Fatty acids, linseed-oil, reaction products with bisphenol A-epichlorohydrin polymer and glycidyl neodecanoate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

repeated dose toxicity: oral, other

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:

From May 24, 2016 to July 09, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Not considered to adversely affect the results or integrity of the study

Qualifier:

according to guideline

Guideline:

other: and OECD guidance document No. 43

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Batch no.: #210162718
Purity 00 % as per the definition of a UVCB substance
Appearance: brown liquid

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The guideline was designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Species and strain: Crl:WI Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D 97633, Sulzfeld, Germany) from SPF colony
Justification of species/strain: the rat is regarded as a suitable species for toxicology and reproduction studies. The Wistar rat was selected due to 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 (CiToxLAB study code: 15/512-220PE).
Number of animals: 48 male, 48 female rats, 4 groups. Each group contained 12 animals/sex. Animals originated from different units, to avoid brother/sister mating. A sufficient number of spare animals were ordered for the study, those animals were allocated to the spare colony of the Test Facility after the in life phase had been finished.
Age of animals: young adult rats, at least 10 weeks old at the start of treatment and 12 weeks at mating.
Body weight range: males: 411-486 g, females: 233-272 g at the start of the treatment; values did not exceed ± 20% of the mean weight for each sex.
Acclimation period: 12 days

Husbandry:
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.6 – 25.7°C (target range: 22±3°C)
Relative humidity: 31 – 69% (target range: 30-70%)
Ventilation: 15-20 air exchanges/hour
Food and water: Ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by Ssniff Spezialdiäten GmbH, (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany) ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.

Route of administration:

oral: gavage

Details on route of administration:

Test substance or vehicle control treated animals were administered the dosing formulations daily on a 7 days/week basis by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 4 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on most recent individual body weights. Dosing of both sexes began after at least 5 days of acclimation and 2 weeks before mating and continued up to the day before necropsy.

Vehicle:

corn oil

Details on oral exposure:

The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from a Dose Range Finding (DRF) study in the rat (CiToxLAB study code 15/512-220PE). The aim was to use a maximum of 1000 mg/kg bw/day or to induce toxic effect(s), but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Based on the results from these preliminary study (where there was no serious toxicity at 1000 mg/kg bw/day), doses of 100, 300 and 1000 mg/kg bw/day were selected for this main study. The oral route was selected, as it is a possible route of exposure to the test item in humans.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC-UV

Details on analytical verification of doses or concentrations:

- The test substance was formulated in the vehicle, as a clear solution at the appropriate concentrations according to the selected dose level and volume in the Pharmacy of CiToxLAB Hungary Ltd.
- Formulations were prepared freshly or within 4 days before use (in that case formulations were stored in a closed container at room temperature), based on the stability assessment results. Stability of the test substance in the vehicle was assessed in the conditions employed on the study during the analytical method validation (CiToxLAB study code: 15/512-316AN). In this study, analysis of the test substance formulation samples in the 10-300 mg/mL concentration range (using corn oil as vehicle) showed no decrease of concentration and were considered as stable for at least 6 days at room temperature.
- Analysis of test substance formulations for concentration and homogeneity was performed using a HPLC-UV method in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken and analysed from test substance formulations on 3 occasions during the treatment period, one set to analyze (which were collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the middle of the vehicle control formulation for concentration measurement.

Duration of treatment / exposure:

Male and 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 post-partum/lactation Day PPD4.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Concentration: 0 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Concentration: 25 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Concentration: 75 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Concentration: 250 mg/mL
Dose volume: 4 mL/kg bw

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Positive control:

No

Observations and examinations performed and frequency:

Clinical observations and functional observation battery (FOB):
- All animals:
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time. 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. Detailed examinations were performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were performed outside the home cage in a standard arena, at similar day times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or 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); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. (On gestational 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).
- Neurotoxicity:
Neurotoxicity assessment was performed on five males and five females per group in the morning and prior to dosing, during the last exposure week (males on Day 27; females on PPD 4). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength (manual and instrumental) and measurement of landing foot splay and fore/hind limb grip strength. Qualitative and quantitative assessments of motor activity were performed. A modified Irwin test was performed as well. Manual assessment of sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity were conducted and the general physical condition and behaviour of animals was 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/or vocalisation were evaluated using a scoring system, where score 0 was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal.
- Body weight measurement:
All adult animals were weighed with an accuracy of 1g for randomization purposes, then on Day 0, at least weekly thereafter and at termination. Parent females were weighed on gestation Days GD0, 3, 7, 14 and 20 and on post-partum Days PPD0 (within 24 hours after parturition), PPD4 and before termination. Body weights of the female animals were additionally taken on gestational Days GD10 and 17 in order to give accurate treatment volumes but these data were not evaluated statistically.
- Food consumption measurement:
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1g at least weekly (on the days of body weight measurements).
- Observation of the delivery process, offspring and nursing instinct:
Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. Any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition. Dams were observed for signs of nest building with the bedding material and for covering their new-borns.
- Clinical pathology:
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. For terminal blood sampling three samples were taken from each selected animal (5 males and 5 females/group), one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry. (Blood smears were prepared (fixed) for all selected animals but not examined).
- Urinalysis:
Urine samples were collected for 16 hours during an overnight period of food deprivation during the last week of the study (Day 27-28 for males and PPD 4-5 for female animals, respectively) from each selected animal by placing the animals in metabolic cages. The evaluation of the urine samples were performed by observation (e.g. appearance, colour) and test strips.

Sacrifice and pathology:

Pathology - terminal procedures and macroscopic evaluation:
- At termination, adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination. Gross necropsy was performed on all animals, irrespective of the date of death. 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. The number of implantation sites and of corpora lutea was recorded in the females as applicable.
- Organ weight measurements. At the time of termination, body weight and the weight of selected organs from all euthanized adult animals were determined (with a precision of 0.01g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus and with a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids).
Paired organs were weighed together except testes and epididymides, which were weighed individually. Individual and/or paired absolute organ weights were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.
- Tissue preservation and microscopic evaluation. The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution; all other organs in 10% buffered formalin solution.
- Histological examinations:
•on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
•all macroscopic findings (abnormalities) except of minor order from all animals (this point includes animal with extremely low reproductive organ weight),
•on the retained reproductive organs (testes, epididymides, prostate, 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,
•additional histopathology evaluation was performed on the retained liver, kidney, small intestine (duodenum, jejunum and ileum) and caecum samples of 5 male and 5 female animals of the Mid and Low dose groups due to test item-related microscopic findings observed at the High dose level.
The retained tissues and organs for histological examination were embedded in paraffin wax, sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Detailed histological examination was performed on all retained organs in the Control and High dose groups and any macroscopic findings (abnormalities) observed in all animals. 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.

Statistics:

Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS v9.3, 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 SPSS PC+4.0 (SPSS Hungary Ltd., Budapest) or by SAS 4.3 (when using Provantis).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Male animals:
No clinical signs were detected in the Control or Low, Mid and High dose (100, 300 and 1000 mg/kg bw/day, respectively) males during the study.
Female animals:
No observation was recorded for any female in the Control group or High dose (1000 mg/kg bw/day) group. In the Low dose group (100 mg/kg bw/day), decreased activity, hunched back, piloerection was observed for one female on Day 40 (GD23), paleness for this animal was also recorded in the period of Days 40-44 (GD23-PPD4). However, these findings were most probably related to a difficult delivery process. In the Mid dose group (300 mg/kg bw/day), decreased activity, hunched back, piloerection and red liquid / red discharge (near the vulva) was observed for one female on Day 38 or Day 39 (PPD2 or PPD3), paleness for this animal was also recorded in the period of Days 38-41 (PPD2-5). However, these findings were most probably related to a difficult delivery process.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related effects were noted on the mean body weight or body weight gain values following daily administration of the test substance at dose levels up to and including 1000 mg/kg bw/day. Significant increases (p<0.05 or p<0.01) were nevertheless observed in the body weight gain of the Low and Mid dose males (100 and 300 mg/kg bw/day, respectively) in the Days 14-21 period when compared to the control. However, due to the lack of dose response and lack of biological relevance, this fact was not considered as a test substance related effect. Significant decrease (p<0.05) compared to the control animals (by approximately 64%) was also observed in the body weight gain of the High dose females (1000 mg/kg bw/day) in the post partum period (PPD0-PPD4). Similar difference (approximately 54% decrease) was observed in the Mid dose females (300 mg/kg bw/day) in the same period compared to the Control group, although the difference in that case remained below the level of statistical significance. However, the observed differences were caused by only one animal (Mid dose group) or two animals (High dose group) showing body weight loss (negative values). Based on the low incidences, the length of the observation period, and as there were no significant differences compare to the control in the complete treatment period, these facts were considered as no treatment related effect.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance related effects in the mean daily food consumption in any test substance treated group when compared to the controls. Statistically significant increases (p<0.05 or p<0.01) were nevertheless observed in the food consumption of the Mid dose (300 mg/kg bw/day) males on the first, third and fourth weeks when compared to the control males. Similar effect was seen in the High dose (1000 mg/kg bw/day) males on the third week. However, based on the absolute level of magnitude and the lack of clear dose response, these facts were considered as biological not relevant changes. No statistically significant differences were seen in the Low, Mid and High dose females (100, 300 and 1000 mg/kg bw/day, respectively) when compared to the control females.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to the controls, there were no differences that could be considered toxicologically significant in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively).
Significantly (p<0.05 or p<0.01) increased relative ratio of neutrophils, monocytes and large unclassified cells was observed the High dose males when compared to the control means, while significantly (p<0.05) decreased relative ratio of lymphocytes and eosinophils was observed in the same group. However, no similar pattern was seen in High dose females except of the significant (p<0.05) decrease in the ratio of eosinophils, and the individual values were comparable with the historical control data. Therefore, none of these finding were attributed to the test substance administration.
Statistically significant (p<0.01) decreased mean prothrombin time value was detected in the High dose (1000 mg/kg bw/day) males. No similar trend was observed in the lower doses for males, nor in any of the groups of females, and the observed values in the High dose males were comparable with the female control data. Furthermore, the values found for both control males and females were below the historical control range indicating a slight variation in the batch of animals. Therefore, the observed minor statistical difference was considered as having no biological relevance, thus it was not considered as a test substance related effect.
For other parameters, evaluation of the mean and individual results in comparison with the control data did not reveal any test-substance related changes, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered incidental or individual findings, which were not related to treatment. These findings were comparable with the expected physiological range or were with no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant changes or adverse effects on the serum chemistry that could be ascribed to the test substance administration in the conditions of this study.
Cholesterol concentration was significantly (p<0.01) higher in the High dose males (1000 mg/kg bw/day) when compared to the Control. Sodium concentration was significantly lower (p<0.01) in the Mid and High dose males (300 and 1000 mg/kg bw/day, respectively) when compared to the Controls. However, the data for these two parameters from all animals were in the normal physiological range and these small differences were not consistent between sexes. Furthermore, in case of cholesterol one sample of the Control group was below the limit of quantification, making the mean control value slightly lower than usual. For these reasons, these observed minor variations were not considered toxicologically significant or related to treatment.
No statistically significant changes were recorded for any parameters in the test substance treated females.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No significant differences were recorded in the volume, pH or specific gravity of the urine samples between the control and any test substance treated groups. The appearance of urine samples as well as the amount of urine crystals observed in the test substance treated groups was comparable to controls.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no test substance related effects in the neurological assessment as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test item treated groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related adverse effects were observed on the organ weight of any dose groups in the study.
Liver weights were higher than control in both sexes in the High dose group (1000 mg/kg bw/day). This was statistically significant both in terms of absolute weight or when adjusted for body and brain weights. A similar statistical difference was noted in male liver weights in the Mid dose group (300 mg/kg bw/day). In Mid dose females, liver weights were increased only when adjusted for body weight. Liver absolute and relative weights (when adjusted for body weight) were also statistically significantly increased in the Low dose females. However, the increases were minor, there was no clear dose response in any sex, and all the recorded absolute weights (except of 2 Mid dose males) were within the historical control range. Furthermore, the liver function remained unaffected as confirmed by the relevant clinical chemistry parameters (AST, ALT). Therefore, these findings were not considered as a test substance related adverse effect.
Statistically significant increase in the kidney weight was only seen in the males. Both absolute weights and relative weights (when adjusted for body weight) were increased in the Mid and High dose groups (300 and 1000 mg/kg bw/day, respectively). No effect was seen at the Low dose level (100 mg/kg bw/day). However, no similar trend was seen in females and the observed individual values (except of two Mid dose males) were within the historical control range. Furthermore no renal function parameters like blood creatinine, urea and electrolytes concentrations were affected; these findings were not considered as being a test substance related adverse effect.
There were no other statistically significant differences among groups in the weights of other organs measured when compared to controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related macroscopic findings were observed up to the dose level of 1000 mg/kg bw/day. Single cyst of left kidney in 1/12 Mid dose female and uterine dilatation in 1/12 High dose female were considered to be incidental and a common change in cycling rats, respectively.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment noted during the assessment of landing foot splay test, grip strength or motor activity measurements. Grip strengths of the forelimb were significantly (p<0.05) higher in the Low and Mid dose (100 and 300 mg/kg bw/day, respectively) females, but without dose dependency. As no similar results were observed in the hind limbs of these females or in the male animals, therefore this difference was not considered to be a test substance related effect. The total travelled distance in the High dose group and the shape of the activity curve (5-minute periods for one hour) were comparable to the control groups for both sexes; all data were within the normal expected range. Thus, there was no test substance related effect on locomotor activity in either sex.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

During the detailed histological examination, test substance-related microscopic findings were observed in the liver, kidney, small intestine (duodenum, jejunum, ileum) and/or caecum in the selected animals of the High dose and/or Mid dose groups (5 animals/sex/group) when compared to the control samples. The hepatic and renal microscopic alterations correlated with increased liver and kidney (in the males only) absolute and/or relative weights.
- Liver: In case of the High dose group (1000 mg/kg bw/day) samples, test substance-related changes were characterized as diffuse (without zonal pattern) microvesicular vacuolation of hepatocytes. The hepatocytes were filled by small clear vacuoles. Minimal severity was noted in 2/5 males and 3/5 females, while slight degree was only seen in 2/5 High dose males. Minimal diffuse microvesicular vacuolation of the hepatocytes was noted in 2/5 females of the Mid dose group (300 mg/kg bw/day). No test substance-related findings were seen by light microscopy in the samples of the Low dose group (100 mg/kg bw/day). 
- Kidney: Test substance-related degeneration was observed in the cortical tubules of some High dose (1000 mg/kg bw/day) animals. Minimal focal/multifocal, unilateral degeneration (with/without casts) was recorded in 2/5 High dose males and 1/5 High dose female. This type of degeneration can be reversible or irreversible. No test substance-related findings were recorded in the Low and Mid dose groups (100 and 300 mg/kg bw/day, respectively).
- Small intestine (duodenum, jejunum and ileum): Slight to moderate vacuolation of the apical part of the mucosal villi was present in 4/5 High dose males and 5/5 High dose females. Large, clear vacuoles were accumulated in the epithelial cells of the villi. There were no signs of degeneration/necrosis or inflammation, except of one High Dose male. Slight mixed cell inflammation altered jejunal and ileal mucosa were observed in this male rat. Minimal vacuolation of the apical mucosal villi of the ileum in 1/5 female occurred in the Mid dose group (300 mg/kg bw/day). No test related-related findings were present in the Low dose group (100 mg/kg bw/day).
- Caecum: Moderate vacuolation in the epithelial cells of the villi affected 1/5 High dose male. No degeneration/necrosis or inflammatory response were microscopically detected. No test substance-related findings were recorded in the Low and Mid dose groups (100 and 300 mg/kg bw/day, respectively).
Furthermore, reproductive organs of males (testes, epididymides, prostate, seminal vesicles with coagulation gland) that failed to sire or having extremely low reproductive organ - testis - weight and females (uterus, cervix, ovary, oviduct and vagina) that failed to deliver healthy pups or being non-pregnant were also examined. No test substance-related microscopic changes were observed in those cases.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: increased liver weight (in HD males+females and MD females) and increased kidney weight (in MD and HD males)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

other: hepatobiliary and urinary

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

Under the conditions of the study, the NOAEL for systemic toxicity due to the test substance was considered to be at 100 mg/kg bw/day.

Executive summary:

A study was conducted to determine the repeated dose toxicity with a reproduction/developmental toxicity screening test of the test substance according to OECD Guideline 422, in compliance with GLP. Male and female Wistar rats received the test substance by oral gavage at concentrations of 0 (vehicle alone: cron oil), 100, 300 and 1000 mg/kg bw/day (in a volume of 4 mL/kg bw). 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 post-partum/lactation Day PPD-4. Analysis of test substance formulations for concentration and homogeneity was performed using a HPLC-UV method. Formulations were considered to be adequate for the study. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. 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. For the adult animals, a detailed histological examination was performed on the selected organs in the Control and High dose (HD) groups as well as on the liver, kidney, small intestine (duodenum, jejunum and ileum) and caecum samples of the Mid and Low dose groups (5 animals/sex/group). Testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries and uterus (including cervix) were also examined. Daily administration of the test substance by oral gavage to Wistar rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/day did not result in adverse changes in clinical signs, neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry, urinalysis or on reproductive parameters during the treatment period under the conditions of this study. Test substance related liver changes were observed in the Mid dose (MD; i.e., 300 mg/kg bw/day) and HD (i.e., 1000 mg/kg bw/day) males and HD dose females with increased weight (approximately 13-18%). Microvesicular vacuolation of hepatocytes was observed in HD males and females, and in MD females. Kidney changes were also recorded in the MD and HD males with increased weight (approximately 10-12%). Unilateral degeneration of the corticular tubules was recorded in a few HD male and female animals. The histopathology evaluation showed no findings in the liver of Low dose (LD) animals or MD males or in the kidney samples of LD and MD animals. Slight to moderate vacuolation in the small intestine (duodenum, jejunum and ileum) was observed in HD males and females, with minimal vacuolation in MD females. No test substance-related findings were present in the LD group. Moderate vacuolation in the caecum was present only in one HD male. Intestinal changes were not accompanied by any degeneration/necrosis or inflammatory response. For details on reproductive toxicity refer to section 7.8.1 of IUCLID or 5.9 of the CSR. Under the conditions of the study, the NOAEL for systemic toxicity due to the test substance was considered to be at 100 mg/kg bw/day (Hargitai, 2016).