Urea, polymer with ethanedial

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction: Key study: Test method OECD 422. GLP study: The NOAEL for parental toxicity after at least 28 days of oral exposure to test item was determined to be 250 mg/kg bw/day in rats (basis for effect: minor changes in clinical chemistry and urinalysis parameters and the ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day dose level) . The NOAEL for the reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day (basis for effect: no treatment related effects noted at the highest dose tested).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 06, 2013 - March 24, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Laboratories, Research Models and Services, Germany GmbH- Age at study initiation: Approx. 10 weeks at starting and 12 weeks at mating.- Weight at study initiation: Males: 362–391 g, Females: 195-225 g- Fasting period before study:- Housing: up to 5 animals per sex and cage type II and/or III polycarbonatem, with lignocel bedding.- Diet (e.g. ad libitum): Ad libitum- Water (e.g. ad libitum): Ad libitum- Acclimation period: 6 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 18.5-23.6 ºC- Humidity (%): 33-68 %- Air changes (per hr): 15-20 air chaiges per hour- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(distilled)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared fresh prior to administration to animals. The amount of the dose volumes was formulated considering the density of the test item and then they were corrected with the purity of the test item in water solution.VEHICLE- Amount of vehicle (if gavage): 1.71 mL/kg bw- Lot/batch no. (if required): 3450611 / 4310612

Details on mating procedure:

- M/F ratio per cage: 1 female and one male of the same dose group (1:1).- Length of cohabitation: Until copulation occurred, for up to 5 days.- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy. Sperm positive females were caged individually. - After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the formulations for concentration and homogeneity was performed using validated spectrophotometric method (CiToxLAB study code 12/331-316AN). Control, top, middle and bottom duplicate samples were taken from test item formulations on 3 occasions, during the first and last weeks and approximately midway during the treatment, one set to analyse and the other for back-up. No test item was identified in the control samples. The test item formulation appeared to be homogenous and had actual concentrations of 99-105% of the nominal concentrations, within the 100±10% acceptable range. These results were considered suitable for the study purposes.

Duration of treatment / exposure:

2 weeks before mating, during the mating, and continued up to and including the day before the necropsy.

Frequency of treatment:

Daily, 7 days per week.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12 animals per sex and per dose (at least 8 pregnant female/group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based the previous repeated dose range finding study in the rat (CiToxLAB study code12/331-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Test item administrated to Wistar rats at 62.5, 250 and 1000 mg/kg bw/day, daily for 7 consecutive days, was not associated with any overt adverse effects that could be clearly ascribed to test item administration other than a transient body weight and food intake effect in the first few days of treatment in males at the High dose. Based on the results, the dose levels selected for the main study were 0, 62.5, 250, 1000 mg/kg bw/day.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes- Time schedule:Twice daily for morbidity and mortality. Once daily for general clinical observations and behavious.On gestation day 13 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). DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Once before the first exposure, and at least weekly.- Observations: 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. No such clinical signs were observed during the study.BODY WEIGHT: Yes- Time schedule for examinations: before treatment for randomization purposes, on Day 0, afterwards at least weekly and at termination. Parental females were weighed on gestation days 7, 14 and 20 and postpartal days 0 (24 h after parturition), 4 and 5.FOOD CONSUMPTION:- Animal food consumption was determined by re-weighing the non-consumed diet on Day 7 and then at least weekly.HAEMATOLOGY: Yes- Time schedule for collection of blood: Immediately prior to scheduled necropsy.- Anaesthetic used for blood collection: Yes, pentorbital anaesthesia.- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Red Blood Cell (erythrocyte), White Blood Cell (leukocyte), Haemoglobin concentration, Haematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Haemoglobin, Mean Corpuscular (erythrocyte) Haemoglobin Concentration, Red Cell (erythrocyte) volume, Platelet (thrombocyte) count, Mean Platelet Thrombocyte volume, Reticulocyte count, Neutrophil, Lymphocyte, Monocyte, Basophil, Eosinophil, Large Unstained Cells, Activated Partial Thromboplastin Time, Prothrombin Time.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Immediately prior to scheduled necropsy.- Anaesthetic used for blood collection: Yes, pentorbital anaesthesia.- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Blood sugar concentration, Total Bilirubin concentration, Urea concentration, Cholesterol concentration, Creatinine concentration, Phosphorus concentration, Sodium concentration, Potassium concentration, Calcium concentration, Chloride concentration, Total Protein concentration, Albumin concentration, Alb/glob ration, Alanine Aminotransferase activity, Alkaline. Phosphatase – activity, Gamma Glutamyltransferase -activity, Bile acids.URINALYSIS: Yes- Time schedule for collection of urine: Prior to scheduled necropsy- Metabolism cages used for collection of urine: Yes (16 hours)- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Leukocyte, Nitrite, pH, Protein, Glucose, Urobilinogen, Bilirubin, Ketones, ERY Blood, Erythrocytes, Specific Gravity, Sediment, Volume, Colour/Appearance.NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: During the last exposure week (males on Day 24, females on PND 4).- Dose groups that were examined: 5 rats per sex and per dose (subgroup A)- Battery of functions tested: sensory activity / grip strength / motor activity.

Oestrous cyclicity (parental animals):

Estrous cyclicity was observed in all female parentals.

Sperm parameters (parental animals):

Parameters examined in all male parental: Spermatogenesis stages in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

PARAMETERS EXAMINEDThe following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, runts (significantly smaller pups), postnatal mortality, presence of gross anomalies, weight gain (weighed on postnatal day 0 and 4), physical or behavioural abnormalities.OTHER PARAMETERS: Duration of gestation (calculated from day 0 of pregnancy).Dams were observed whether they form a nest from the bedding material and cover their new-borns or not. Efficiency of suckling.

Postmortem examinations (parental animals):

SACRIFICE- Male animals: All surviving animals, after at least 14 days post-mating period.- Maternal animals: All surviving animals, at post-natal day 5.GROSS PATHOLOGY: YesAll animals were necropsied. 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 corporea lutea was recorded in the females as applicable.ORGAN WEIGHTS: YesIn all animals, uterus (with and without cervix), vagina, testes, epididymides, prostate, seminal vesicles with coagulating glands, brain. ovaries and pituitary were measured.For 5 rats per sex and group (subgroup B), heart, kidneys, liver, spleen, thymus and adrenals were also weighted. Paired and individual organ weights were summarised. Relative organ weight (to body and brain weight) were calculated.HISTOPATHOLOGY: YesThe weighed organs and all organs showing macroscopic lesions of all adult animals were preserved.For 5 rats per sex and group (subgroup B), the following organs and tissues were preserved:Gross findings, Adrenal glands, Animal identification, Aorta (thoracic and abdominal), Brain, Clitoral gland / Preputial gland, Epididymes, Eye with the optic nerve, Oesophagus, Femur with marrow incl. joint, Heart, Kidney, Large intestine, External lachrymal gland, Harderian gland, Liver, Lungs with bronchi, Lymph nodes, Larynx, Nasopharynx, Ovaries with oviduct, Pancreas, Pituitary, Prostate, Salivary glands, Sciatic nerve, Seminal vesicles (with coagulating glands), Skeletal muscle (quadriceps), Skin/subcutis with mammary, gland area, Small intestine, Spinal cord (cervical, lumbar, and thoracic levels), Spleen, Sternum with marrow, Stomach, Testis, Thymus, Thyroid with parathyroid gland, Tongue, Trachea, Urinary bladder, Uterus, Vagina.Detailed histological examination were performed in the control and high dose groups and all macroscopic findings from all animals.Special attention were paid to evolution 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 insterstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Postmortem examinations (offspring):

SACRIFICEAll F1 offspring was terminated on Day 4 post-partum. GROSS NECROPSYGross necropsy consisted of external examination for gross abnormalities.

Statistics:

The statistical evaluation was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Reproductive indices:

Male mating index, female mating index, male fertility indey, gestion index.

Offspring viability indices:

Survival index, pre-implantation mortality, intrauterine mortality, total mortality, post-natal mortality, sex-ratio.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs related to treatment.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males treated at 1000 mg/kg, transient lower body weight and body weight gain values were observed during the pre-mating period. The mean body weight value at termination was comparable to the control. The mean body weight values of females in all test item treated groups were comparable to the control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Compared to control, lower food consumption values, attaining statistical significance were noted for males at 1000 mg/kg (High dose) during the first week of the treatment. These changes were generally correlated with the body weight and body weight gain values. In addition, slightly lower food consumption was observed during the mating period.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant differences between the control and any of treated groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Higher bile acid concentration, attaining statistical significance were noted in High dose male and female animals. Compared to the control higher potassium and serum urea concentration (Urea) were observed in Mid and High dose female animals. No signs of kidney injury were observed during pathology evaluation.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urobilinogen and urinary bilirubin, calcium oxalate crystals as well as dark yellow discoloration of the urine was presented in high dose males. In high dose females and in one from the mid dose, calcium oxalate crystals were also presented in the urine. Dark yellow discoloration of the urine was observed in one mid dose female. Semi quantitative measurement of protein in the urine showed a slight elevation in high dose, when compared to the control.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related ulcers of the non-glandular mucosa of the stomach were microscopically observed in 4/7 High dose males and 4/7 High dose females, and were in correlation with necropsy. There was no evidence of test item-related histological findings in the High dose animals or macroscopic observations from all groups in the reproductive organs. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogonic cells representing different phases of the development and differentiation of the spermatozoa were similar in control and high dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both control and high dose females.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the oestrus cycle or reproductive parameters.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogonic cells representing different phases of the development and differentiation of the spermatozoa were similar in control and high dose males.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation to the test item administration.Mating indices = 100% in all groups.Fertility indices = 83% in control, 92% in mid dose, and 100 in low and high doses. Gestation indices = 90% in control, 100% in low, mid and high doses.Test item administration did not have impact on the duration of mating period. There was no effect of treatment noted during gestation, parturition or the post-partal period. Mean duration of pregnancy was similar to control (22-24 days). All the parturitions were normal. Number of mean corporea lutea and implantation sites were similar in the treated group compared to control. There were no item related effects on pre/post-implantation, post-natal or mortality values.

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

250 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical biochemistry

urinalysis

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

Any adverse effect considered treatment related or toxicologically significant.No external abnormalities were detected at the clinical or external macroscopic examination.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Test item administration did not lead to mortality. The sex ratios were similar in the control and treated groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect of treatment on the offspring body weight or body weight gain.

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 specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no abnormalities at the external examination of all pups surviving to post-natal day.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No abnormal behaviour of the pups was noted.

Developmental immunotoxicity:

not specified

VIABILITY (OFFSPRING)Test item administration did not lead to mortality. The sex ratios were similar in the control and treated groups.CLINICAL SIGNS (OFFSPRING)Any adverse effect considered treatment related or toxicologically significant. No abnormal behaviour of the pups was noted. No external abnormalities were detected at the clinical or external macroscopic examination. BODY WEIGHT (OFFSPRING)There was no adverse effect of treatment on the offspring body weight or body weight gain.GROSS PATHOLOGY (OFFSPRING)There were no abnormalities at the external examination of all pups surviving to post-natal day.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction / developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

gross pathology

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reproductive ability assessment and indices

	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Paired males - females	12/12	12/12	12/12	12/12
Pregnant females	10/12	12/12	11/12	12/12
Pregnant females, with stillborn	1/10	0/12	0/11	0/12
Male-female Mating Index (%)	100	100	100	100
Male-female Fertility Index (%)	83	100	92	100
Gestation Index (%)	90	100	100	100

Evaluation of the gestation, parturition and post-partal period

Females	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Number of pregnant females	10	12	11	12
Number of delivered dams	10	12	11	12
Duration of pregnancy (days, mean)	22.50	22.33	22.82	22.42
Number of corpora lutea / dams (mean)	19.50	20.17	18.82	19.25
Number of implantations / dams (mean)	17.10	16.25	14.82	16.58
Number of dams with live pups day 0	9	12	11	12
Number of dams with live pups day 4	9	12	11	12
Pre-implantation mortality (%)	12.10	18.56	22.31	13.82
Intrauterine mortality (%)	19.86	7.46	15.26	9.13
Post-natal mortality (%)	0.00	3.69*	1.56	6.88
Total mortality (%)	19.86	10.94	16.64	14.18

Mortality, clinical observations and survival of pups

Females	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Number of liveborns (total) on PND0	141	181	145	181
Number of viable pups (mean) on PND0	14.10	15.00	13.18	14.75
Number of viable pups (mean) on PND4	14.10	14.50	12.91	14.33
Number of viable pups (total) on PND0	141	180	145	177
Number of viable pups (total) on PND4	141	174*	142	172*
Survival index PND0	90.00	99.54	100.00	96.67
Survival index PND4	100.00	96.31*	98.44	93.12
Sex ratio (%) on PND0	46.24	39.71	45.85	53.94
Sex ratio (%) on PND4	46.24	39.24	45.70	52.19

Conclusions:

The NOAEL for parental toxicity after at least 28 days of oral exposure to test item was determined to be 250 mg/kg bw/day in rats (basis for effect: minor changes in clinical chemistry and urinalysis parameters and the ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day dose level) . The NOAEL for the reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day (basis for effect: no treatment related effects noted at the highest dose tested).

Executive summary:

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed in accordance with OECD Guideline 422 following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels, 62.5, 250 and 1000 mg/kg bw/day. The control animals were treated with the vehicle only (distilled water). 12 Male and female Wistar rats per group were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period, up to and including postpartum/lactation Day PPD4. 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. In addition, the reproductive performance and indices, 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 PND4. At termination, necropsy with macroscopic examination was performed. Selected organs were subjected to histopathology examination. No mortality was observed during the study. There were no clinical signs related to treatment. There was no effect of treatment noted during evaluation of the functional observation battery, grip strength, foot splay or motor activity. In males treated at 1000 mg/kg, transient lower body weight and body weight gain values were observed during the premating period. The mean body weight value at termination was comparable to the control. The mean body weight values of females in all test item treated groups were comparable to the control. There was no effect of treatment noted during evaluation of the reproductive parameters. There were no adverse effects on the F1 offspring viability, clinical signs or development. Single or multiple ulcers of the non-glandular mucosa of the stomach (4/12 High dose males and 4/12 High dose females) were observed as treatment-related macroscopic findings. There was no effect of treatment on organ weights. Test item related microscopic findings were found at 1000 mg/kg bw/day (High dose). In the stomach, ulcers of the non-glandular mucosa were microscopically observed in 4 High dose males and 4 High dose females. There were no microscopic findings related to treatment at 62.5 or 250 mg/kg bw/day. Lower food consumption values were noted for males at 1000 mg/kg during the first week of the treatment. These changes generally correlated with the body weight and body weight gain values. There was no effect of treatment on haematology and blood clotting parameters. Increases were noted in potassium and/or urea and bile acids in high dose males and females. Calcium oxalate crystals were noted in high dose males and females, with increases in urobilinogen and bilirubin in males.

Based on the minor changes in clinical chemistry and urinalysis parameters and the ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day dose level, the no adverse effect level (NOAEL) was determined to be 250 mg/kg bw/day in male and female rats after at least 28 days of test item treatment by oral gavage. Moreover, since no treatment related effects were noted during the evaluation of the reproductive paramenters and F1 offsprings, the NOAEL for the reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day.

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:

The study is a GLP compliant and has Klimisch score 1.

Additional information

Toxicity to reproduction: Key study: This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed in accordance with OECD Guideline 422 following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels, 62.5, 250 and 1000 mg/kg bw/day. The control animals were treated with the vehicle only (distilled water). 12 Male and female Wistar rats per group were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period, up to and including postpartum/lactation Day PPD4. Single or multiple ulcers of the non-glandular mucosa of the stomach were observed as treatment-related macroscopic findings. Test item related microscopic findings were found at 1000 mg/kg bw/day (High dose). In the stomach, ulcers of the non-glandular mucosa were microscopically observed in High dose males and females. Increases were noted in potassium and/or urea and bile acids in high dose males and females. Calcium oxalate crystals were noted in high dose males and females, with increases in urobilinogen and bilirubin in males. There was no effect of treatment noted during evaluation of the reproductive parameters. There were no adverse effects on the F1 offspring viability, clinical signs or development. Based on these results the NOAEL for parental toxicity was determined to be 250 mg/kg bw/day in rats and the NOAEL for reproduction/developmental toxicity was determined to be 1000 mg/kg bw/day.

Extended one-generation toxicity study: Data waiving (study scientifically not necessary/other information available): In accordance with Column 1 of REACH Annex IX, the extended one-generation reproductive toxicity study does not need to be conducted since the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test did not indicate adverse effects on reproductive organs or tissues and any other parameter related to reproduction nor development.

Effects on developmental toxicity

Description of key information

Pre-natal developmental toxicity: Key study: Test method OECD 414. GLP study: In this prenatal developmental toxicity study performed with the test item in rats (oral route), no significant toxicological changes on dams, embryos or foetuses were observed up to the highest dose tested and therefore, the NOAEL for maternal toxicity, embryotoxicity, foetotoxicity and teratogenecity was determined to be 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 June 2016 to 21 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hannover Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Age at study initiation: at least 11 weeks old at mating
- Weight at study initiation: 193-252 g (the variation did not exceed ± 20% of the mean weight)
- Fasting period before study: No
- Housing: Successfully mated animals were housed individually. Deep wood sawdust was use as bedding to allow digging and other normal rodent activities. Nest building material was also added into the cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-24.8°C
- Humidity (%): 31-63%
- Air changes (per hr): 15-20 air exchanges per hour
- Photoperiod (hrs dark / hrs light): 12 hrs light / 12 hrs dark

IN-LIFE DATES: From: 21 June 2016 To: 21 July 2016

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (distilled water) at the appropriate concentrations according to the dose level and volume selected. The test item was formulated considering the density of the test item and then it was corrected with the purity of the test item in the applied water solution. Formulations were prepared fresh prior to administration to animals.

VEHICLE
- Amount of vehicle (if gavage): 1.79 mL/kg body weight
- Lot/batch no. (if required): 809 0416

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis of test item formulations for concentration and/or homogeneity was performed by an UV-spectrophotometric method in the Analytical Laboratory of CiToxLAB Hungary Ltd.
Control, top, middle and bottom duplicate samples were taken from test item formulations two times during the study (approximately during the first and last week of treatment): one set to analyse and one set as a back-up.
All formulations were found to be in the range of 91-101% of the nominal concentrations and were homogenous. No test item was detected in the control (vehicle) formulation.
These results were considered suitable for the study purposes.
Similarly, duplicate samples were taken on each occasion from the control (vehicle) formulation for concentration measurement.
Stability of the NOPCOTE 1661 in the vehicle (distilled water) was assessed during the method validation study (CiToxLAB study code: 12/331-316AN). According to the results, the test item is stable in the vehicle in the concentration range of 2.894 (w/w)% - 46.300 (w/w)% on the day of preparation.

Details on mating procedure:

After acclimation (5 days), the females were paired according to their oestrus cycle (examined shortly before start of pairing) with males in the morning for approximately 2 hours (1 male : 1 female) until at least 24 sperm positive females/group are attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm positive females were separated and caged individually.

Duration of treatment / exposure:

6-14 days pc.

Frequency of treatment:

Daily, 7 days per week.

Duration of test:

Approximately 1 month, from first mating of animals to last necropsy on gestation day 20.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 (control, low and high dose), 25 (mid dose).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were set based on available data, including the results of a Dose Range Finding (DRF) study by oral gavage in rats (CiToxLAB study code: 12/331-220PE) and another combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats (CiToxLAB study code: 12/331-220P), with the aim of inducing toxic effect(s), but no death or suffering at the highest dose and a NOAEL at the lowest dose.
In those studies, doses of 62.5, 250 and 1000 mg/kg bw/day were administered by oral gavage to wistar rats. Administration at high dose level for at least 28 consecutive days was associated with minor changes in clinical chemistry and urinalysis parameters in males and females. In addition, ulcers of the non-glandular mucosa of the stomach were noted in 4/12 males and 4/12 females. No adverse effect was found on reproduction/developmental parameters at this high dose level. No adverse effects or test item related histopathology findings were observed at the low or mid dose levels.
Based on these results, the doses selected for this study were 100, 300 and 1000 mg/kg bw/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-side clinical observations were made at twice daily (at the beginning and end of each working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At GD6 and then weekly.
- Observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, 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), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
In addition, on 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, which is considered to confirm implantation).

BODY WEIGHT: Yes
- Time schedule for examinations: GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION: Yes
Food was measured on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.
Food consumption was calculated for each interval, including GD0-6, GD6-20 and GD0-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
Dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed.
Stomach of all dams was retained in 10% buffered formalin solution.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Placentas were examined macroscopically

Fetal examinations:

- External examinations (including great arteries): Yes: all per litter.
- Soft tissue examinations: Yes: half per litter. The abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomya mixture then after fixation the body was micro-dissected by means of a dissecting microscope. Skeletal examinations: Yes: half per litter. After fixation the skeletons were stained nd examined by means of a dissecting microscope.
- Head examinations: Yes: half per litter. The heads were examined by Wilson's free-hand razor blade method.
- Others: Individual weight.

Statistics:

The statistical evaluation of data was performed using the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest).
The heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out.
If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied.
If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test.
The Chi squared test was used for comparing group incidences against the controls.

Indices:

Maternal Data:
-Number of animals at test start, no. of animals surviving, no. of pregnant animals, no. of animals with total intrauterine mortality
-Clinical signs (by gestation day)
-Mortality (by gestation day)
-Body weight and body weight gain: mean ± S.D.
-Corrected body weight on GD20 (body weight-gravid uterine weight), corrected body weight gain and corrected net body weight gain: mean ± S.D.
-Net body weight change (Body weight on GD20 minus body weight on GD6 minus gravid uterine weight): mean ± S.D.
-Food consumption: mean ± S.D.
-Gross pathology findings
-Gravid uterine weight: mean ± S.D.

Caesarean Section and Necropsy Data:
-Number of corpora lutea: mean ± S.D.
-Number of implantations: mean ± S.D.
-Number and percentage of live foetuses: mean ± S.D.
-Number and percentage of intrauterine mortality: mean ± S.D.
Classified according to time of death: preimplantation loss, postimplantation mortality, Early and late embryonic, as well as foetal death
-Preimplantation loss: %, group mean
-Postimplantation loss: %, group mean

Foetal Data:
-Sex distribution: %, group mean
-Foetal body weight (accuracy 0.01 g): mean * S.D.
-External abnormalities/litter: %, group mean
-Visceral abnormalities/litter: %, group mean
-Skeletal abnormalities/litter: %, group mean

Historical control data:

See Section “Any other information on results incl. tables”

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects or systemic clinical signs were noted in any dose groups.
Fur thin and/or alopecia observed in some control and test item treated animals were considered as irrelevant findings.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no unscheduled mortality during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant changes compared to the control were observed in the mean body weights or body weight gain values in any dose group.
No statistical significance in any of the dose groups when compared to the control group was observed for corrected body weight gain and corrected net body weight gain values (when results were adjusted by the uterus weight), although the high dose group had lower values (by approximately 8-9%).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No statistical significant differences compared to the control in the mean daily food consumption of dams were observed in any period for any of the dose groups, although in the last observation period (GD18-20) lowed food consumption (by approximately 21%) was recorded in the High dose group.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No observations were recorded for any animals in the study except of recording the fur thin.
The stomach of all animals was carefully examined, but no findings were observed in any control or test item treated animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

The study makes no reference of any abortion.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups.
No test item effect was observed in preimplantation loss of the test item treated groups when compared to the control.
There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The total intrauterine mortality was comparable with the control, no statistically significant differences were observed.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

There was no statistically significant difference in foetal death in any group compared to the control.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Ninety-seven females (24 animals in the Control, Low and High dose groups and 25 animals in the Mid dose group) were mated in the study. The number of confirmed pregnant, evaluated dams was 24, 24, 24 and 21 in the Control, Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) and High dose (1000 mg/kg bw/day) groups, respectively.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

other: No adverse effect observed up to the highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For the mean body weight/foetus significant difference was observed in the Low and High dose groups when compared to the control. However the slightly higher mean foetal body weight values showed no dose response and all values were within the historical range, hence the statistical differences were not considered as a test item related effect.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The total number of retarded foetuses (runts) as well as the number of affected litters in the test item treated groups was comparable with the control value.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of viable foetuses was comparable with the control mean.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The weight of foetuses per litter in any of the dose groups did not differ significantly from the control mean value.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No external malformations were recorded in the study. External variation was recorded for one foetus in the High dose group, but based on the isolated occurrence it was considered incidental, ascribed to individual variability and not related to treatment (although concurrent control data did not contain this observation but it was included in the historical control database).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All of the skeletal findings were consistent in general nature and the observed incidence was within the study concurrent control data or the existing historical control data, therefore they were considered as incidental findings.
No skeletal malformations were recorded in the study.
In one case (wavy ribs), an increased incidence of changes was observed in the absolute number of findings in the Mid and High dose groups with a frequency of 8.8% and 11.6% for foetuses, and 29.2% and 33.3% for litters, respectively. The difference gained statistical significance (p<0.05) in the High dose group when compared to control (3.6% for foetuses and 16.7% for litters). However, this observation is a common background finding and occurred with a comparable incidence in the historical control (9.9% for foetuses and 30.8% for litters). Furthermore, the incidence in the individual control groups of the historical control database showed this variation in a 4.1-18.6% range for foetuses and 8.7-56.5% for litters. Therefore, this finding was not considered as a test item related adverse effect.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All of the visceral findings were consistent in general nature and the observed incidence was within the study concurrent control data or the existing historical control data or were considered as incidental background finding, therefore they were considered not related to the test item treatment.
There were no malformations in the Mid or High dose groups. A low incidence malformation (one foetus) in the Low dose group was ascribed to incidental background findings, unrelated to treatment.
The number of foetuses with variation was statistically higher (p<0.05) in the Low dose group, consequently the number of intact foetuses was significantly lower (p<0.05) in the same group when compared to the control. This fact was caused by the significantly (p<0.05) increased number of foetuses with thymic cord variation in the Low dose group. In case of this visceral variation, although the occurrence (5.7% for foetuses, 16.7% for litters) in the Low dose group was higher rate than in the actual control group (0.7% for foetuses, 7.1% for litters), but it was comparable to the rate of the HC database (2.7% for foetuses, 11.3% for litters). Furthermore, there was no dose response, so this fact was not considered as a test item related effect.

Other effects:

no effects observed

Description (incidence and severity):

No abnormalities were observed on the placentas of any animals in the Control, Low, Mid or High dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: No treatment related adverse effect observed in any of the parameters at the highest dose tested

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: rib

Description (incidence and severity):

There were no effects of the test item on external, visceral and/or skeletal development of foetuses in the study which could be related to the test item administration. Increased incidence of the skeletal variation of wavy ribs in the Mid and High dose groups was comparable with the relevant historical control data.

Key result

Developmental effects observed:

no

Table 1: Summary of the dose formulation analysis

Nominal concentration (mg/mL)	Measured concentration (mg/mL)	Percentage of the nominal concentration (%)
Analytical sampling #1(27 June 2016)
Control	not detectable	-
4.54% w/w	4.44 ± 0.105	98
13.63% w/w	12.4 ± 0.293	91
45.4% w/w	43.8 ± 1.04	97
Analytical sampling #2(12 July 2016)
Control	not detectable	-
4.54% w/w	4.54 ± 0.319	100
13.63% w/w	13.8 ± 0.449	101
45.4% w/w	45.8 ± 1.395	101

Table 2: Summary of pregnancy data

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of mated females	24	24	25	24
Number of non-pregnant females	0	0	1	2
Number of females with ≤ 5 implantation sites	0	0	0	1
Number of evaluated females on GD20 (Caesarean section)	24	24	24	21

Table 3: Summary of the intrauterine evaluation

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated dams	24	24	24	21
Mean number of corpora lutea	12.21	11.13	11.67	11.57
Mean number of implantations	11.67	10.71	10.71	10.81
Preimplantation loss, mean	0.54	0.42	0.96	0.76
Preimplantation loss (%), mean	4.71	3.58	8.21	6.43
Early embryonic loss, mean	0.21	0.25	0.08	0.10
Early embryonic loss (%), mean	2.21	2.33	0.79	0.76
Late embryonic loss, mean	0.00	0.04	0.17	0.00
Late embryonic loss (%), mean	0.00	0.33	1.50	0.00
Dead foetuses, mean	0.00	0.00	0.00	0.00
Dead foetuses (%), mean	0.00	0.00	0.00	0.00
Postimplantation loss, mean	0.21	0.33	0.25	0.10
Postimplantation loss (%), mean	2.21	3.04	2.29	0.76
Total intrauterine mortality, mean	0.75	0.75	1.21	0.86
Total intrauterine mortality (%), mean	6.46	6.54	10.17	7.14
Viable foetuses, mean	11.46	10.42	10.46	10.71

Table 4: Examination of viable foetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of examined litters	24	24	24	21
Viable foetuses, mean	11.46	10.42	10.46	10.71
Male foetuses, mean	5.88	5.42	5.13	5.76
Female foetuses, mean	5.58	5.00	5.33	4.95
Total number of foetuses	275	250	251	225
Total number of male foetuses	141	130	123	121
Total number of female foetuses	134	120	128	104
Sex ratio (males), %	50.96	52.00	49.33	53.48
Mean foetal weight / litter (g)	3.406	3.509	3.422	3.483
Mean body weight / foetus (g)	3.400	3.509^**DN	3.418	3.486^**DN
Number of foetuses with retarded body weight	12	6	11	10
Number of affected litters (Runts)	10	4	9	8

DN:’s multiple range test, *: p<0.05, **: p<0.01

Table 5: Summary table of the external abnormalities

	Dose (mg/kg bw/day)				HC data
	Control	100	300	1000
Total number of examined litters	24	24	24	21	485
Total number of examined foetuses	275	250	251	225	4955
Total number of intact (normal) foetuses	275	250	251	224	--
Number of foetuses with malformation	0	0	0	0	--
Number of foetuses with variation	0	0	0	1	--
External malformations
No external malformations were observed
External variations
Pale foetus	--	--	--	1 / 1	1 / 1

Notes: Numbers represent the number of abnormalities / number of affected litters. HC: historical control

Table 6: Summary table of the visceral abnormalities

	Dose (mg/kg bw/day)				HC data
	Control	100	300	1000
Total number of examined litters	24	24	24	21	485
Total number of examined foetuses	138	123	126	113	2484
Total number of intact (normal) foetuses	135	113^*CH	118	108	--
Number of foetuses with malformation	0	1	0	0	--
Number of foetuses with variation	3	9^*CH	8	5	--
Visceral malformations
Liver lobe (caudate process), absent	--	1 / 1	--	--	--
Visceral variations
Thymic cord	1 / 1	7 / 4^*CH	3 / 3	3 / 3	66 / 55
Renal papilla, small	--	--	1 / 1	1 / 1	18 / 16
Ureter, convoluted	--	1 / 1	1 / 1	--	3 / 3
Brachiocephalic trunk, short	2 / 2	1 / 1	3 / 3	1 / 1	43 / 35

Notes: Numbers represent the number of abnormalities / number of affected litters. HC: historical control. CH: Chi²test, *: p<0.05, **: p<0.01

Table 7: Summary table of the skeletal abnormalities

	Dose (mg/kg bw/day)								HC data
	Control		100		300		1000
Total number of examined litters	24		24		24		21		484
Total number of examined foetuses	137		127		125		112		2467
Total number of intact (normal) foetuses	123		116		104		92		--
Number of foetuses with malformation	0		0		0		0		--
Number of foetuses with variation	14		11		21		20		--
Skeletal malformations
No skeletal malformations were observed
Skeletal variations
Skull, 2 or more bones incomplete ossification		6 / 5		3 / 3		12 / 5		7 /4	474 / 326
Ossified sternebra (3 or less)		1 / 1		4 / 2		3 / 3		2 / 1	46 / 37
Sternebra(s), misaligned		--		1 / 1		--		--	6 / 6
Ribs, wavy		5 / 4		5 / 4		11 / 7		13 / 7^*CH	243 / 149
Vertebra, dumbbell or asymmetric or unilateral ossification (2 or more)		--		--		2 / 1		--	79 / 73
Vertebra, unossified		--		1 / 1		--		--	2 / 1
Vertebra, bipartite ossification		1 / 1		1 / 1		1 / 1		--	24 / 24
Pubis, unossified		--		1 / 1		--		--	6 / 6
Carpal ossified ≤ 2.5		--		1 / 1		--		--	4 / 4
Tarsal ossified ≤ 3.5		1 / 1		2 / 2		--		1 / 1	75 / 53

Notes: Numbers represent the number of abnormalities / number of affected litters.HC: historical control.CH: Chi²test, *: p<0.05, **: p<0.01

Conclusions:

In this prenatal developmental toxicity study performed on the test item, no significant toxicological changes on dams, embryos or foetuses were observed and NOAEL for maternal toxicity, NOAEL for embryotoxicity, NOAEL for foetotoxicity and NOAEL for teratogenecity were determined to be 1000 mg/kg bw/day.

Executive summary:

A prenatal developmental toxicity study was performed in accordance with OECD 414 guideline (GLP study) in order to assessthe effects of the test item on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. After mating, the pregnant dams were treated daily by oral (gavage) administration at 3 dose levels, 100, 300 and 1000 mg/kg bw/day, from gestation day GD6 up to and including GD19. Caesarean section and necropsy with macroscopic examination was performed on GD20. Control dams were treated with the vehicle (distilled water) only. The number of confirmed pregnant, evaluated dams was 24 in the Control, 24 in the Low, 24 in the Mid and 21 in the High dose groups, respectively.

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain, individual food consumption and maternal reproductive parameters associated with uterine, for dams, and weight, external, visceral and skeletal examinations, for foetuses. Placentas were examined macroscopically.

There were no unscheduled mortality or treatment related clinical signs in the study. No significant differences in the body weight, body weight gain and daily food consumption of dams in any treated group were observed. There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 1000 mg/kg bw/day. There were no effects on the early or late embryonic loss in the test item-treated evaluated dams. The mean number of viable foetuses, the mean foetal body weight per litter and the sex ratio did not differ significantly from the control mean value in any of the treated groups. There were no effects of the test item on external, visceral and/or skeletal development of foetuses in the study which could be related to the test item administration. Increased incidence of the skeletal variation of wavy ribs in the Mid and High dose groups was comparable with the relevant historical control data.

In conclusion, from the observations made in the dams and their foetuses, there were no significant toxicological changes on embryos or foetuses, and therefore, the NOAEL for maternal toxicity, NOAEL for embryotoxicity, NOAEL for foetotoxicity and NOAEL for teratogenecity were determined to be 1000 mg/kg bw/day.

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:

The study is a GLP compliant and has Klimisch score 1.

Additional information

Pre-natal developmental toxicity: Key study: A prenatal developmental toxicity study was performed in accordance with OECD 414 guideline (GLP study) with Hannover Wistar rats in their first pregnancy. After mating, the pregnant dams were treated daily by oral (gavage) administration at 3 dose levels, 100, 300 and 1000 mg/kg bw/day, from gestation day GD6 up to and including GD19. Caesarean section and necropsy with macroscopic examination was performed on GD20. Control dams were treated with the vehicle (distilled water) only. The number of confirmed pregnant, evaluated dams was 24 in the Control, 24 in the Low, 24 in the Mid and 21 in the High dose groups, respectively. There were no significant differences compared with the control or test-item related-changes in any of the parameters observed o measured, mortality, clinical signs, body weight, body weight gain, daily food consumption, reproductive parameters of dams, early or late embryonic loss, number of viable foetuses, foetal body weight per litter and the sex ratio, and external, visceral and/or skeletal development of foetuses. Increased incidence of the skeletal variation of wavy ribs in the Mid and High dose groups was comparable with the relevant historical control data. In conclusion, no significant toxicological changes on dams, embryos or foetuses were observed. and NOAEL for maternal toxicity, NOAEL for embryotoxicity, NOAEL for foetotoxicity and NOAEL for teratogenecity were determined to be 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) no. 1272/2008.