Registration Dossier

Administrative data

Endpoint:
developmental toxicity
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD 422 (Combined Repeat Dose Toxicity Study with the Reproduction/Developmental Toxicity 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.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Storage Conditions:Stable at ambient temperature (10-30°C). Store in darkness; may be used/formulated in light.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar HanTM:RccHanTM:WIST strain
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

Administration / exposure

Route of administration:
oral: gavage
Details on 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.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as Day 0 of pregnancy.
- Further matings after unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): housed individually in a cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Any other deviations from standard protocol:no
Duration of treatment / exposure:
6 - 8 weeks
Frequency of treatment:
Daily
Duration of test:
6-8 weeks
Doses / concentrationsopen allclose all
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
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
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.

Examinations

Maternal examinations:
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.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other:
Fetal examinations:
External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
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)
Indices:
Offspring viability indices including; viability index, lactation index and sex ratio.
Historical control data:
Historical control data was available to the Study Director.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
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):
At all dosages, food consumption was lower than control during Days 4-7 of lactation, but group mean values showed no dosage relationship. Food consumption was again lower than control for treated animals during the second week of lactation, with differences being most notable at 750 mg/kg bw/day. These observed differences in food intake of the lactating females may reflect lower demand from the smaller litters for the treated females.
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 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):
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.
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.
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 female at 350 mg/kg bw/day and one female at 750 mg/kg bw/day. 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. 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. 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 100 mg/kg bw/day and both sexes at 350 and 750 mg/kg bw/day, absolute and body weight-relative liver weights were statistically significantly higher than control. For this organ, body weight relative values are generally considered to be the more accurate indicator of any toxicological effect. For males, all individual relative weight exceeded the historical control at 350 and 750 mg/kg bw/day compared to 2/5 in the control group. For females, all individual relative weight exceeded the historical control at 100, 350 and 750 mg/kg bw/day, however, so did 4/5 control values. The increase in liver weight may represent adaptive changes in liver metabolism but as there was no evidence of histopathological change detected for this organ, this finding was considered not to represent an adverse effect.
For females at 350 and 750 mg/kg bw/day, absolute and body weight-relative thyroid weights were statistically significantly higher than control, although mean values showed no dosage relationship. At 350 mg/kg bw/day, 11/12 absolute and 10/12 relative weights and at 750 mg/kg bw/day 9/11 absolute and 7/11 relative weights, exceeded the respective historical control range compared to only 3/12 absolute and 2/12 relative weights in the control group. Follicular hypertrophy was observed in the thyroid of some females receiving 350 or 750 mg/kg bw/day and may be the underlying cause of the observed differences in thyroid weights. In the absence of any dosage relationship this finding was considered most likely to be incidental and not to represent an adverse effect of 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 females at 750 mg/kg bw/day, absolute and body weight-relative adrenal weights were statistically significantly higher than control, however mean values were adversely influence by one female that showed an atypically low adrenal weight. Excluding this animal, all individual absolute and body weight relative weights were within the historical control range. In the absence of any histopathological change this finding was considered to be incidental and unrelated to treatment.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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; at 350 and 750 mg/kg bw/day this finding correlated with a non-dosage dependent increase in organ weight. No histopathological changes in the thyroid were apparent for males and whilst a direct stand-alone change in the thyroid gland cannot be ruled out, it is considered to be less likely when occurring only in females. Additionally, thyroid activity is increased in females which are pregnant/lactating, therefore, this finding, in view of the lack a true dose-response, may represent an uneven incidence of normal variability for these female animals.
Other incidental findings detected at microscopic evaluation of the tissues did not indicate any effect of treatment at 750 mg/kg bw/day. In particularly, 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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Although post-implantation loss and post-natal survival were unaffected by treatment, at 100, 300 & 750 mg/kg bw/day, the mean number of implantations was lower than control leading to lower litter size from birth.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
other: Lower numbers of implantations

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean values for total litter weights, offspring body weights and body weight changes, a summary incidence of clinical signs, ano-genital distance and visible nipple counts (male offspring) are given in Tables 13, 17, 17 and 19. Individual values and observations are given in Appendices 13, 16, 17 and 18.
At 750 mg/kg bw/day, mean body weight of the offspring was similar to control on Day 1, however subsequent offspring body weight gain was slightly lower than control, leading to lower mean offspring body weight at termination on Day 13 of age. This lower gain at 750 mg/kg bw/day occurred despite the lower litter size at this dosage, compared to control. Litter weight at this dosage 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 mean offspring body weight on Day 1 and subsequent mean offspring body weight gain to Day 13. Litter weights were lower than control throughout, but this was considered to reflect the slightly lower litter size at both dosages compared to control.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at 100, 350 or 750 mg/kg bw/day.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, the mean number of implantations was lower than control leading to lower litter size from birth until termination on Day 13. Although one female total litter loss post partum at this dosage, offspring survival for remaining litters was good and there was considered to be no effect of maternal treatment on post-implantation loss or post-natal survival.
At 100 and 300 mg/kg bw/day, the mean number of implantations was also lower than control but, to a lesser extent, than observed at 750 mg/kg bw/day. This resulted in slightly lower litter size from birth although post-implantation loss and post-natal survival appeared unaffected by treatment.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Lower litter size and lower offspring growth

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)
Treatment related:
no
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Any other information on results incl. tables

Table 3. 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 

1(F)

5

975.8

 

434.2

 

933.0

 

423.0

 

963.2

 

285.0

 

401.0

 

0.0

50.8

 

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(F)

5

794.8

 

212.6**

 

883.0

 

343.2

 

1018.0

 

413.8

 

351.4

 

0.0

32.0

 

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 4. Summary Incidence of Sensory Reactivity Assessments (Scores).

Group/ sex

No of animal /Group

Grasp response

 

Vocalisation

 

Toe

pinch

Tail

pinch

Fingerapproach

 

Touchescape

 

Pupilreflex

 

Blinkreflex

 

Startlereflex

 

1

5

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

2

5

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

3

5

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

4

5

0.0

0.0

0.0

0.2

0.0

0.0

0.0

0.0

0.0

Table 5. Group Mean Body Weight and Body Weight Gains Values

Group

Body Weight (g)

 

Increase in Body Weight (g)

 

Day Numbers Relative to Start Date

Gestation

Lactation

Day Numbers Relative to Start Date

Abs gain

% gain

Gestation

Cumulative Body Weight Change (g)Days

 

Lactation

 

Cumulative Body Weight Change (g)Days

 

1

8

15

0

7

14

20

1

4

7

14

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

211.5

 

217.8

 

224.7

 

227.4

 

254.5

 

280.2

 

339.0

 

257.8

 

270.8

 

276.0

 

307.8

 

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

211.5

 

217.2

 

227.5

 

226.9

 

251.0

 

273.8

 

325.8

 

251.6

 

267.5

 

277.7

 

303.1

 

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

206.6

 

214.3 

 

223.5

 

220.9

 

249.8

 

272.4

 

324.7

 

251.0

 

265.8

 

277.7

 

297.9

 

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

211.5

 

215.0

 

222.3

 

220.3

 

241.8

 

265.8

 

315.8

 

249.5

 

261.9

 

268.5

 

290.0

 

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 6. Group Mean Food Consumptions and Efficiency

Group/ sex

Day Numbers Relative to Start Date

 

Group/ sex

Day Numbers Relative to Start Date

 

Gestation

Lactation

 

1 - 8

8 - 15

15 -22

29 – 36

36 - 43

1 - 8

8 - 15

0 - 7

7-14

14-20

1 - 4

4 - 7

7-14

 

1(M)

23.8

23.8

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

22.6

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

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

23.7

25.6

25.8

4(F)

14.6

16.9

19.7

24.2

24.6

34.1

42.0

44.6

 

Table 7. Summary of Reproductive Index

Group

Mating Index (%)

Number of females showing evidence of regular estrous cycling Number of females showing di-estrus

Pregnancy Index (%)

Female with living offspring

Parturition Index (%)

1

100

12

12

100

12

100

2

100

12

12

100

12

100

3

100

12

12

100

12

100

4

100

12

11#

100

12@

100@

# One female found dead on Day 50 (Day 7 of lactation) prior to scheduled necropsy

@ = Includes one female which had a total litter loss

Table 8. Group Mean Litter Size

Group

Number of ImplantationSites

Total Number of Offspring Born 

Number of Live Offspring

Day 1

Day 4 BC

Day 4 AC

Day 7

Day 13

1

12.5

11.9

11.9

11.9

10.0

9.9

9.9

2

11.0

9.8

9.8

9.8

8.4

8.4

8.3

3

10.3

10.0

10.0

9.8

8.4

8.4

8.4

4

9.5

8.6

8.6

8.5

7.7

7.7

7.6

 

Table 9. Group Mean Litter Values for Implantation Losses, Survival Indices and Offspring Sex Ratio

Group

Post implantation loss (%)

Live birth index (%)

Viability index 1 (%)

Viability index 2 (%)

Sex ratio (% males) Post partum day

At birth

1

4BC

4AC

7

1

4.7

100.0

100.0

99.1

45.6

45.6

45.6

51.4

52.0

2

12.0

100.0

100.0

98.5

41.0

41.0

41.0

46.1

46.1

3

1.9

100.0

97.9

100.0

51.4

51.4

51.7

57.0

57.0

4

8.4

99.2

98.9

98.9

40.4

40.8

44.1

44.1

44.1

Table 10. Group Mean Litter Values for Offspring Weights and Litter Weight

Group

Offspring Weight (g)

Litter Weight (g)

Offspring Body Weight Change (g)

Cumulative Offspring Body Weight Change (g)

Day 1

Day 4 BC

Day 4 AC

Day 7

Day 13

Day 1

Day 4 BC

Day 4 AC

Day7

Day 13

Day 1-4 BC

Day 1-4 AC

Day 4-7 AC

Day 7-13

Day 1-7

Day 1-13

M

F

M

F

M

F

M

F

M

F

M

F

M

F

M

F

M

F

M

F

M

F

1

5.97

5.67

8.86

8.43

8.87

8.43

13.91

13.36

26.67

25.83

25.83

68.63

101.58

132.55

255.0

2.90

2.77

2.90

2.77

5.04

4.93

12.76

12.47

7.94

7.70

20.70

20.16

2

6.28

6.01

9.06

8.89

9.07

8.91

13.91

13.92

26.81

26.81

25.83

58.51

83.59

112.58

214.77

2.78

2.88

2.79

2.90

2.79

5.00

12.90

12.98

7.63

7.90

20.53

20.88

3

6.11

5.82

8.69

8.33

8.70

8.34

13.17

12.57

25.56

24.61

25.83

58.68

81.00

107.21

208.71

2.58

2.52

2.58

2.52

2.58

4.23

12.39

12.04

7.05

6.75

19.44

18.79

4

6.18

5.72

8.32

7.79

8.32

7.81

11.96

11.24

22.15

21.24

25.83

49.49

65.55

88.06

163.51

2.13

2.06

2.13

2.09

2.13

3.46

10.19

10.01

5.78

5.51

15.97

15.52

Table. 11. Group Mean Litter Values for Offspring Ano-Genital Distance

Group

Male

Female

Ano-genital Distance (mm)

Body weight on Day 1 (g)

Normalised Ano-genital Distance (mm)

% Male Offspring Showing Visible Nipples

Ano-genital Distance (mm)

Body weight on Day 1 (g)

Normalised Ano-genital Distance (mm)

1

3.18

5.97

1.76

0.0

1.50

5.67

0.84

2

3.36

6.28

1.82

0.0

1.56

6.01

0.86

3

3.30

6.11

1.81

0.0

1.65

5.82

0.92

4

3.56

6.18

1.94

0.0

1.75

5.72

0.98

Table 12. Group Mean Blood Chemical and Haematology Values

Blood chemistry parameters

Female

Group 1

Group 2

Group 3

Group 4

P mmol/l

1.74

1.60

1.46

1.44

ASAT IU/l

106.2

95.4

135.4

101.4

ALAT (IU/l)

104.0

82.6

121.4

125.4

AP (IU/l)

199.4

184.2

245.2

258.4

Creat mg/dl

0.662

0.584

0.536*

0.538**

Chol mg/dl

113.8

142.2

212.2**

200.0**

Bili

mg/dl

0.076

0.088

0.112*

0.120**

Bile acid

μmol/l

23.30

28.32

53.92

50.22

Urea

mg/dl

58.2

52.4

55.8

52.6

Glucose

mg/dl

128.8

124.4

117.8

128.4

Tot. Prot.

g/dl

5.672

5.408

5.010**

4.772**

Abumin

g/dl

3.22

3.08

2.68**

2.52**

A/G

Ratio

1.324

1.336

1.136

1.132

Na+mmol/l

145.4

146.4

144.2

144.2

K+mmol/l

5.418

5.144

5.348

4.864

Cl-mmol/l

102.4

99.6

99.6

100.0

Ca++mmol/l

2.614

2.588

2.478

2.340**

Hematology parameters

Female

Group 1

Group 2

Group 3

Group 4

Hbg/dl

14.30

14.18

13.88

14.02

RBC

10^12/l

6.928

7.252

6.738

7.472

Hct

%

40.40

40.36

39.36

40.66

MCH

pg

20.68

19.64

20.68

18.74*

MCV

f1

58.46

55.56

58.58

54.48

MCHC

g/d1

35.40

35.22

35.12

34.32

WBC

10^9/l

4.78

5.50

6.34*

6.10*

Neut

10^9/l

1.510

1.908

2.278

1.936

Lymp

10^9/l

3.242

3.578

4.062*

4.166*

Mono

10^9/l

0.000n

0.000n

0.000n

0.000n

Eco

10^9/l

0.000n

0.014n

0.000n

0.000n

Bas

10^9/l

0.000n

0.000n

0.000n

0.000n

CT

Seconds

8.58

8.74

8.82

8.78

PLT10^9/l

598.0

613.0

592.2

591.6

APTTSeconds

15.22

14.42

15.90

15.06

Retics%

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

Female

Group 1

Group 2

Group 3

Group 4

Terminal Bodyweight 

307.8

303.1

297.9

290.0

Adrenals

 

0.08984

0.0984

0.08634

0.06886*

Relative weight 

0.030

0.030

0.029

0.024*

Brain (Including Cerebrum,Cerebellum And Pons

 

1.89094

1.88286

1.85480

1.86090

Relative weight

0.631

0.634

0.617

0.629

Heart

1.26666

1.21634

1.24060

1.26134

Relative weight

0.418

0.409

0.413

0.423

Kidneys

1.92546

1.86024

2.11782

1.96906

Relative weight

0.639

0.626

0.702

0.662

Liver

14.9901

16.2430**

18.3088**

19.9560**

Relative weight

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 0.01688

 

0.01684

 

0.01973

 

0.01516

 

Relative weight

0.005

0.006

0.007

0.005

Spleen

0.78504

0.79592

0.76730

0.62180

Relative weight

0.214

0.264

0.254

0.207

Thymus

0.26014

0.28096

0.27406

0.27402

Relative weight

0.087

0.095

0.091

0.091

Thyroid/Parathyroid 

0.02671

0.03178

0.03821*

0.03509*

Relative weight

0.009

0.010

0.013*

0.012*

Uterus & Cervix (And Oviducts)

0.54190

0.57278

0.50861

0.59657

Relative weight

0.177

0.192

0.171

0.171

Applicant's summary and conclusion

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. At 750 mg/kg bw/day, offspring growth was lower than control, therefore, the NOAEL for the offspring was considered to be 350 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.

Lower numbers of implantations were apparent for females at 750 mg/kg bw/day and as the etiology of this finding was unknown, the NOAEL for reproduction was considered to be 350 mg/kg bw/day.  

At 750 mg/kg bw/day, offspring growth was lower than control although there was no accompanying effect on offspring survival or development; the NOAEL for the offspring was considered to be 350 mg/kg bw/day.