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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effect on reproductive performance as well as offspring was observed in the combined repeated dose and reproduction / developmental screening test.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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: OECD Guidance document No. 43

GLP compliance:

yes

Limit test:

no

Justification for study design:

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 4 post-partum.

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

Vehicle:

corn oil

Details on 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.
- 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.

Details on 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 5 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.

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

Details on study schedule:

- Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating), and then euthanized and subjected to necropsy examination.
- Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.
- All F1 offspring were terminated on Day 4 post-partum. In order to allow for overnight fasting of dams prior to urine collection on PPD 5, the offspring were euthanized on PPD/PND 4 and the dams on PPD/PND 5.

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 g/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

Parental animals: Observations and examinations:

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.

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0) and on PND 4, with accuracy of 0.01 g. All litters were checked daily for the number of viable and dead pups. Evidence of suckling was observed by the presence of milk in the pups' stomach. All pups were culled on PND 4.

Postmortem examinations (parental animals):

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.

Postmortem examinations (offspring):

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

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).

Reproductive indices:

Male Mating Index %, Female Mating Index %, Male Fertility Index %, Female Fertility Index%, Gestation Index %

Offspring viability indices:

Survival Index %, Pre-implantation mortality %, Intrauterine mortality%, Total mortality %, Sex ratio % (females)

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

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

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Reproductive organs: No test substance-related microscopic changes were noted in the reproductive organs of the High dose group (1000 mg/kg bw/day). The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar in histological structure in both Control and High dose females.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Reproductive organs: No test substance-related microscopic changes were noted in the reproductive organs of the High dose group (1000 mg/kg bw/day). Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were similar in Control and High dose males.

Reproductive performance:

no effects observed

Description (incidence and severity):

- Reproductive ability assessment and indices:
There were no significant differences between the Control and test substance treated groups with regard to reproductive ability, mating fertility or gestation. The mating indices were normal in all test substance treated groups (100%) for both males and females. The fertility indices were also in the normal range (92-100%) for all test substance treated males and females. The gestation index was 92% in the Low dose group (100 mg/kg bw/day) and 100% in the Mid and High dose groups (300 and 1000 mg/kg bw/day, respectively).
Test substance administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within up to 5 days of pairing (cohabitation) in each case. The mean duration of mating was 2.6, 3.1, 2.3 and 2.2 days in Control, Low, Mid and High dose groups, respectively.
- Evaluation of the gestation, parturition and post-partum period:
There was no effect of treatment noted during gestation, parturition or the post-partum period. Delivery lasted more than three hours for one Low dose (100 mg/kg bw/day) female. This relatively long parturition was considered to be incidental, and not related to the treatment. The number of corpora lutea and number of implantation sites, as well as the duration of pregnancy was comparable to the control mean in all dose groups. There were no significant differences or effects that could be ascribed to treatment on the pre-implantation, intrauterine, post-natal or total mortality values (litter mean and %) at up to and including 1000 mg/kg bw/day.

Refer to RDT section 7.5.1. for detailed results on systemic toxicity

Key result

Dose descriptor:

NOAEL

Remarks:

for reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

for systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on kidney, liver and small intestine at higher doses

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

Other effects:

no effects observed

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Overall, there were no treatment-related effects on the mortality and viability of pups on PND 0 and PND 4. The number of viable pups was significantly (p<0.01) decreased in the Low dose group on PND 0 and in the Mid dose groups on PND 4, but due to the lack of dose response, these facts were considered as animal variability, unrelated to the treatment.
Autolysis was observed only in one Low dose litter (#2506) at the delivery, but in that litter all the 12 pups were autolyzed. However, based on the lack of dose response, this fact was considered as an incidental occurrence, not related to the test substance.
Higher number of pups died during the postnatal period in the Mid dose group (300 mg/kg bw/day) when compared to the control, but there was no dose dependence, furthermore all those cases were recorded in a single litter. Thus, this fact was not considered as a test substance related effect, but just an incidental event.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on the offspring body weight on PND0 or PND4 following administration of the test substance at 100, 300 or 1000 mg/kg bw/day. Significantly increased (p<0.01) body weights (mean value of all pups) were seen in the Low dose group (100 mg/kg bw/day) on PND 0 and PND4 when compared to the control; however, the difference was weaker when the mean litter weight were compared (p<0.05 on PND 0 and no statistical significance was detected on PND4). As no similar trend was seen in the Mid (300 mg/kg bw/day) and High (1000 mg/kg bw/day) dose groups and due to the lack of toxicological relevance, this fact was not considered as a treatment related effect.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The sex ratio on PND 0 and PND 4 was comparable to control values at up to and including 1000 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross findings were observed in the F1 generation in any dose group (PN0-4). Eleven pups died (same dam)without any observed macroscopic changes.
Based on the external evaluation, all the pups except of three were normal. Two Low dose pups (both in the same litter) had small threadlike tail, while for one stillborn Mid dose pup anencephaly, mandible small maxilla and protruding tongue were recorded. These findings are known types of spontaneous malformation. Based on the low incidence and lack of dose response these events were considered as not related to the test substance treatment.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No adverse effects observed following treatment with the test substance

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the conditions of the study, the NOAEL for reproductive and development toxicity due to the test substance was considered to be 1000 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: corn 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. In addition, the reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND-4. 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 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. 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. Daily administration of the test substance did not result in adverse nor substance-related changes in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period in any dose groups. There were no effects on the F1 offspring viability, clinical signs, development or at macroscopic observations in any dose groups. No test substance-related microscopic changes were noted in the reproductive organs in any dose groups. Under the conditions of the study, the NOAEL for reproductive and development toxicity due to the test substance was considered to be the highest tested dose of 1000 mg/kg bw/day (Hargitai, 2016).  

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline compliant study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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: corn 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. In addition, the reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND-4. 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 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. 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. Daily administration of the test substance did not result in adverse nor substance-related changes in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period in any dose groups. There were no effects on the F1 offspring viability, clinical signs, development or at macroscopic observations in any dose groups. No test substance-related microscopic changes were noted in the reproductive organs in any dose groups. For details on systemic toxicity observed in this study, refer to the section 7.5 of IUCLID or 5.6 of the CSR. Under the conditions of the study, the NOAEL for reproductive and development toxicity due to the test substance was considered to be the highest tested dose of 1000 mg/kg bw/day (Hargitai, 2016).

Effects on developmental toxicity

Description of key information

No adverse effect on reproductive performance as well as offspring was observed in the combined repeated dose and reproduction / developmental screening test.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline compliant study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

See above reproductive toxicity section for effects on offsprings.

Justification for classification or non-classification

Based on the results of a combined repeated dose toxicity and reproductive/developmental screening study (OECD 422), the test substance does not meet the criteria for classification for this endpoint according to CLP (Regulation 1272/2008/EC).​