Registration Dossier

Administrative data

Description of key information

NOAEL (systemic toxicity) = 750 mg/kg bw/d; OECD 422; S. Fulcher. (2019)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18 May 2018 - 11 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day dietary study in the Crl:CD(SD) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day dietary study in the Crl:CD(SD) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Nalco/8247-22B
- Expiration date of the lot/batch: 31/ 01/2020
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable): n/a
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: formulations stable for at least one day
- Solubility and stability of the test substance in the solvent/vehicle: soluble in Polyethylene glycol 400
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: No
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Not reported
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: Females 190 to 244g and male 333 to 356g
- Housing: TMakrolon Type II (pre-test) / III (main study), with wire mesh top. Granulated soft wood bedding
- Diet (e.g. ad libitum): rodent diet 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK
- Water (e.g. ad libitum): mains drinking water was supplied from polycarbonate bottles
- Acclimation period: At least 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): At least 15 changes/ hour
- Photoperiod (hrs dark / hrs light): 12 h: 12 h (light: dark)
- IN-LIFE DATES: 28 June 2018 and 12 July 2018 (Phase 1), and 13 July 2018 and 27 July 2018 (Phase 2).
Route of administration:
oral: gavage
Details on route of administration:
Oral Gavage
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400
Details on oral exposure:
Gavage using a stainless steel cannula attached to a disposable plastic syringe
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services under Envigo study number VJ90QJ. Results showed the formulations to be stable for at least one day. Formulations were therefore prepared and used daily.

No analysis was conducted to determine the concentration of the test item formulation. It is considered that the purpose or integrity of the study was unaffected by the lack of information in relation to dosing formulations.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Phase I
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
Phase I
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Phase I
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
Phase II
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Phase II
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
The test item was administered daily at dosages of 500, 750 and 1000 mg/kg bw/day, for up to fourteen consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner to Phase 1 animals with 4 mL/kg of Polyethylene glycol 400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted on Days 4, 8 and 11.
Positive control:
No
Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not indicated
- Time schedule:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before dosing, up to thirty minutes after dosing and one hour after dosing, and four hours after dosing except during weekend.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Days -2 (Phase 1 only) and -1 (Phase 2 only), 1, 4, 8, 11 and 15.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was recorded for each cage group for Days -2 to 1 (Phase 1 only), Days -1 to 1 (Phase 2 only), 1 to 4, 4 to 8, 8 to 11 and 11 to 15

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Daily for each cage group.
.

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

HAEMATOLOGY: No
- Time schedule for collection of blood: n/a
- Anaesthetic used for blood collection: n/a
- Animals fasted: n/a
- How many animals: n/a

CLINICAL CHEMISTRY: No
- Time schedule for collection of blood: n/a
- Animals fasted: No
- How many animals: n/a

URINALYSIS: No
- Time schedule for collection of urine: n/a
- Metabolism cages used for collection of urine: n/a
- Animals fasted: n/a

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/s
- Dose groups that were examined: n/a

OTHER: no
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: No
Other examinations:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Phase 1:
Adverse clinical signs including decreased respiratory rate, pilo-erection, lethargy, hunched posture, pallor of the extremities and dehydration were apparent for two female animals at 1000 mg/kg bw/day and were killed in extremis on day 9.
Increased post-dosing salivation was observed on a few occasions during the second week of treatment for males at 750 and 1000 mg/kg bw/day with similar increased post-dosing salivation being observed on single occasions for one female at 750 and another at 1000 mg/kg bw/day.
Noisy respiration was observed for one control female, one male at 500 mg/kg bw/day, all males and one female at 750 mg/kg bw/day and two males at 1000 mg/kg bw/day on isolated occasions during the study and was considered to reflect occasional difficulties dosing particular animals rather than any systemic effect of treatment.

Phase 2: Increased salivation was observed for all females at both 750 and 1000 mg/kg bw/day mainly during the second week of treatment. Noisy respiration was observed for one female at 750 mg/kg bw/day on three occasions.
Mortality:
mortality observed, treatment-related
Description (incidence):
Phase 1: At 1000 mg/kg bw/day, two females were killed in extremis on Day 9 due to adverse clinical signs, including decreased respiratory rate, pilo-erection, lethargy, hunched posture, pallor of the extremities and dehydration. The remaining female at this dosage was terminated due to these deaths but no clinical signs or macroscopic necropsy findings were apparent for this animal.

Phase 2: There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Phase 1: Weight lost observed in female at 750 and 1000 mg/.kg bw/day during days 4 - 8.

Phase 2: At 1000 mg/kg bw/day, all females showed body weight losses between Days 8-11 and 11-15, resulting in an overall group mean body weight losses for the fourteen day treatment period and initial mean body weight loss between Days 1-4 at 750 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Phase 1: At 1000 mg/kg bw/day, females showed lower consumption compared to control during the first week of treatment, prior to their termination on Day 9. At 750 mg/kg bw/day, initial food consumption (Days 1-4) for females was slightly lower than control.

Phase 2: At 1000 mg/kg bw/day, lower food consumption was apparent for females during Days 1-4 and again during Days 11-15. At 750 mg/kg bw/day, low food consumption was observed for females during Days 1-4.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Phase 1: At 1000 mg/kg bw/day, food conversion efficiency for both sexes was lower than control during Days 4-8, lower food conversion efficiency was also noted at 750 mg/kg bw/day during day 4 - 8.

Phase 2: At 1000 mg/kg bw/day, lower food conversion efficiency for females during day 1- 4 and the second week of treatment with further observation in 750 mg//kg bw/day on day 1- 4.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Phase 1: At 750 & 1000 mg/kg bw/day, water consumption of females appeared higher than control during Days 1-2, 3-4 and 4-5. Lower water intake was subsequently observed during Days 6-7 and 7-8, but may have been influenced by the declining food intake/clinical condition of the animals.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Phase 2: Macroscopic necropsy findings were restricted to brown colored contents in the stomach for two females at 750 mg/kg bw/day and all females at 1000 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Macroscopic necropsy findings restricted to brown colored contents in female stomach
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: No effect observed
Organ:
other: No effects observed
Conclusions:
Based on the results of this study, it is considered that dose levels of 0 (control), 100, 350 and 750 mg/kg bw/day, utilizing a dose volume of 6 mL/kg, would be appropriate for further investigation of effects of the Test Item on reproduction or toxicity.
Executive summary:

Dose Range Finder - DRF for OECD 422 (2018) - The purpose of this test was to assess the systemic toxicity potential of α-phenyl-1H-benzimidazole-2-methanol following repeated exposure to 3 males and 3 femals Wistar Han™:RccHan™:WIST strain ras at 1500, 750 and 1000 mg/kg bw/day at a treatment volume of 4 mL/kg. While control groups received vehicle (Polyethylene glycol 400)  alone.  In phase two, 2 further groups of three female rats were then administered the test item by gavage at dose levels of 750 and 1000 mg/kg bw/day at a treatment volume of 6 mL/kg for fourteen consecutive days. The findings would be used to select suitable dose levels for a subsequent combined repeat dose toxicity study with reproductive/ developmental toxicity screening study (OECD 422).

A summary of adult responses to the test item are described below;

 

Mortality - Two females at 1000 mg/kg bw/day of phase 1 were killed in extremis on Day 9 due to adverse clinical signs, including decreased respiratory rate, pilo-erection, lethargy, hunched posture, pallor of the extremities and dehydration. No mortality was observed during phase 2.

 

Clinical signs - Phase 1: decreased respiratory rate, pilo-erection, lethargy, hunched posture, pallor of the extremities and dehydration in 2 females. at 1000 mg/kg bw/day.

Phase 2: Increased salivation was observed for all females at both 750 and 1000 mg/kg bw/day mainly during the second week of treatment.  Noisy respiration was observed for one female at 750 mg/kg bw/day on three occasions.

  

Body weight - Phase 1:  At 1000 mg/kg bw/day, all females showed notable body weight loss between Days 4-8 prior to their termination on Day 9 due to adverse clinical signs. The weight lost was also apparent in female at 750 mg/kg bw/day from day 4-8. No effect of treatment on body weight gain for either sex at 500 mg/kg bw/day or for males at 750 mg/kg bw/day.    

Phase 2:  At 1000 mg/kg bw/day, all females showed body weight losses between Days 8-11 and 11-15, resulting in an overall group mean body weight losses for the fourteen day treatment period. At 750 mg/kg bw/day females showed initial mean body weight loss between Days 1-4.

Food consumption and efficiency - Phase 1:  At 1000 mg/kg bw/day, females showed lower consumption compared to control during the first week of treatment, prior to their termination on Day 9. Initial food consumption (Days 1-4) for females was slightly lower at dose group 750 Mg/kg bw/day.  For males at this dosage, initial food intake (Days 1-4) was slightly lower than control.Phase 2:  At 1000 mg/kg bw/day, lower food consumption was apparent for females during Days 1-4 and again during Days 11-15.  At 750 mg/kg bw/day, low food consumption was observed for females during Days 1-4.

Phase 1:  At 1000 mg/kg bw/day, food conversion efficiency for both sexes was lower than control during Days 4-8.  At 750 mg/kg bw/day, food conversion efficiency for females was lower than control during Days 4 8.  

Phase 2: Food conversion efficiency was lower in female treated at 750 & 100 mg/kg bw/day in female only.

Water consumption - Phase 1: high water consumption was only observed in female  750 & 100 mg/kg bw/day.    Phase 2: Phase 2: No consistent dose-related effect was seen on water consumption at 750 and 1000 mg/kg bw/day, however, values appeared generally higher compared to controls in Phase 1.

Necropsy - Phase 1: No macroscopic abnormalities were apparent for surviving animals at terminal necropsy at 500, 750 or 1000 mg/kg bw/day.  

Phase 2: Macroscopic necropsy findings were restricted to brown colored contents in the stomach for two females at 750 mg/kg bw/day and all females at 1000 mg/kg bw/day.

Based on the results of this study, it is considered that dose levels of 0 (control), 100, 350 and 750 mg/kg bw/day, utilizing a dose volume of 6 mL/kg, would be appropriate for further investigation of effects of the Test Item on reproduction or toxicity.

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
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
Deviation from target value for relative humidity (RH) from 50 ± 20% to 76%, but the study was unaffected.
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Version / remarks:
2008
Deviations:
yes
Remarks:
Deviation from target value for relative humidity (RH) from 50 ± 20% to 76%, but the study was unaffected.
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 489 “In vivo Mammalian Alkaline Comet Assay”
Version / remarks:
2016
Deviations:
yes
Remarks:
Deviation from target value for relative humidity (RH) from 50 ± 20% to 76%, but the study was unaffected.
GLP compliance:
yes (incl. certificate)
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 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
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
1. Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
2. Low
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
3. Intermediate
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
4. High
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
5. Positive Control
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
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse systemic effect observed in both sexes
Remarks on result:
other: No adverse effects observed
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)
System:
other: No effects observed

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

 

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

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

5

7

14

15

16

17

18

19

1(F)

Animals alive

 

12

12

12

12

12

12

12

12

Animals Normal

 

12

12

12

12

12

11

12

12

2(F)

Animals alive

 

12

12

12

12

12

12

12

12

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

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.2

0.4

12

0.0

0.0

12

0.0

0.0

12

Group 2

100 mg/kg bw/day

 

Mean

S.D

N

0.0

0.0

12

0.2

0.4

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.2

0.6

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

Group 3

350 mg/kg bw/day

 

Mean

S.D

N

0.0

0.0

12

0.1

0.3

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.3

0.5

12

0.3

0.8

12

0.0

0.0

12

Group 4

750 mg/kg

Mean

S.D

N

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.3

0.5

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

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

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.4

0.5

12

0.0

0.0

12

0.0

0.0

12

Group 2100 mg/kg bw/day

 

Mean

S.D

N

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.2

0.4

12

0.0

0.0

12

0.0

0.0

12

Group 3

350 mg/kg bw/day

 

Mean

S.D

N

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.3

0.7

12

0.0

0.0

12

0.0

0.0

12

Group 4

750 mg/kg

Mean

S.D

N

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.0

0.0

12

0.2

0.4

12

0.1

0.3

12

0.0

0.0

12

0.0

0.0

11

0.5

0.5

11

0.1

0.3

11

0.0

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

 

1 - 8

8 - 15

15 -22

22 - 29

29 – 36

36 - 43

1 - 43 1 - 43

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

 

1 - 8

8 - 15

29 – 36

36 - 43

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

 

1 -2

2 - 3

5 - 6

6 -7

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

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

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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The endpoint is concluded based on a single key study with a Klimish rating of 1.
System:
other: The NOAEL was the highest dose tested.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

During the treatment period mean levels of thyroxine (T4) in adult males were statistically significant higher than control at 100, 350 and 750 mg/kg bw/day, although mean values showed no dosage-relationship at the lower dosage levels. However, there was no effect on organ weight and no evidence of histopathology, therefore the effect was considered incidental and unrelated to treatment. Absolute and body weight-relative organ weight (liver & kidney in both sexes and thyroid in female) were higher than control but without any evidence of histopathology. These changes were considered adaptive alteration to liver metabolism not to represent an adverse effect of treatment. Therefore the substance was not considered toxic up to the highest dose tested and as such the mode of toxicity was not relevant.

Additional information

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.

At 750 mg/kg bw/day, Female 94 was found dead on Day 50 of the study, after having shown hunched posture, pallor of the extremities, apparent hypothermia and piloerection on Day 49.  Histopathology examination revealed 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 underlying reason for mortality and this isolated atypical occurrence was considered to be incidental and unrelated to treatment.

There were no clinical signs observed that indicated any systemic effect of treatment for either sex at 100, 300 or 750 mg/kg bw/day.

Behavioral assessments were unaffected by treatment at 100, 350 and 750 mg/kg bw/day. Functional performance tests were unaffected by treatment at 100, 350 and 750 mg/kg bw/day.

Sensory reactivity assessments were unaffected by treatment at 100, 350 and 750 mg/kg bw/day.

There was no effect of treatment on body weight gain throughout the study for either sex, including for females during gestation and lactation phases, at 100, 350 or 750 mg/kg bw/day.

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.  The observed differences in food intake for these females was considered to reflect lower demand due to smaller litter size for the treated females.

There was no effect of treatment on food conversion efficiency of either sex during the pre-pairing phase of the study or for males during the post-pairing phase at 100, 350 or 750 mg/kg bw/day.  Visual inspection of water bottles throughout the study did not indicate any effect of treatment for either sex at 100, 300 or 750 mg/kg bw/day. There was no effect of treatment on estrous cycles of females and matting performance were unaffected with all female achieving pregnancy at 100, 350 or 750 mg/kg bw/day. Gestation length was unaffected by treatment at 100, 350 or 750 mg/kg bw/day.

At 750 mg/kg bw/day, the mean number of implantations was lower than control leading to lower litter size from birth, however, there was no effect of maternal treatment on post-implantation loss or post-natal survival. At 100 and 300 mg/kg bw/day, implantation count was lower than control but, to a lesser extent, than observed at 750 mg/kg bw/day, and resulted in slightly lower litter size from birth, although post-implantation loss and post-natal survival were unaffected by treatment. Sex ratio for the offspring was unaffected by maternal treatment at 100, 350 or 750 mg/kg bw/day. At 750 mg/kg bw/day, offspring body weight gain was slightly lower than control, despite the lower litter size at this dosage, leading to lower mean offspring body weight at Day 13 of age.  Litter weight at 750 mg/kg bw/day, was lower than control throughout, initially reflecting the lower litter size and additionally the lower weight gain as lactation progressed.   At 100 or 350 mg/kg bw/day, mean offspring body weight on Day 1 and subsequent mean offspring body weight gain to Day 13 were unaffected by maternal treatment.  Litter weights were lower than control throughout lactation but this reflected the slightly lower litter size at these dosages compared to control. At 750 mg/kg bw/day, there was a higher incidence of small offspring consistent with the lower offspring growth at this dosage, but otherwise, offspring clinical signs appeared unaffected by maternal treatment at all doses.  Mean ano-genital distance for offspring (both sexes) generally appeared longer than control at 100, 350 or 750 mg/kg bw/day, but all normalized for body weight values, for either sex,  were within the normal historic range and this finding was considered to be incidental and of no toxicological significance.   Visible nipple count for male offspring on Day 13 post partum was unaffected by maternal treatment at all treatment dose.

There was no adverse effect of treatment on blood chemistry and hematological parameters at all tested dose.The mean levels of thyroxine (T4) in adult males were statistically significant higher than control at all treatment groups, this effect was not observed in offspring.

At 750 mg/kg bw/day, there was a higher incidence of small offspring at necropsy, consistent with the lower offspring growth at this dosage, but otherwise, offspring necropsy findings appeared unaffected by maternal treatment. Necropsy findings did not indicate any adverse effect of treatment.

There was no adverse effect of treatment on organ weights and histopathology did not reveal any evidence of adverse effects at treatment dosage. It was noted that absolute and body weight-relative thyroid weights were statistically significantly higher than control for females at 350 and 750 mg/kg bw/day.  This increase in thyroid weights appeared to reflect follicular hypertrophy in the thyroid of some females at these dosages, however, neither the incidence of this microscopic finding nor the differences in mean thyroid weights showed any dosage relationship and this finding was considered not to represent an adverse effect of treatment.

Comet assay investigations during the study did not show any treatment induced DNA damage in the jejunum, glandular stomach or liver under the conditions of the test at 100, 350 and 750 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity for the adult animal was considered to be 750 mg/kg bw/day.

Justification for classification or non-classification

The substance does not meet the criteria for classification for specific target organ toxicity - repeated exposure (STOT-RE) in accordance with Regulation (EC) No 1272/2008 (CLP).