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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
(Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 August 2019 - 24 October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Condensation products of phenol and salicylaldehyde
EC Number:
947-320-5
Molecular formula:
Variable as UVCB substance
IUPAC Name:
Condensation products of phenol and salicylaldehyde
Test material form:
solid: flakes
Details on test material:
Description: Red-reddish brown solid flake
Storage conditions: Controlled room temperature (15-25 °C, ≤70 % relative humidity)

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to the Test Facility’s experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for Dose Range Finding study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adults, approximately 12 weeks old at the start of treatment and 14 weeks old at mating.
- Weight at study initiation: Males: 424 – 488 g, females: 245 – 314 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
- Housing: Type II and III polycarbonate cage type. Rodents were group-housed, up to 2 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed (with pups), respectively.
- Diet: ad libitum
- Water: tap water from the municipal supply, as for human consumption from 500 mL bottles, ad libitum.
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY
- Food: the food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Water: water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8200 Veszprém, József Attila u. 36., Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 – 26.3 °C (target range 22 ± 3 °C)
- Humidity (%): 30 – 76 % (target range 30 – 70 %)
- Air changes (per hr): 15 – 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The dosing solutions were administered to the test material or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight.
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was formulated at appropriate concentrations in the vehicle (as a visibly stable homogenous formulation). Formulations were prepared up to 8 days before use (formulation were kept closed, at room temperature until use).

VEHICLE
- Justification for use and choice of vehicle: Propylene glycol was selected as vehicle for this study based on the formulation and analytical trials. The same vehicle was used in the Dose Range Finding (DRF) study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
> Stability of the test material in the vehicle was assessed in the conditions employed on the study during the analytical method validation. In that study, the formulation samples in the 0.5-25 mg/mL concentration range (using propylene glycol as vehicle) were proven as being stable for at least 8 days when stored at 20 ± 5°C.

> Analysis of test material formulations for concentration and homogeneity was performed using a validated HPLC-UV method:
- Sampling: Duplicate samples of approximately 0.5 mL, accurately weighed were taken from the top, middle and bottom of the test material formulations three times during the study (during the first and last weeks and approximately midway during the treatment). Similarly, two sets of duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
- Analytical method: HPLC-UV
- Analytical conditions:
Instrument: Dionex Ultima 3000 UHPLC with UV detection
Column: Waters Acquity UPLC; BEH C18; 100 x 2.1 mm, 1.7 µm
Column temperature: 50°C
Eluent A: water
Eluent B: Acetonitrile
Flow rate: 0.3 mL/min
Elution: Isocratic, 65:35 (A:B)
Detection: 230 nm
Run time: 10 min
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating). Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. The first day of dosing of each animal was regarded as Day 0.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (group 1)
Dose / conc.:
4 mg/kg bw/day (nominal)
Remarks:
Low Dose (group 2)
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Mid Dose (group 3)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
High Dose (group 4)
No. of animals per sex per dose:
12 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELETION RATIONALE / PRELIMINARY STUDY
In the preliminary study, dose-dependent test material related adverse effects were observed in all test-material treated groups (75, 150 and 250 mg/kg/day) with a continuous body weight loss with reduced food consumption in the Mid and High dose males. Also, the degree of the increases in the liver weight relative to the body weight in Mid and High dose females, the doses of 150 and 250 mg/kg bw/day were considered unacceptable for a main study. The effects observed in the 75 mg/kg bw/day dose group were considered to be below the MTD (Maximum Tolerable Dose) and not high enough for the High dose level for the Main study. The choice of 100 mg/kg bw/day for the Main study, based on available data from the preliminary study, was considered to be justified. Based on this information and using a factor of 5, the doses of 4, 20 and 100 mg/kg bw/day were deemed suitable for the purpose of the study; to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.

ANIMAL ASSIGNMENT
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day 0). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.

OTHER
- Fasting period before blood sampling for clinical biochemistry: overnight
- Mating procedure: mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male from the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 14 days. A vaginal smear was prepared daily during the mating period and stained with 1 % aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for signs of morbidity and mortality. General clinical observations were performed daily (during the pre-treatment and treatment period).
- No general clinical observations were made on those days when detailed clinical observations were made (except on one case on 23 October 2019).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the start of the pre-exposure period and once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment.
Observations included:
- Changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern).
- Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
- Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable.
- On gestation day GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during the pre-exposure period, then on Day 0 for randomisation purposes, afterwards at least weekly and at termination.
- Parental females were weighed on gestation Days GD0, 3, 7, 10, 14, 17 and 20 and on post-partal Days PPD0 (within 24 hours after parturition), PPD4, 7, 10, 13 and 14 (before termination).
- The body weight of the female animals measured on gestational Days GD3, 10 and 17 as well as PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

FOOD CONSUMPTION: Yes
- Animal food consumption was determined weekly by re-weighing the non-consumed diet with a precision of 1 g (on the days of body weight measurements). Food mean consumption values (g/animal/day) were calculated and reported.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: 5 male and 5 female animals/group.
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy.
- Animals fasted: Yes (overnight)
- How many animals: 5 male and 5 female animals/group. (Since in the control group included pregnant animals, the Study Director decided not to sample the non-pregnant High dose females for clinical pathology due to a lack of comparative controls.)
- Parameters checked in table [No.2] were examined.

PLASMA/SERUM HORMONES/LIPIDS: THYROID HORMONE ANALYSIS
- Time of blood sample collection: samples were taken over a minimum period during the morning on the day of sampling.
- Method: venepuncture (sublingual, in case of adult animals) or decapitation (in case of pups).
- Animals: from up to two pups per litter on PND4, from all dams and at least two pups per litter on PND14 (females) / PND13 (pups), from all adult males at termination.

URINALYSIS: Yes
- Time schedule for collection of urine: immediately prior to scheduled necropsy.
- Metabolism cages used for collection of urine: Yes (for approximately 16 hours)
- Animals fasted: Yes (overnight)
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the last exposure week (males on Day 23; pregnant females on PPD9-10; non-pregnant females (High dose group) on Day 45).
- Dose groups that were examined: All (5 males and 5 females/group)
- Battery of functions tested: quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength. Additionally, sensory activity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted and the general physical condition and behaviour of animals were tested. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex and vocalisation were evaluated. Locomotor activity assessment was conducted using an Automatic Monitoring System (SMART v. 2.5, Harvard Apparatus, Germany).

IMMUNOLOGY: No

OTHER: Yes
- Oestrous cycle monitoring
- Observation of the delivery process, offspring, and nursing instinct
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- At termination, all adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.
- Special attention was paid to all rats which failed to mate or litter after pairing, to establish potential causes relative to potential treatment related effects.
- Males were killed on Day 29, High dose females were killed on Day 29 (preterminal euthanasia) and on Days 41-46 (terminal euthanasia) and low and mid dose females were killed on PPD14. Offspring were killed on PND4 (culled animals) or on PND13.
- Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically.
- Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.

- Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
- The number of implantation sites and of corpora lutea were recorded in female animals as applicable.
- Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities, where possible. The anaesthetic product was diluted for pups’ euthanasia as required.

ORGAN WEIGHTS: Yes
At the time of termination, body weight and the weight of the following organs from all adult animals were determined:
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus,
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids.
Testes and epididymides were weighed individually.

HISTOPATHOLOGY: Yes (see table 4)
For the adult animals, a detailed histological examination was performed as follows:
- On the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/Control and 5 males and 5 females (although they were not pregnant and not totally comparable with the pregnant controls) plus the 2 females of High dose group which failed to mate.
- All organs where macroscopic findings (abnormalities) were seen.
- Retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups and of all males that failed to sire and all females that failed to deliver healthy pups.

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Due to the lack of pregnancy and histological findings at the High dose group, further histopathological examination and a detailed peer review was made on the following reproductive tissues of adult animals:
- Mid and low dose female reproductive organs, compared with Control (uterus, cervix, ovary, oviduct and vagina);
- Ovaries, testis and epididymis of all Control, Low, Mid and High Dose Animals;
- Secondary sex organs of males from Control, Low, Mid and High dose.
Statistics:
- Data were recorded on the appropriate forms and then tabulated using the Microsoft Office Word and/or Excel, or collected using the software PROVANTIS v.9, as appropriate.
- Group means and standard deviations were calculated from numerical data obtained in the study. The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 (when using Provantis) or with the program package SPSS PC+4.0 (SPSS Hungary Kft., Budapest), as follows:

SPSS PC + 4.0
- Heterogeneity of variance between groups: Bartlett's test.
- Not a significant result: one-way analysis of variance (ANOVA).
- Significant result: Duncan's Multiple Range test is used to assess the significance of inter-group differences.
- Significant heterogeneity: the normal distribution of data is examined by Kolmogorow-Smirnow test.
- Non-normal distribution: non-parametric method of Kruskal-Wallis One-Way analysis of variance
- Normal distribution: inter-group comparisons performed using Mann-Whitney U-test.
- Non-continuous data: Chi-squared test.

SAS 9.2
- Normality and heterogeneity of variance between groups: Shapiro-Wilk and Levene tests.
- No significant heterogeneity: Anova / Ancova (one-way analysis of variance) test.
- Positive result: Dunnett’s (Multiple Range) test.
- Significant heterogeneity: non-parametric analysis is required.
- A Kruskal-Wallis analysis of variance is used after Rank Transformation. If there is a positive result, the inter-group comparisons are performed using Dunn test.
- Non-continuous data: Cochran-Armitage test for trend. Chi-squared test is used for statistical differences relative to control.
- Pathology data (macroscopic and microscopic data): Cochran-Armitage test for trend is applied, then if appropriate, the Chi-squared test homogeneity test.
- If significance is plausible based on a user-defined value (0.05): pairwise test of each treatment group versus the control group.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related clinical signs were observed during the study. There were no abnormal clinical signs in any animal during the study, excluding one female in the Mid dose group where a whole body tonic convulsion was observed on sporadic occasions between Day 26 to Day 43. In the absence of any other similar findings at this dose level or higher, this finding was considered to be incidental, and not related to the test material.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed during the study.
Two High dose females were euthanized (on Day 29), because they had no evidence of positive mating. Tissues were examined to try to identify any reason for lack of mating/pregnancy. No cause could be identified after microscopic examination and the uterus of one of these animals had signs of oestrus present.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES
In the first 7 days of treatment the Low dose had a transient, statistically lower mean weight gain (7.8 g) compared to Controls (19.8 g), however this was fully compensated during the study and hence this observation was not considered as adverse.
The males of the Mid dose group lost body weight (a negative weight gain) in the first 7 days (-8.6 g) but gained weight thereafter; males of the High dose group lost body weight in the first 7 days ( 14.5 g), and lost further body weight (-8.8 g) from day-7 to day-14 of the study. The Mid dose gained some weight during the second week (10.5 g), which was 51% less (p<0.05) than the concurrent controls (21.6 g) over the same period. Despite some compensatory weight gain in the last two weeks of dosing, at the end of the 28-day period the Mid dose males had a slightly lower (4.9%) mean body weight (481.4 g) than the controls (506.2 g), total weight gain for mid-dose males was about 50% of the controls, mainly attributed to a significant weight loss in the first 7 days.
The male High dose animals gained some weight in the second half of the dosing period, but the mean body weight remained below the starting weight for the entire treatment period (Days 0 - 28). These animals displayed no clinical signs of toxicity and were not recorded as being thin or abnormal in any way. However, the body weight effect on High dose males was clearly adverse. The initial significant effect on Mid dose males in the first one to two weeks of the study, was followed by above control body weight gains in weeks 3 and 4 of the study (overall greater weight gain than controls from Day 7 to end of study). Hence, their weight effect was considered to be transient, but the significant loss in body weight in the first 7 days is a clear adverse effect.
The degree of effect seen in the High dose group was a final body weight 12.4 % below controls with 122.6 % less weight gained overall when compared with controls. The degree of body weight effect in this study was close to the 10 % guideline value, but the overall loss of weight during the study was considered to exceed the MTD.
At the Mid dose the Day 28 body weights were 4.9 % below controls, which is fully acceptable for a standard toxicology study though this was statistically significant. However, considering the body weight loss in the first week of dosing and the 51 % reduction in weight gain during the second week, despite recovery gains for the remaining period, this affect was considered to be adverse but not severe as overall weight gain for the total dosing period was 49.4 % less than controls.

FEMALES
In the first 7 days of treatment (pre-mating period) the Low dose had a lower mean weight gain than controls (5.7 g and 16.3 g for the low dose and controls, respectively) but it was not statistically significant and during the second week, the weight gain was very similar to controls (approx. 3 g for low dose and controls).
The Mid and High dose groups lost body weight during the first week of the treatment period (-4.8 g and -13.2 g in the Mid and High dose, respectively), but both groups gained weight in the second week. By the end of the pre-mating period, the females of the control group had gained an average of 19.1 g bodyweight, low-dose females had gained 8.7 g, mid-dose females had lost an average of 1.2 g and high-dose females had lost an average of 0.3 g, compared to body weights at the start of dosing.
During the mating period, Control and Low-dose females generally recorded gains in body weight; Mid and High dose females showed static bodyweights or body weight losses. However, negative values are very common in the historical control database (~20 % of rats) so this group difference was not an adverse effect.
As the High dose females were not pregnant and therefore had no offspring, body weight data for pregnancy and lactation periods were not comparable with controls. Only the Low and Mid dose body weight and body weight gain values of the treated females were evaluated statistically during the gestation and lactation periods.
Group mean body weights of the Mid dose females were statistically significantly lower than controls on day 0, 14 and 20 of gestation. During the gestation period the Mid dose had significantly lower weight gain (by 25%, p<0.01) than Controls, such that group mean body weight of Mid dose females (387.3 g) was 14.8% lower than Control (454.6 g) on day-20 of gestation. The low dose was not affected.
During the lactation period the Control, Low and Mid groups gained a similar weight. However, group mean body weight for Mid-dose females remained significantly lower than controls for the whole period (days 0 to 13, 13.7% to 11.2% lower than controls, respectively).
The observed body weight or body weight gain differences between the female groups showed no adverse effects during gestation or lactation in the Low dose, though markedly less weight was gained during gestation (-25 %) in the Mid dose. - This affect was considered as adverse. The High dose female data was not comparable with controls due to a lack of pregnancy in this group, but during pre-mating period there was a significant but transient body weight loss which though not as severe as the effect in males at the same dose, was still considered to be an adverse effect of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
MALES
The Low dose were unaffected by treatment; Mid dose males were generally lower (-0.6 - 32.3 %) than controls and the overall intake was slightly but statistically significantly lower (-16.3 %) than control; High dose males had significantly lower ( 34.8 %) food intake throughout the study.

FEMALES
The Low dose had slight but statistically significant lower food intake as a total in the first 14 days (-16.2 %) and during gestation (-10.3 %), however the percentage differences were relatively small and were not considered to represent an adverse effect of treatment. The Mid dose group had food intake values of about 20-40 % lower than controls during each phase of the study, this was considered to be an adverse effect of treatment. High dose females had an intake of over 60 % lower than control in the first week and almost 50 % lower for the total of the first 2 weeks which was considered as an adverse effect (no further comparisons were made since this group were not pregnant and not comparable with control).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related adverse changes were observed in the haematology parameters.
High dose females were not pregnant during this study, therefore the Study Director decided parameters were not comparable with pregnant controls, so the Low and Mid dose group were sampled only.
Decreased thromboplastin time (APTT) achieved statistical significance in High dose males, but data was well within the historical control range, so it was considered to be unrelated to treatment, further partial thromboplastin time was similar in all groups.
Slightly decreased MCHC and increased numbers of Large Unstained Cells (LUC) achieved statistical significance in Low and Mid dose females respectively, but also within historical control range. The significant differences were considered to be incidental as there was no relationship with dose, and therefore these differences were considered to not reflect an effect of the test material.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
For all statistically significant differences, either the histopathology results (with non-adverse adaptive responses) confirm a lack of treatment related adverse effects, or they were considered to be incidental, with no relationship with dose and/or all recorded values were near or within the historical control ranges. Hence, it is considered that the test material had no adverse effects on Clinical Chemistry parameters, statistical differences were considered to incidental and unrelated to treatment.
High dose females were not pregnant during this study, therefore the Study Director decided parameters were not comparable with pregnant controls, so the Low and Mid dose group were sampled only.
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis: There were no significant differences between the control and test material treated male animals for T4 hormone analysis. The test material had no effect on measured thyroid hormone levels.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No clear test material-related changes were observed in the urinalysis parameters. The significant differences were considered to be incidental, not reflecting an effect of the test material.
High dose females were not pregnant during this study, therefore the Study Director decided parameters were not comparable with pregnant controls, so the Low and Mid dose group were sampled only.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted during the assessment of grip strength, foot splay or locomotor activity.
Whilst fore and hind grip strength was reduced in Mid and High dose males when compared with controls, changes lacked statistical significance or dose response and remained within the historical control range. The occasional statistically significant differences in grip strength compared to control in the Low and Mid dose females were not considered as test material related effects as the observed values were in the middle of the historical control range, with control values close to the maximum value of the historical control data.
All males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. High dose males appeared to be slightly less active than controls, but High dose females appeared to be slightly more active; but all mean results were within the historic control range. There was no statistical significance between the test material treated animals and the Control when evaluating the overall distance travelled. The test material was not considered to have had a significant effect on locomotor activity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weights of Mid and High Dose animals were statistically significantly lower (by 5.5% or 15.0% in males and 14.9% or 27.2 % females, respectively, though high dose females were nulliparous and not comparable to the lactating controls)
As High dose females were nulliparous, organ weights were not statistically analysed for this group being non-comparable to controls, but the mean and percentage difference to control were tabulated and evaluated by the Study Director.

MALE ORGAN WEIGHTS
- Prostate: Statistically significantly lower than Control in the High Dose group. The absolute prostate weights were lower by 57.2 %, related to body weights by 49.7 % and by 56.7 % when brain-related, respectively. In the Mid dose males, the absolute prostate weights were statistically significantly lower than Control by 16.9 %, and by 15.7 % when brain-related, respectively.
- Seminal vesicle: Statically significantly lower than Control in the High Dose group. The absolute seminal vesicle weights were lower by 52.6 %, related to body weights by 44.2 % and by 52.0 % when brain-related, respectively. In the Mid dose group the absolute seminal vesicle weights were statistically significantly lower than Control by 14.9 %, but not statistically lower when adjusted for body or brain.
- Liver: Statistically heavier than control at the High dose group, when adjusted for body weight; the adjusted weight was above the Historical Control range. Weights were only minimally different and no histopathological differences were observed; this profile is common for substances causing a non-adverse, slight hepatic hypertrophy though none was evident on the study.
- Other: There were statistically significantly changes in the organ weights that were attributed to the 15 % lower bodyweight, where organs that are normally in the same body weight ratio were ~15 % lower absolute weight, but not statistically different when adjusted for body weight. These included kidney and heart. Other organs were not statistically different for absolute weight, but larger than control when adjusted for the reduced body weight, such as brain, spleen, adrenal and testes. The brain adjusted weights were generally similar is statistical differences to the absolute weights.

FEMALE ORGAN WEIGHTS
No treatment-related adverse effects on organ weight were observed in Mid and Low dose females when compared with controls. The body weights of the Mid dose females were about 15% lower than control, so similarly to the cases with smaller males, a range of organs showed statistical differences related to body weight differences. However, these sporadic, statistically significant differences (absolute and/or relative to body and brain, of the adrenals, kidneys, uterus and ovaries) lacked any histopathology changes.
The High dose females were about 27 % lower bodyweight, taking this into account, there were no organs showing evident changes in weight due to a direct test material effect. The lack of any histopathological changes in organs at the High dose confirms this conclusion.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
PRETERMINAL EUTHANASIA
Two High dose females were pre-terminally euthanized on Day 29 because they did not mate during the mating period. No test material-related macroscopic findings were seen at necropsy. The uterine body and horns of one animal was dilated, filled with clear fluid and was considered as background change.
- NON PREGNANT FEMALES
Necropsy did not show any test material related change in the non-pregnant animals.

TERMINAL / PARENTAL GENERATION
Test material-related changes were observed in the prostate and seminal vesicles of High dose males. The prostate was small in 6/12 and seminal vesicles were small in 4/12 High dose males. These changes correlated with the organ weight changes, with the mating data and microscopic findings. All other changes were considered incidental or a common background finding.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
PRETERMINAL EUTHANASIA
No test material-related microscopic findings were observed. In the uterus of the animal with the dilated uterine body and horns, signs of oestrus was seen and considered to be normal background for non-pregnant animals and consistent with other sex organ histology for this animal.
- NON PREGNANT FEMALES
Organs from the reproductive system (ovary, oviduct, uterus, cervix) were microscopically examined from these animals and did not reveal any test material-related changes.

TERMINAL
Males:
Test material-related findings were observed in the prostate and seminal vesicles of the High dose males, corresponding with the macroscopic observations and the organ weight data. In the prostate of 12/12 High dose males diffuse atrophy was seen with predominantly mild/moderate severity. Increased incidence and severity of focal/multifocal inflammatory cell infiltrate was seen in High dose males (6/12 with predominantly mild/moderate severity) compared to the 3/12 in Control males with minimal severity. In the seminal vesicles of 10/12 High dose males diffuse atrophy was seen with predominantly mild/marked severity.
Other than these High dose male findings in secondary sex organs, there was no evidence for systemic toxicity in the organs or tissues of High dose males or females.

Females:
The ovaries and uterus of High dose animals were not different from tissues of normal non-pregnant rats. The ovaries and uterus of Mid and Low dose animals were not different from tissues of normal pregnant rats. The ovary, uterus and vagina histology were checked against the vaginal smear taken at necropsy to check for any signs of desynchrony. Generally, there were no unusual findings although a non-specific change of an increased incidence of mucification in the vagina was identified; in these cases the degree of mucification was slightly above the normal control range. A Mild increase in mucification was observed in 9/12 Mid dose females, a similar change in the vagina was seen in 2/12 High dose females. None were seen in the Low dose. This change could indicate abnormal cycles, but it is not a clear adverse effect and was not associated with any other detectable changes in sex organs. This mucification did not reflect the proestrus phase.
Mid and Low dose males and Low dose females had normal reproductive tissues with no differences in incidences or findings compared with controls.
All other changes were seen in control and/or treated animals, or without meaningful differences in severity and incidence, therefore were regarded as incidental or a common background.

There were no indications from the female reproductive tissues of the High dose group as to why there were longer time to mating and no implantations at this dose level. The High dose males showed persistent body weight loss, and small secondary sex organs with evidence of atrophy. The atrophy of prostate and seminal vesicles would be expected to cause significantly reduced semen fluid quantity and/or quality in High dose males. The cause of the reduced size/atrophy was considered to be correlated with body weight loss; this is a common correlation.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Reproductive parameters were adversely affected; females were acyclic before and during pairing, mating was delayed and despite positive mating, there were no implantations; microscopically female reproductive organs/tissues were normal except for a mild increase in vaginal mucification of unclear significance. It is unclear whether effects in High dose females could represent a direct effect of treatment or a secondary effect of body weight. High dose males had smaller prostate and seminal vesicles which were atrophic; the body weight loss was considered to be sufficient to cause the effect in secondary sex organs, hence the male sex organ effect was not ascribed to a direct effect of the test item. The testes and epididymis were unaffected at the High dose.
The changes in male secondary sex organs were sufficiently significant that they would be expected to have affected the quantity and quality of seminal fluid in the semen. The lack of any implantations or pregnancy at the High dose could be related to the male effects, or possibly to observed oestrous cycle changes (with no evidence of adverse histological changes in reproductive tissues) and delayed time to mating observed. Since both sexes had body weight loss with apparent changes in reproductive system/function, it is unclear whether effects in both males and females may have contributed to the adverse finding of no achieved pregnancies.
The lower number of Mid dose pups born is considered to be of equivocal relationship with treatment. At the Low dose there was no indication of effects of treatment.
There were no endocrine changes seen in nipple retention, anogenital distance, thyroid gland weights or thyroid hormone level.
Details on results:
In High dose males, a significant loss of bodyweight, which did not recover during the 28-day period of the study, and atrophy of secondary sex organs was observed. In High dose females there were also significant body weight losses during the first week followed by an increased weight gain for the following 14 days, but from this point the body weights were practically static. Although there are no comparable concurrent non-pregnant female body weights, the lack of weight gain from about the 3rd week of treatment is considered to be a treatment effect. Changes in oestrous cycling (persistent dioestrus/pseudo pregnancy) and increased time to mating, with no implantations in any of the High dose female animals may have been influenced by body weight, although the degree of weight change does not conclusively imply a causal effect.
Histology of all primary sex organs of both sexes were normal. Histologically a mild increase in vaginal mucification was found in High dose females without dose response, which was of unclear significance; considering the successful mating at the mid dose (which also had mucification), and a lack of any other microscopic abnormalities in the reproductive tract of High dose females. Whether effects on oestrous cyclicity at the high dose reflected a direct effect of test item or secondary effect of the body weight loss is unclear.
In High dose males, the main observed effect was on the secondary sex glands with prostate and seminal vesicle atrophy evident. Whilst this may have contributed to the reproductive failure as it could be expected to reduce semen fluid quantity/quality, semen was visible in the vaginal smears. Furthermore, as females were acyclic before and during mating, indicating a lack of ovulation for fertilisation, this may have also contributed to the infertility at the high dose.
In the Mid dose group, all measured parameters were within a range for control animals but some statistical differences were seen between the concurrent controls and the Mid dose for body weight, prostate and seminal vesicle weights (but no histological evidence of any change) and a slightly lower number of pups born. In evaluating the significance of the lower number of pups born, there were no significant effects on associated factors: The number of corpora lutea, implantations and prenatal mortalities were all in the normal range, with no evidence of an effect of treatment; hence the reason for low number of pups born (p<0.05) is not clearly an effect of treatment. However, the difference cannot be definitively declared to be unrelated to treatment considering the systemic toxicity also apparent in these females. Based on this evaluation, this statistical difference was not considered as clearly adverse but a treatment effect is considered to be equivocal. Mid dose as well as High dose had an increased incidence of vaginal mucification, but the expert opinion of the histopathologist and peer reviewer was that this was not a clearly adverse effect. Similarly, the male secondary sex organ weight differences in Mid dose animals were relatively small and there were no histological changes, hence these statistical differences were not considered to be adverse. Overall, at the Mid dose, there were a number of apparent trends in endpoints related to reproduction, but careful analysis of the data in relation to current and historic controls, did not identify any change which could be considered clearly as an adverse effect of the test item. The statistically lower number of pups born is considered of equivocal relationship with treatment; taking a conservative approach, this finding should be considered as a potential effect when assessing the NOAEL.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Dose formulation analysis

All test material formulations were shown to be homogeneous. The measured concentrations of test material evaluated for each test material treatment group varied between 98.3 % and 105.0 % of the nominal concentrations. The relative standard deviation (RSD) was below 5 % in each case. No test material was detected in the control samples. These results were within the acceptable ranges (85 % - 115 %) and were considered suitable for the study purposes.

Selected body weight and body weight gains in males

Parameter (g)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Mean body weight

Day 0

454.9

455.4

455.5

454.8

Differences from control (%)

0.1

0.1

0.0

Mean body weight

Day 7

474.8

463.2

446.9**

440.3**

Differences from control (%)

-2.4

-5.9

-7.2

Mean body weight

Day 14

496.3

484.2

457.4**

431.5**

Differences from control (%)

-2.5

-7.8

-13.1

Mean body weight

Day 21

499.0

492.6

465.8**

435.0**

Differences from control (%)

-1.3

-6.7

-12.8

Mean body weight

Day 28

506.2

506.0

481.4*

443.3**

Differences from control (%)

0.0

-4.9

-12.4

Mean body weight gain

Day 7 – 28†

31.5

42.8

34.5

2.9

Differences from control (%)

26.4

9.5

-91.0

Mean body weight gain

Days 0 – 28

51.3

50.6

25.9**

-11.6**

Differences from control (%)

-1.3

-49.4

-122.6

Note: *= p<0.05, **= p<0.01; Dunnett two sided test,=Not analysed statistically.

Body weight changes in females during the pre-mating and mating period

Parameter (g)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Mean body weight

Day 0

280.3

280.6

279.9

280.2

Differences from control (%)

0.1

-0.1

-0.1

Mean body weight

Day 7

296.7

286.3

275.2

267.0

Differences from control (%)

-3.5

-7.2

-10.0

Mean body weight

Day 14

299.4

289.3

278.8

279.9

Differences from control (%)

-3.4

-6.9

-6.5

Mean body weight

Day of sperm positive smear

308.3

292.2

277.3*

277.0*

Differences from control (%)

-5.2

-10.0

-10.2

Note: *= p<0.05, **= p<0.01; Dunnett two sided test

Days(s) relative to start date.

Body weight changes in females during the gestation period

Parameter (g)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Mean body weight

Day 0

308.3

292.2

277.3*

277.0*

Differences from control (%)

-5.2

-10.0

-10.2

Mean body weight

Day 7

340.5

322.1

307.8

290.3

Differences from control (%)

-5.4

-9.6

NA

Mean body weight

Day 14

378.6

354.5

329.8**

291.6†

Differences from control (%)

-6.4

-12.9

NA

Mean body weight

Day 20

454.6

435.6

387.3**

287.4

Differences from control (%)

-4.2

-14.8

NA

Note: *= p<0.05, **= p<0.01; Dunnett two sided test,=Not analysed statistically as animals were not pregnant.

Day(s) relative to mating.

Food consumption of females during the premating period

Parameter (g/animal/day)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Days 0-7

17.87

13.87

10.45**

6.55**

Differences from control (%)

-22.4

-41.5

-63.4

Days 7-14

19.00

17.04

15.82*

13.19**

Differences from control (%)

-10.3

-16.7

-30.6

Days 0-14

18.43

15.45**

13.14**

9.87**

Differences from control (%)

-16.2

-28.7

-46.4

Note: *= p<0.05, **= p<0.01; Dunnett two sided test

Day(s) relative to start date.

Food consumption of females during the gestation period

Parameter (g/animal/day)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Days 0-7

22.7

20.47

18.69**

15.31

Differences from control (%)

-10.1

-17.9

NA

Days 7-14

24.99

22.40*

19.49**

15.76

Differences from control (%)

-10.3

-22.0

NA

Days 14-20

27.63

24.77*

21.54**

14.27

Differences from control (%)

-10.4

-22.0

NA

Days 0-20

25.01

22.44*

19.83**

15.16

Differences from control (%)

-10.3

-20.7

NA

Note: *= p<0.05, **= p<0.01; Dunnett two sided test,=Not analysed statistically as animals were not pregnant.

Day(s) relative to mating.

Selected haematology parameters in males

Haematology (males)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Activated Partial Thromboplastin Time (sec)

12.62

12.14

11.82

11.16 *

Differences from control (%)

-3.8

-6.3

-11.6

Historical control data (min – max)

8.4 - 14.4

Note: *= p<0.05; Dunnett two sided test.

Selected haematology parameters in females

Haematology (females)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL)

33.52

32.94 *

33.06

-

Differences from control (%)

-1.7

-1.4

-

Historical control data (min – max)

32.1 – 37.3

Large Unstained Cells

(%)

0.38

0.44

0.64 *

-

Differences from control (%)

15.8

68.4

-

Historical control data (min – max)

0.1 – 2.1

Note: *= p<0.05; Dunnett two sided test.

Selected clinical chemistry parameters in males

Clinical chemistry
(males)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Cholesterol concentration (mmol/L)

1.162

1.204

1.428+

1.716++

Differences from control (%)

3.6

22.9

47.7

Historical control data (min – max)

1.16 – 2.13

Creatinine concentration (μmol/L)

58.30

62.96

59.12

79.08 **

Differences from control (%)

8.0

1.4

35.6

Historical control data (min – max)

38.4 – 79.6

Calcium concentration (mmol/L)

2.556

2.548

2.752 **

2.782 **

Differences from control (%)

-0.3

7.7

8.8

Historical control data (min – max)

2.31 – 2.87

Total Protein concentration (g/L)

51.38

51.66

51.48

55.26 **

Differences from control (%)

0.5

0.2

7.6

Historical control data (min – max)

50.4 – 59.7

Albumin concentration (g/L)

28.08

28.84

28.58

30.64 **

Differences from control (%)

2.7

1.8

9.1

Historical control data (min – max)

26.1 – 33.7

Chloride

(mmol/L)

98.28

98.16

98.26

101.24+

Differences from control (%)

-0.1

0.0

3.0

Historical control data (min – max)

97.2 – 107.9

Note: += p<0.05, ++= p<0.01; Dunn two sided test.

 **= p<0.01; Dunnett two sided test.

Selected clinical chemistry parameters in females

Clinical chemistry (females)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Total Bilirubin concentration (μmol/L)

6.26

6.62

9.00 *

-

Differences from control (%)

5.8

43.8

-

Historical control data (min – max)

2.9 – 8.1

Note: *= p<0.05; Dunnett two sided test.

Significant differences in urinalysis parameters in males

Urinalysis (males)

Groups/Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Urine bacteria

1.0

2.8+

2.4

3.0+

Note:+= p<0.05; Dunn two sided test.

Significant differences in urinalysis parameters in females

Urinalysis (females)

Groups/Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

pH

6.00

6.20

7.20+

-

Specific gravity

1.0260

1.0250

1.0150+

-

Note:+= p<0.05; Dunn two sided test.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study the NOAEL for repeated dose toxicity of the test material in adults was considered to be 4 mg/kg bw/day.
There were accessory sex organ changes but these are considered as a secondary effect to the systemic toxicity, rather than an indication of specific organ toxicity. As such, in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to repeated dose toxicity.
Executive summary:

The repeated dose oral toxicity of the test material was investigated in accordance with the standardised guideline OECD 422 under GLP conditions. The study was conducted with repeated (daily) administration of the test material (0, 4, 20 and 100 mg/kg bw/day) by oral gavage to Wistar rats. The study included a reproductive / developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 13 post-partum. Dose levels were based on the results of a dose range finding study. During the study, groups of 12 male and 12 female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13.

Parameters measured during the study included signs of morbidity and mortality twice daily, daily general and/or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessments, including a functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until post-natal day (PND13).

At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals and/or offspring. The thyroxine (T4) levels in the Day 13 pups and adult males were also assessed. For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

Under the conditions of the study, daily administration of the test material did not result in test material-related mortality and no treatment related clinical signs were noted.

The body weight, body weight gain, and food consumption of the High dose animals were significantly and adversely affected. Males lost body weight in the first 14 days of treatment, with some recovery, but the mean weight remained below the starting weight at termination at the High dose. Male High dose body weight on Day 28 was 12.4 % below controls. Females of the High dose also lost weight in the first 7 days, but regained their original mean weight at about day 14. However, weight gain was lower than control; from about the third week of treatment, High dose females failed to continue gaining bodyweight. Since High dose females were not pregnant, subsequent statistical comparison of their body weight with the pregnant control females was not made. Markedly lower food intake of High dose animals of both sexes reflected the body weight effects.

Mid dose males and females also lost body weight in the first 7 days. Whilst some weight was gained thereafter, in males overall weight gain was still 50 % lower than controls, and in females, lower gain was evident in the mating, gestation and lactation periods resulting in a mean body weight approximately 12 % lower than controls by the time of termination. The body weight effect was considered to be transient but adverse in Mid-dose group males and females. Food intake values of the Mid dose reflected the body weight in both sexes. There was no significant effect on the Low dose for body weight or food intake.

At the functional observation battery (FOB) there were no adverse changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups. No clearly adverse test material-related findings were seen in the clinical pathology parameters of animals that were assessed. There were no effects on thyroid hormone levels of adult males.

There was a significant effect of treatment on reproductive parameters in High dose animals. Oestrous cycling data for High dose females showed 11/12 females were in a state of persistant dioestrous/pseudopregancy before and during pairing, and mating was delayed beyond historical control maximum periods. Although, 10/12 females recorded sperm positive smears during the mating period (lower than controls), no High dose females were pregnant and at necropsy there was no evidence of uterine implantations, indicating that the 10 mated females probably did not have fertilised ova. Considering the adverse effects on body weight in the first 7 days, and lower body weights that were evident in the pre-mating and mating period for females of this group, it is unclear whether effects on oestrous cyclicity could be a direct effect of treatment or a secondary effect of body weight.

There were no differences between the control and Low or Mid dose groups with regard to mating, reproductive ability, gestation indices or duration of pregnancy.

There were no effects on the number of Low dose pups born, or live born, or on F1 offspring viability, clinical signs or at observations following euthanasia. At the Mid dose, the number of pups born and born alive was statistically less than the controls, but individual values remained within the normal historical control range. There were statistical differences in pup growth (body weight gain) at the low and mid-dose levels; however, body weight and body weight gain values were within the normal range, and there was not a dose response; therefore, this was not considered to be a treatment effect. Considering the changes in reproductive parameters at the high dose, a test material effect on the number of pups born and live born pups in the Mid dose group could not be excluded; however, the relationship with the test material was considered to be equivocal since this dose level was also responsible for an adverse effect on body weight in adult females. It was considered that the test material did not affect the Mid dose pup post-natal development/survival.

There were no treatment related changes in endocrine sensitive endpoints in the pups at any of the dose levels (anogenital distance, nipple retention, thyroid gland weights, thyroid hormone level).

There were no adverse effects of treatment on organ weights of either sex at any dose level other than for lighter male secondary sex glands (prostate and seminal vesicles) at the high dose; higher liver weights in High dose males were considered to be adaptive though there was no histopathological change and statistical differences in kidney and other organ weights were considered as secondary to body weight differences.

At histopathology, High dose prostate and seminal vesicles showed atrophy, reflecting the organs weight effects; this was considered secondary to the body weight loss with low food intake throughout the study in males. There was no evidence of a direct effect of the test material on the other reproductive organs in males, with the testes and epididymis normal. There were no histological changes seen in the Mid or Low dose secondary sex organs.

Careful microscopic examination of all the female reproductive organs/tissues found no adverse effect of treatment in the High dose group. Histologically, a mild increase in vaginal mucification was found in Mid and High dose groups which was of unclear significance and lacked clear dose response.

In High dose males, the main observed effect was on the secondary sex glands with prostate and seminal vesicle atrophy evident. Whilst this may have contributed to the reproductive failure as it could be expected to reduce semen fluid quantity/quality, semen was visible in the vaginal smears. Furthermore, as females were acyclic before and during mating, indicating a lack of ovulation for fertilisation, this may have also contributed to the infertility at the high dose.

Other than the histological effects seen in High dose male secondary sex organs, there were no histological adverse changes attributed to treatment with the test material in any group of either sex (the sex organs of all groups were examined).

Based on transient effects on body weight/food intake at the mid dose, and persistent body weight/food intake and secondary male sex organ effects at the High dose the NOAEL for repeated dose toxicity of the test material in adults was considered to be 4 mg/kg bw/day.