	25.1	24.8	3(F)	15.7	16.6	20.5	24.0	24.9	32.4	39.4	49.3
4(M)	24.6	23.6	25.6	25.8	4(F)	14.6	16.9	19.7	24.2	24,6	34.1	42.0	44.6

Table 11. Group Mean Food Efficiency

Group/ sex	Weeks Relative to Start Date				Group/ sex	Weeks Relative to Start Date
Group/ sex	1 -2	2 - 3	5 - 6	6 -7	Group/ sex	1 -2	2 - 3
1(M)	12.9	9.1	8.3	5.1	1(F)	5.7	5.8
2(M)	14.3	9.7	5.6	5.8	2(F)	4.6	9.1
3(M)	15.8	10.9	6.5	6.5	3(F)	7.1	7.8
4(M)	14.3	10.5	5.8	7.4	4(F)	3.3	6.1

Table 12. Group Mean Blood Chemical and Haematology Values

Blood chemistry parameters	Male				Female
Blood chemistry parameters	Group 1	Group 2	Group 3	Group 4	Group 1	Group 2	Group 3	Group 4
P mmol/l	2.00	2.38*	2.48*	2.52*	1.74	1.60	1.46	1.44
ASAT IU/l	99.6	84.0	83.8	90.0	106.2	95.4	135.4	101.4
ALAT (IU/l)	54.6	53.0	45.8	50.8	104.0	82.6	121.4	125.4
AP (IU/l)	160.4	157.0	154.6	200.8	199.4	184.2	245.2	258.4
Creat mg/dl	0.670	0.654	0.610	0.638	0.662	0.584	0.536*	0.538*
Chol mg/dl	78.4	86.2	99.6*	135.0**	113.8	142.2	212.2**	200.0**
Bili mg/dl	0.060	0.062	0.104**	0.134**	0.076	0.088	0.112*	0.120**
Bile acid μmol/l	17.32	23.50	16.32	48.56*	23.30	28.32	53.92	50.22
Urea mg/dl	44.2	41.0	37.8	39.0	58.2	52.4	55.8	52.6
Glucose mg/dl	143.6	129.4	126.0	123.0	128.8	124.4	117.8	128.4
Tot. Prot. g/dl	6.012	6.352	6.330	6.588	5.672	5.408	5.010**	4.772**
Abumin g/dl	3.30	3.48	3.52	3.60	3.22	3.08	2.68**	2.52**
A/G Ratio	1.204	1.214	1.264	1.202	1.324	1.336	1.3136	1.132
Na+mmol/l	148.2	147.8	148.4	151.6	145.4	146.4	144.2	144.2
K+mmol/l	5.522	5.188	4.882	5.328	5.418	5.144	5.348	4.864
Cl-mmol/l	101.6	100.6	101.6	101.8	102.4	99.6	99.0	100.0
Ca++mmol/l	2.462	2.592	2.544	2.470	2.614	2.588	2.478	2.340**

Hematology parameters	Male				Female
Hematology parameters	Group 1	Group 2	Group 3	Group 4	Group 1	Group 2	Group 3	Group 4
Hbg/dl	16.04	16.12	15.78	15.56	14.30	14.18	13.88	14.02
RBC 10^12/l	8.566	8.970	8.602	8.910	6.928	7.252	6.738	7.472
Hct %	45.02	45.20	44.90	44.24	40.40	40.36	39.36	40.66
MCH pg	18.70	18.00	18.38	17.50**	20.68	19.64	20.68	18.74*
MCV f1	52.38	50.36	52.16	49.70*	58.46	55.56	58.58	54.48
MCHC g/d1	35.62	35.72	35.18	35.18	35.40	35.22	35.12	34.32
WBC 10^9/l	7.12	7.56	9.18*	9.30*	4.78	5.50	6.34*	6.10*
Neut 10^9/l	1.170	1.330	1.368	1.122	1.510	1.908	2.278	1.936
Lymp 10^9/l	5.894	6.185	7.744*	8.106*	3.242	3.578	4.062*	4.166*
Mono 10^9/l	0.000n	0.000n	0.000n	0.014n	0.000n	0.000n	0.000n	0.000n
Eco 10^9/l	0.058	0.046	0.072	0.058	0.000n	0.014n	0.000n	0.000n
Bas 10^9/l	0.000n	0.000n	0.000n	0.000n	0.000n	0.000n	0.000n	0.000n
CT Seconds	9.84	912	9.06	9.84	8.58	8.74	8.82	8.78
PLT10^9/l	528.4	668.4**	692.8**	759.6**	598.0	613.0	592.2	591.6
APTTSeconds	16.26	14.56	16.22	13.34	15.22	14.42	15.90	15.06
Retics%	2.70	3.00	3.40*	3.36*	4.08	4.78	4.66	4.58

Table 13. Mean Serum T4 Concentrations (pg/mL)

Group	Adult Terminal Male	Males offspring on Day 13 of age	Females offspring on Day 13 of age
1	43800	39800	40200
2	52400*	41400	43200
3	54200**	39100	41800
4	57100**	33900	36700

Table 14. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

Organ	Male				Female
Organ	Group 1	Group 2	Group 3	Group 4	Group 1	Group 2	Group 3	Group 4
Terminal Bodyweight	402.6	399.5	419.1	410.0	307.8	303.1	297.9	290.0
Adrenals	0.07604	0.08212	0.08306	0.08738	0.08944	0.08984	0.08634	0.06886*
Relative weight	0.019	0.021	0.020	0.023	0.030	0.030	0.029	0.024*
Brain (Including Cerebrum,Cerebellum And Pons	2.03794	1.96792	2.11446	2.11988	1.89094	1.88286	1.85480	1.86090
Relative weight	0.503	0.503	0.523	0.545	0.631	0.634	0.617	0.629
Epididymides	1.57071	1.57443	1.57373	1.65603
Relative weight	0.392	0.397	0.379	0.406
Heart	1.20340	1.16954	1.05046	1.25526	1.26666	1.21634	1.24060	1.26134
Relative weight	0.297	0.304	0.260	0.319	0.418	0.409	0.413	0.423
Kidneys	2.53500	2.28878	2.63396	2.83520*	1.92546	1.86024	2.11782	1.96906
Relative weight	0.625	0.591	0.653	0.723*	0.639	0.626	0.702	0.662
Liver	14.7494	12.9958	16.7185*	18.9891**	14.9901	16.2430**	18.3088**	19.9560**
Relative weight	3.623	3.306	4.131*	4.807**	4.971	5.451**	6.043**	6.669**
Ovaries					0.08398	0.10168**	0.10708**	0.09728**
Relative weight					0.027	0.034**	0.036**	0.033**
Pituitary	0.01346	0.01462	0.01453	0.01428	0.01688	0.01684	0.01973	0.01516
Relative weight	0.003	0.004	0.003	0.003	0.005	0.006	0.007	0.005
Prostate	0.59454	0.65658	0.6887	0.74909*
Relative weight	0.149	0.164	0.166	0.184*
Seminal Vesicles (With Coagulaƒtion Gland)	1.83467	1.80241	2.13610*	2.10727*
Relative weight	0.457	0.453	0.515*	0.522*
Spleen	0.79592	0.84392	0.81226	0.81232	0.64332	0.78504	0.76730	0.62180
Relative weight	0.196	0.218	0.200	0.207	0.214	0.264	0.254	0.207
Testes	3.89569	3.88659	3.90728	3.99370
Relative weight	0.970	0.979	0.940	0.979
Thymus	0.44948	0.26398	0.43702	0.47466	0.26014	0.28096	0.27406	0.27102
Relative weight	0.111	0.066	0.108	0.119	0.087	0.095	0.091	0.091
Thyroid/Parathyroid	0.03972	0.04271	0.04116	0.04364	0.02671	0.03178	0.03821*	0.03509*
Relative weight	0.010	0.011	0.010	0.011	0.009	0.010	0.013*	0.012*
Uterus & Cervix (And Oviducts)					0.54190	0.57278	0.50861	0.49657
Relative weight					0.177	0.192	0.171	0.171

Conclusions:

Based on the results of this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of α-phenyl-1H-benzimidazole-2-methanol to the adult animal was considered to be 750 mg/kg bw/day.

Executive summary:

OECD 422 (2019) - In a combined repeat dose toxicity study with reproductive toxicity screening with Comet Assay (OECD 422), α-phenyl-1H-benzimidazole-2-methanol was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks for males and for approximately eight weeks for females (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 350 and 750 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene Glycol ) over the same period. A further three males were dosed with N-Nitroso-N-methylurea (vehicle: distilled water) for two consecutive days to act as a positive control group for the comet assay

Treatment at dosages of 100, 350 and 750 mg/kg bw/day was well tolerated with no adverse effect on body weight gain, food consumption, food conversion efficiency or water consumption for either sex throughout the study. Food consumption was lower than control for treated females from Day 4 of lactation, with differences being most notable at 750 mg/kg bw/day during the second week of lactation; however, this was considered to reflect lower demand from the smaller litters for treated females, in comparison to their control counterparts, rather than any effect of treatment on the lactating females. Post-dosing salivation was observed for the majority of animals at 750 mg/kg bw/day and this was also apparent for fewer animals and to a lesser extent at 350 mg/kg bw/day. Increased post-dosing salivation is frequently observed when animals are dosed via the oral gavage route and is generally considered to reflect slight distaste or irritancy of the dosing formulations rather than any systemic effect of treatment. Isolated occasions of noisy respiration were observed for all dose groups, including control, although the highest incidence was at 350 mg/kg bw/day and 750 mg/kg bw/day during the study. The higher incidence of noisy respiration at 350 and 750 mg/kg bw/day may have been influenced by the increased post-dosing salivation observed at these higher dosages and this finding was considered to reflect slight distaste of the test item formulations and/or occasional difficulties in dosing isolated animals rather than any systemic effect of treatment.

The only unscheduled adult death on the study occurred at 750 mg/kg bw/day, when a female was found dead on Day 50. This animal had previously shown total litter loss post-partum and hunched posture, pallor of the extremities, apparent hypothermia and piloerection had been apparent on the day prior to death. Necropsy revealed enlarged liver, spleen and right adrenal, a pale area on the liver and thin appearance of the non-glandular region of the stomach and raised limiting ridge and additionally, a pale mass was observed in the right ventricle of the heart during tissue processing. Histopathology examination revealed abscessation in the lungs and marked inflammatory change in the heart with the presence of bacterial colonies. It was considered that sepsis, as a result of complications of pregnancy, was the underlying cause of death and this isolated atypical occurrence was considered to be unrelated to treatment.

Behavioral and function observations did not indicate any underlying neurological effect of treatment for either sex at 100, 350 or 750 mg/kg bw/day.

There was no adverse effect of treatment on hematology or blood chemistry parameters at 100, 350 or 750 mg/kg bw/day. Some intergroup differences in these parameters did attain statistical significance when compared to control but, in the absence of any supporting histopathological change, they were considered to be of little toxicological significance.

Necropsy of adults revealed a small number of liver findings (eg enlarged, mottled appearance, pale area) for females at 100 and 350 mg/kg bw/day and both sexes at 750 mg/kg bw/day and liver weights were statistically significantly higher than control for females at 100 mg/kg bw/day and both sexes at 350 and 750 mg/kg bw/day. However, histopathological evaluation of the liver did not reveal any evidence of hepatic change and, while some adaptive alteration to liver metabolism may have occurred, these findings were considered not to represent an adverse effect of treatment.

For females at 350 and 750 mg/kg bw/day, absolute and body weight-relative thyroid weights were statistically significantly higher than control, but mean values showed no dosage relationship. At both dosages, the majority of individual absolute and body weight-relative weights exceeded the respective historical control range, but again, these incidences showed no dosage response. Follicular hypertrophy was present in the thyroid glands of 1/12, 9/12 and 7/11 females at 100, 350 and 750 mg/kg bw/day respectively but showed no true dose response and this follicular hypertrophy may be the underlying cause of the observed differences in the thyroid weights at 350 and 750 mg/kg bw/day. No histopathological changes in the thyroid were apparent for males and, whilst a direct stand-alone change in the thyroid gland is possible, it is considered to be less likely when occurring only in females. Thyroid activity is increased in females which are pregnant/lactating and, in view of the lack a true dose-response, these findings was considered to reflect an uneven incidence of normal variability for these female animals within the study rather than an adverse effect of treatment.

All other statistically significant differences in organ weights observed during the study were not supporting by any accompanying histopathological change and were considered to be of little toxicological significance and not to represent an adverse effect.

There was no effect of treatment on estrous cycles of females, mating performance or gestation length at 100, 350 or 750 mg/kg bw/day. Although, there was no effect on fertility, as assessed by the number of females that achieved pregnancy at 100, 350 or 750 mg/kg bw/day, the number of implantations was lower than control at 750 mg/kg bw/day. The number of implantation were also lower than control at 100 and 350 mg/kg bw/day but the differences were slight and probably reflect normal biological variation. Histopathological evaluations of reproductive tissues at 750 mg/kg bw/day did not reveal any test item-related microscopic findings for the reproductive tissues and, in particular, there were no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle or for follicles and corpora lutea in the ovaries. The lower number of implantations at 750 mg/kg bw/day is therefore unexplained, and while it this may also reflect normal variation, an effect of treatment cannot be discounted.

Subsequent post-implantation and post-natal survival of the offspring was unaffected by treatment at all dosages. One female at 750 mg/kg bw/day did show total litter post partum but this was considered to reflect a decline in the clinical condition for this animal due to sepsis, as a result of complications of pregnancy, and therefore the high offspring mortality for this particular litter was considered to be unrelated to maternal treatment. Mean offspring body weight on Day 1 of age was similar to control at all dosages but, body weight gain at 750 mg/kg bw/day was slightly lower than control, despite the lower litter size at this dosage, leading to lower mean offspring body weight at termination on Day 13 of age. A number of small offspring were noted during clinical observations and at necropsy, consistent with this lower gain and litter weight at 750 mg/kg bw/day was lower than control throughout, initially reflecting the lower litter size and subsequent the lower litter size and weight gain. At 100 or 350 mg/kg bw/day, there was no effect of maternal treatment on offspring body weight gain to Day 13; litter weights were lower than control throughout, but this reflected the slightly lower litter size at both dosages.

Mean offspring ano-genital distance (actual and normalized) for males at 100 mg/kg bw/day and both sexes appeared longer than control at 100, 350 or 750 mg/kg bw/day; mean values for males at 100 and 350 mg/kg bw/day showed no dosage relationship. For female offspring at 100 mg/kg bw/day, measured ano-genital distance was longer than control, but was similar to control when normalized for body weight. All individual litter values for ano-genital distance, normalized for body weight, were within the historical control range at 100, 350 or 750 mg/kg bw/day. As no normalized for body weight values for either sex were outside the normal historic range, an effect of treatment on ano-genital distance appeared unlikely and this finding was considered to be incidental and of no toxicological significance. Evaluation of visible nipple count for male offspring on Day 13 post partum did not reveal any effect of maternal treatment at 100, 350 or 750 mg/kg bw/day.

For the additional comet investigation, there were no significant increases in the percentage tail intensity or median percentage tail intensity for the jejunum, glandular stomach or liver at 100, 350 or 750 mg/kg bw/day and the test item was, therefore, considered to not induce DNA damage in these tissues under the conditions of the test.

The mean levels of thyroxine (T4) in adult males were statistically significant higher then control at 100, 350 and 750 mg/kg bw/day, although mean values showed no dosage-relationship at the lower dosage levels. These differences from control for T4 levels in males occurred in the absence of any effect on organ weight or evidence of histopathological change for the male thyroid and, therefore, this finding was considered to be incidental and unrelated to treatment.

Under the condition of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of α-phenyl-1H-benzimidazole-2-methanol to the adult animal was considered to be 750 mg/kg bw/day.