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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Extended-one generation reproductive toxicity study (OECD 443, rat) (ToxiCoop ZRT, 2021)
NOAEL for toxicity (P/ F1 adult male and female): 51 mg/kg bw/day


NOAEL for reproductive performance (male and female): 180 mg/kg bw/day


NOAEL for F1 Offspring: 180 mg/kg bw/day


 


Reproduction/developmental toxicity screening test (OECD TG 421, rat) (WIL, 2006):


NOAEL (parental): 50 mg/kg/day


NOAEL (reproduction): >= 75 mg/kg/day


NOAEL (developmental): 50 mg/kg/day

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 August 2020 to 25 May 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
15 July 2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS
- Premating exposure duration for parental (P0) animals: 10 weeks
As requested by the Agency
- Basis for dose level selection: Based on a pre-test conducted with MEKP in DMP as diluent; furthermore, results of the subacute, subchronic toxicity study as well as screening study on toxicity to repdoduction (OECD 421) with the registered substance were taken into consideration
- Exclusion of extension of Cohort 1B
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B
Not required based on available data
- Exclusion of developmental immunotoxicity Cohort 3
Not required based on available data
- Route of administration
Worst case route of exposure: oral (gavage)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P0 males and females: not older than 9 weeks
- Weight at study initiation: (P) Males: 252 – 289 g; Females: 142 – 176 g
- Fasting period before study: no
- Housing:
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females will be housed individually.
Males after mating: 2 animals/cage
F1 offspring (after weaning): 2 animals of the same sex/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 70 %
- Air changes: > 10 per hr
- Photoperiod: 12/12 hrs dark / hrs light
Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of Test Facility and used within 3 days.
Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and measured on 5 occasions. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 17, 34 and 75/60 mg/mL (P)/ 12.75, 25.5 and 45 mg/mL (F1)
- Amount of vehicle: 3 mL (P)/ 4 mL (F1)
- Lot/batch no. 2003-4180
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: single
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentrations in the samples varied within the range from 92% to 101 % in comparison to the nominal values.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front (Toxi-Coop study no: 552-100-4500).
A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery from sunflower oil was 95 % of nominal concentrations at 2 mg/mL and 200 mg/mL. The test item was stable at 2 mg/mL and 200 mg/mL concentrations for at least 24 hours at room temperature and for three days in a refrigerator 5 ± 3 °C.
Duration of treatment / exposure:
Overall treatment period was 124-125 days for male parental animals.
Overall treatment period was up to 115-123 days for dams and 103 days for not mated, non-pregnant or not delivered female animals.
F1 offspring in Cohort 1A were administered up to post-natal day 98-110 (approximately 14-16 weeks).
F1 animals of Cohort 1B were administered up to post-natal day 106-116 (approximately 15-17 weeks).
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 16-18 weeks
Dose / conc.:
51 mg/kg bw/day (actual dose received)
Dose / conc.:
102 mg/kg bw/day (actual dose received)
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Remarks:
The dose level was reduced from 225 mg/kg bw/day to 180 mg/kg bw/day on Day 46 because of increased mortality (eight of 48 animals) on study Days 3, 6, 27, 32, 44 and 45.
No. of animals per sex per dose:
24 P0 / sex / dose
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Rationale for animal assignment: random
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: same as weighing

BODY WEIGHT: Yes
- Time schedule for examinations:
Parental males were weighed on the first day of dosing (day 0) and weekly thereafter.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partum days 0 (within 24 hours after parturition), 4, 7, 14 and 21. Body weight of the female animals will be additionally weighed on gestation day 10 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight data will be reported individually for adult animals.
F1 animals selected for follow-up examinations were weighed on post-natal day 22, then twice a week during the two weeks following weaning, and once weekly thereafter.
For selected F1offspring, the body weight was recorded on the day when they attain puberty (completion of balano-preputial separation or vaginal patency).
Fasted body weight was measured on the day of necropsy for all animals (P and F1).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily by visual inspection

URINALYSIS
The following parameters were evaluated in selected test animals of P and F1 Cohort 1A generation:
Nitrite (NIT), pH, Glucose (GLUC), Urobilinogen (UBG), Bilirubin (BIL), Ketone (KET), Blood, Leucocytes (LEU), Specific Gravity (SG), Protein (PROT), Volume (VOL), Sediment (SED), Colour, Clarity

HEMATOLOGY/BLOOD COAGULATION
The following parameters were measured in all selected animals of P and F1 Cohort 1A generation:
White blood cell (leukocyte) count (WBC), Red blood cell (erythrocyte) count (RBC), Hemoglobin concentration (HGB), Hematocrit (HCT), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) hemoglobin (MCH), Mean Corpuscular (erythrocyte) hemoglobin concentration (MCHC), Platelet (thrombocyte) count (PLT), Reticulocytes (RET), Differential white blood cell count, Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT).

CLINICAL CHEMISTRY
The following parameters were measured in all selected animals of P and F1 Cohort 1A generation:
Alanine Aminotransferase activity (ALT), Aspartate Aminotransferase activity (AST), Total Bilirubin concentration (TBIL), Creatinine concentration (CREA), Urea concentration (UREA), Glucose concentration (GLUC), Cholesterol concentration (CHOL), Sodium concentration (Na+), Potassium concentration (K+), Albumin concentration (ALB), Total protein concentration (TPROT)

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4, TSH) as follows:
- from 10 parent male animals/sex/ group at termination
- from 10 P dams on post-partum day 22
- from 10 adult F1 Cohort 1A male and female animals/group at termination

SEXUAL MATURITY
Sexual maturity of selected F1 animals (Cohort 1A and Cohort 1B) was examined by observing balano-preputial separation on postnatal day 25 or vaginal patency (between postnatal days 25 and 39). The body weight was determined on the day when balano-preputial separation or vaginal patency was completed.
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears from each parental female animal daily for two weeks before the mating starts.
Vaginal smears was also prepared and estrous cycle was monitored daily during the mating period until evidence of copulation.
Vaginal smear was also prepared on the day of the necropsy of parental animals.
Vaginal smears were examined for all F1 Cohort 1A females selected for follow-up examinations after the onset of vaginal patency until the first cornified smear is recorded thus determining the time interval between these events.
Estrous cycle of F1 adult female animals was examined for a period of two weeks commencing on PND77 and PND84 in Cohort 1A and Cohort 1B, respectively, including necropsy days.
Vaginal smears was stained with 1 % aqueous methylene blue solution. After drying, the smears were examined with a light microscope.
Sperm parameters (parental animals):
Sperm parameters were determined in all control, mid and high dose male animals in P generation and in all control and high dose male animals in F1 generation Cohort 1A.

The one-side testes and epididymides were used for examinations. The weights of one-side testes and epididymides were determined and recorded.

Sperm from the ductus deferens was collected for evaluation of sperm motility and morphology at the necropsy. Both numbers of motile and immotile sperms were recorded. Two samples were prepared from each animal. For the determination of the sperm motility, the mean percentage of motile sperms was determined. A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails). The epididymis was used for enumeration of cauda epididymis sperm reserves. The total number of sperm in homogenization was enumerated. The testis and epididymidis were frozen and enumeration was performed later.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups on PND13, FT3, FT4 and TSH in surplus pups at PND 4 (pooled by litters) and in pups not selected for Cohorts on post-natal day 22.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after weaning of offspring.
- Maternal animals: All surviving animals at least up to and including PND 21.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively, for high dose and control animals:
Organ weights (all parental (P) animal and all adult F1 animals of Cohort 1A):
 uterus (with oviducts and cervix)
 ovaries
 testes
 epididymides
 prostate (dorsolateral and ventral parts combined)
 seminal vesicles with coagulating glands as one units (with their fluids)
 brain
 liver
 kidneys
 heart
 spleen
 thymus
 pituitary
 thyroid glands (post-fixation)
 adrenal glands
List of organs preserved and examined microscopically (all parental (P) animal and all adult F1 animals of Cohort 1A):
 Adrenal glands
 Bone with marrow and joint (femur)
 Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
 Eyes (lachrymal gland with Harderian glands and optic nerve)
 Mammary gland (male and female)
 Heart
 Kidneys
 Large intestines (caecum, colon, rectum),
 Liver
 Lungs (with main stem bronchi; inflation with fixative and then immersion;)
 Lymph nodes (submandibular, mesenteric; popliteal for Cohort 1 animals)
 Muscle (quadriceps)
 Esophagus
 Pancreas
 Pituitary
 Salivary glands (submandibular)
 Sciatic nerve
 Sexual organs (testes, epididymides, seminal vesicle with coagulating gland, prostate ovaries, uterus with oviduct, vagina)
 Small intestines (duodenum, ileum, jejunum including Peyer’s patches)
 Spinal cord (at three levels: cervical, mid-thoracic and lumbar)
 Spleen
 Stomach
 Thymus
 Thyroid + parathyroid
 Trachea
 Urinary bladder
In animals of Cohort 1B, the weight of following organs were determined:
 uterus (with oviducts and cervix)
 ovaries
 testes
 epididymides
 prostate (dorsolateral and ventral parts combined)
 seminal vesicles with coagulating glands as one unit (with their fluids)
 brain
 pituitary
In adult F1 animals in Cohort 1B, the uterus (with oviducts and cervix), ovaries, testes, epididymides, prostate (dorsolateral and ventral parts combined), seminal vesicles with coagulating glands as one unit (with their fluids), brain and pituitary were processed up to block stage for later examinations if necessary.

SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS
At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female Cohort 1A animals from each group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected) as follows:
- weighing of the lymph nodes associated with and distant from the route of exposure (submandibular and popliteal lymph nodes);
- splenic lymphocyte subpopulation analysis: CD4+ (Helper T cells) and CD8+ (Cytotoxic T cells) T lymphocytes, B lymphocytes and natural killer (NK) cells were identified by a validated flow cytometry method. Immuno-staining and immunophenotyping of spleen lymphocytes were carried out after preparation of single cell suspensions from one half of each provided spleen. For further information please refer to the attached background material.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations macroscopic and microscopic examination.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera
HISTOPATHOLOGY / ORGAN WEIGTHS
For 10 male and 10 female F1 pups per group, not selected for Cohorts – from as many litters as possible – brain, spleen and thymus were weighed and preserved in 4 % buffered formaldehyde solution. In addition mammary tissues were preserved in the same solution.
Statistics:
The statistical evaluation of appropriate data 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 assess the significance of inter-group differences. Getting significant results 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 if feasible. Frequency of toxic response, pathological and histopathological findings by sex and dose were calculated.
Reproductive indices:
Copulatory Index (Measure of animals ability to mate):
Males: Number of males with confirmed mating / Total number of males cohabited x 100
Females: Number of sperm positive females / Total number of females cohabited x 100

Fertility Index (Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant):
Males: Number of males impregnating a females / Total number of males with confirmed mating x 100
Females: Number of pregnant females / Number of sperm positive females x 100

Gestation Index (Measure of pregnancy that provides at least one live pup):
Number of females with live born pups / Number of pregnant females x 100
Offspring viability indices:
Formulas for Calculation of Pup Mortality and Sex Ratio Indices:

Post-implantation mortality: Number of implantations – Number of liveborns / Number of implantation x 100

Post-natal mortality: Number of liveborns – Number of live pups on PND 13 / Number of liveborns x 100

Survival Index: Number of live pups on PND 13 / Number of liveborns x 100

Sex ratio: Number of pups examined – Number of pups males (females) / Number of pups examined x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Daily Observations
Clinical signs related to the test item administration were observed in parental male and female animals at 102 and 180/225 mg/kg bw/day in a dose related manner and incidence.

Dead and moribund animals
One female animal at 51 mg/kg bw/day (1/24) was euthanized due to its moribund state on Day 45. Decreased activity, hunched back, piloerection, swelling under the left fore-limb and abnormal limb position of left fore-limb were observed on Days 44 and 45. Necropsy observation revealed signs of mis-gavage causing moribund state.
In one male animal at 102 mg/kg bw/day (1/24), opalescent left eye between Days 111 and 117, and dyspnea on Day 117 were observed and this animal was found dead on Day 118.
Dyspnea was detected in one (1/2) female animal at 102 mg/kg bw/day on Days 66 and 67 and this animal was found dead on Day 68. One dam (1/2) at 102 mg/kg bw/day showed dyspnea, piloerection, marked activity decrease and sanguineous nose on lactation day 2 and died on lactation day 3.
At 180/225 mg/kg bw/day, nine male (9/24) and five (5/24) female animals were found dead following variable number of treatments between Days 6 and 100.
Nuzzling up the bedding material (8/9), salivation (3/9), noisy breathing (2/9) and dyspnea (1/9) were noted for dead male animals at 180/225 mg/kg bw/day. Most of male animals (7/9) died without showing severe preceding signs (dyspnea or noisy breathing).
In dead female animals 180/225 mg/kg bw/day, noisy breathing (1/3), salivation (1/3) and nuzzling up the bedding material (2/3) were observed during the pre-mating period.
During the gestation period, noisy breathing (1/2) dyspnea (1/2), piloerection (1/2), salivation (1/2) and nuzzling up the bedding material (2/2) were observed in dead female animals at 180/225 mg/kg bw/day.
These clinical signs (dyspnea, noisy breathing, decreased activity, piloerection and sanguineous nose) were presumably related to the test item administration.

Surviving animals
Male animals in control group were normal during the entire observation period.
In one dam in the control group, alopecia was detected on the forelimbs and on the right side of the abdomen between lactation day 7 and 21 as well as between lactation day 9 and 21, respectively. All other female animal were normal in the control group.
At 51 mg/kg bw/day, scar on the skin of the neck (right side; 1/24; Days 69-96) and alopecia behind the left ear (1/24, Days 111-123) were seen in the male animals.
All female animals were normal at 51 mg/kg bw/day during the entire observation period.
At 102 mg/kg bw/day, dyspnea (1/23; Day 122), noisy breathing (1/23; Days 112-123) and alopecia behind ears (1/23; Day 112-124) were noted for single male animals.
In the female animals at 102 mg/kg bw/day, salivation (1/23) during the pre-mating period (Days 11, 12) and noisy breathing (1/21 dam) on lactation day 14 were observed transiently.
At 180/225 mg/kg bw/day, male animals showed dyspnea (3/15), noisy breathing (5/15), nuzzling up the bedding material (15/15) and salivation (7/15).
In the female animals at 180/225 mg/kg bw/day, dyspnea (1/21), nuzzling up bedding material (21/21), salivation (3/21) and scars on the skin (2/21; on the scapula or back) were observed during the pre-mating period.
Nuzzling up bedding material was noted for one not mated and one non pregnant female animal at 180/225 mg/kg bw/day during the post-mating period.
During the gestation period, nuzzling up bedding material (17/17), salivation (1/17), noisy breathing (1/17), dyspnea (1/17), piloerection (1/17) and scars on the skin of the scapula or back (2/17) were observed in female animals at 180/225 mg/kg bw/day.
Dyspnea (1/17), salivation (1/17) and small scars on the back (1/17) were seen in single dams at 180/225 mg/kg bw/day during the lactation period.
These clinical signs (dyspnea, noisy breathing, decreased activity, piloerection and sanguineous nose) were presumably related to the test item administration.
Alopecia and scars on the skin are common findings in experimental rats of this strain. These were observed with low incidence in the control (female), at 51 and 102 mg/kg bw/day (male) and at 180/225 mg/kg bw/day (female).
Opalescent eye – observed in one male animal at 102 mg/kg bw/day – is also a species-specific ocular alteration occurring in non-treated animals.
For incidences during the detailed weekly clinical observations please refer to the attached tables.
Mortality:
mortality observed, treatment-related
Description (incidence):
Test item-related mortality was observed in parental animals at 102 mg/kg bw/day (1/24 M, 2/24 F) and 180/225 mg/kg bw/day (9/24 M, 5/ 24 F).
Based on clinical signs, necropsy findings and results of histological examinations, these animals died probably as a consequence of reflux in the laryngopharyngeal area during the oral gavage of the highly irritant test item.
One female animal at 51 mg/kg bw/day (1/24) was euthanized in moribund condition on Day 45. Clinical observations and necropsy findings refer to technical mistake at the treatment (mis-gavage) supported by histopathological findings as cause of death.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development was undisturbed in parental (male and female) at 51, 102 and 180/225 mg/kg bw/day during the entire treatment period.
Statistical significances with respect to the control were detected sporadically as follows:
Male animals
51 mg/kg bw/day: lower mean body weight gain between Days 56-63 and Days 104-111; higher mean body weight gain between Days 63-69
180/225 mg/kg bw/day: lower mean body weight gain between Days 42-49; higher mean body weight gain between Days 14-21, Days 49-56, Days 56-63
Female animals
Pre-mating period:
51 mg/kg bw/day: higher mean body weight gain between Days 63-69
102 mg/kg bw/day: lower mean body weight gain between Days 21-28 and Days 49-56; higher mean body weight gain between Days 63-69;
Gestation period:
102 mg/kg bw/day: lower mean body weight on gestation day 7 (-4%);
180/225 mg/kg bw/day: lower mean body weight on gestation day 7 (-5 %);
Lactation period:
51 mg/kg bw/day: lower mean body weight on lactation day 14 (-4 %);
102 mg/kg bw/day: lower mean body weight on lactation days 0, 4, 7 and 14 (up to max. 5%);
180/225 mg/kg bw/day: lower mean body weight on lactation days 4 and 7 (up to max. 5%);;
The mean body weight was comparable with the control in male animals at 51, 102 and 180/225 mg/kg bw/day during the entire observation period, in female animals at 51, 102 and 180/225 mg/kg bw/day during the premating period, at 51 mg/kg bw/day during gestation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean daily food consumption was not adversely affected in parental male or female animals at 51, 102 or 180/225 mg/kg bw/day.
The mean daily food consumption was similar to the control in male animals at 51 and 102 mg/kg bw/day during the study.
Statistical significance with respect to the control was detected at the lower or higher mean daily food consumption of parental male animals at 180/225 mg/kg bw/day between Days 0-7, 21-28 and 49-56 (higher) and between Days 0-7 and Days 42-49 (lower).
The mean daily food consumption was similar to the control in parental female animals at 51 mg/kg bw/day during the pre-mating, gestation and lactation period.
In the parental female animals, the mean daily food consumption was statistically significantly lower at 102 mg/kg bw/day between Days 0-7 and Day 49-56 and 180/225 mg/kg bw/day between Days 0-7, comparing to the control.
During the gestation period, the mean daily food consumption was below the control at 102 mg/kg bw/day between gestation days 0-7 (up to 9 %) and 7-14 and at 180/225 mg/kg bw/day between gestation days 7-14 and 17-21 (up to 7 %).
The mean daily food consumption was comparable in the control and test item administered groups during the lactation period.
These slight differences with respect to the control were of low degree and not consistent during the treatment period. Therefore, these were not considered to be toxicologically relevant.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related adverse changes in the examined hematological or blood coagulation parameters in parental male or female animals at 51, 102 or 180/225 mg/kg bw/day.
In the male animals at 51 mg/kg bw/day, statistical significance was detected at the higher mean percentage of eosinophil granulocytes (EOS) and at a slightly shorter mean prothrombin time (PT) when compared to the control.
Statistical significance with respect to the control was observed at higher mean percentage of monocytes (MONO), at the lower mean hemoglobin concentration (HGB) and lower mean corpuscular hemoglobin content (MCH) and lower mean corpuscular hemoglobin concentration (MCHC) in male animals administered with 102 mg/kg bw/day.
At 180/225 mg/kg bw/day, higher mean white blood cell count (WBC), lower mean red blood cell count (RBC) and hemoglobin concentration (HGB), lower mean hematocrit (HCT), higher mean corpuscular volume (MCV) and lower mean corpuscular hemoglobin concentration (MCHC), elevated mean percentage of reticulocytes (RET) and slightly shorter mean prothrombin time were observed in male animals when compared to the control.
In female animals at 51 mg/kg bw/day, statistical significance was detected at the higher mean percentage of monocytes (MONO), lower mean percentage of red blood cell count (RBC), higher mean corpuscular volume (MCV) and at the slightly longer mean prothrombin time (PT) when compared to the control.
The mean prothrombin time exceeded the control in female animals at 102 mg/kg bw/day.
At 180/225 mg/kg bw/day, higher mean percentage of monocytes, lower mean percentage of red blood cell count, higher mean corpuscular volume and mean corpuscular hemoglobin content (MCH) and slightly longer mean prothrombin time were observed in female animals when compared to the control.
The differences with respect to the control group reached statistical significance at the mentioned parameters. However, the individual values (EOS, MONO, MCH, MCHC, WBC, HCT, MCV, MCH, PT) met the historical control range (i.e., were within or close to the range) in male and female animals, except for RET in male animals and RBC in female animals at 180/225 mg/kg bw/day.
There was no dose relevance in the degree of changes (EOS, MONO, RBC, MCH, MCHC, PT in male animals, MONO, RBC, MCV, PT in female animals).
Therefore, the differences in these parameters were considered to have little or no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The examined clinical chemistry parameters were not adversely affected in parental male or female animals at 51, 102 or 180/225 mg/kg bw/day.
In male animals, a higher mean urea concentration (statistically significantly different to control at 51 and 180/225 mg/kg bw/day) and a lower mean glucose concentration (statistically significantly different to control at 180/225 mg/kg bw/day) were detected. However, all values were within historical control values. A slight elevation of mean activity of alanine aminotransferase was observed at 51 mg/kg bw/day.
In female animals, a dose related statistically significant elevation the mean urea concentration compared to control was detected in all treatment groups. However, all values of treated animals were within the historical control range and statistical difference is based on a low value in the control group compared to historical control data.
Statistically significant differences with respect to the control were detected at the lower mean concentration of potassium (K+) at 51 mg/kg bw/day and at the higher mean activity of alanine aminotransferase (ALT) and lower mean concentration of creatinine (CREA) at 225/180 mg/kg bw/day.
Although findings were either of low degree or did not show a dose-response relationship, treatment-related changes in the high dose animals cannot be fully excluded.
Endocrine findings:
no effects observed
Description (incidence and severity):
The thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals or in PND22 F1 offspring at any dose levels.
The FT3, FT4 and TSH levels were comparable in male animals and in PND22 pups in the control, 51, 102 and 180/225 mg/kg bw/day groups.
Statistically significant difference was detected at the higher mean FT3 level at 102 and 180/225 mg/kg bw/day and higher mean FT4 concentrations at 180/225 mg/kg bw/day and lower mean TSH level at 102 mg/kg bw/day in female animals. The degree of changes was minor and individual and mean values were well within the historical control ranges. In the absence of related changes in reproductive performance, organ weight or histopathology observations, these findings in female animals were considered to be toxicologically not relevant.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in the examined urine parameters in parental male or female animals at 51, 102 or 180/225 mg/kg bw/day.
The examined urine parameters were comparable in the parental male and female animals in the control and 51, 102 and 180/225 mg/kg bw/day groups.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology investigation did not reveal toxic or other test item-related lesions of investigated genital organs of the experimental male and female parental animals at 51, 102 or 180/225 mg/kg bw/day doses.
Squamous cell hyperplasia was observed in the mucous membrane of non-glandular stomach – with dose dependent incidence – at 102 mg/kg bw/day (11/23) and 180/225 mg/kg bw/day (21/24) in connection with the local effect of high and medium doses of the test item.
In dead animals at 102 and 180/225 mg/kg bw/day (male and female), histological examinations revealed lesions referring to systemic toxicity: serious congestion in the lungs, collapse of fluid balance, accumulation of large amount of enteral fluid in the stomach, dilatation of small and large intestines. Local lesions were observed in the stomach (squamous cell hyperplasia in the mucous membrane of non-glandular stomach)
The investigated organs of the male reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic for the sexually mature organism in all parental male animals in the control and 180/225 mg/kg bw/day groups (24/24 for both groups) as well as in not mated males and in males not impregnated females at 51 or 102 mg/kg bw/day (2/2 at 51 mg/kg bw/day and 1/1 at 102 mg/kg bw/day).
In the female animals at 180/225 mg/kg bw/day and control groups and in not mated, non-pregnant female animals at 51 mg/kg bw/day (2/2) or 102 mg/kg bw/day (1/1) of parental generation, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
In dead male animals, dilatation of small and large intestines (1/1 at 102 mg/kg bw/day and 3/9 at 180/225 mg/kg bw/day), pulmonary congestion (1/1 at 102 mg/kg bw/day and 5/9 at 180/225 mg/kg bw/day) and squamous cell hyperplasia (8/9 at 180/225 mg/kg bw/day) were detected as test item-related lesions.
Test item-related lesions were detected in dead female animals, too:
- dilatation of small and large intestines (5/5 at 180/225 mg/kg bw/day),
- pulmonary congestion (1/2 at 102 mg/kg bw/day and 3/5 at 180/ 225 mg/kg bw/day)
- squamous cell hyperplasia (1/2 at 102 mg/kg bw/day and 5/5 180/225 mg/kg bw/day)
- dilatation of stomach (1/2 at 102 mg/kg bw/day, 4/5 at 180/225 mg/kg bw/day)
Additionally, renal pyelectasia (1/2 female at 102 mg/kg bw/day, 2/9 male at 180/225 mg/kg bw/day), renal congestion (1/2 female at 102 mg/kg bw/day and 1/5 female at 180/225 mg/kg bw/day), purulent necrotic bronchitis (1/2 female at 102 mg/kg bw/day), thymic hemorrhage (1/2 female at 102 mg/kg bw/day and 1/5 female at 180/225 mg/kg bw/day) and dermal inflammation (1/5 female at 180/225 mg/kg bw/day) were detected in dead animals. These findings were probably in connection with the death of animals (renal congestion) or were individual ones (pyelectasia, purulent bronchitis, thymic hemorrhage, dermal inflammation).
In moribund female animal (1/1 at 51 mg/kg bw/day), serous purulent inflammation in the skin and inflammation in the salivary gland were observed in accordance with necropsy findings as individual lesions due to the para-gastric treatment.
Surviving animals
Squamous cell hyperplasia in the mucous membrane of non-glandular stomach was detected in a dose related manner in surviving male animals (11/23 at 102 mg/kg bw/day and 13/15 at 180/225 mg/kg bw/day) and in female animals (2/2 at 102 mg/kg bw/day and 19/19 at 180/225 mg/kg bw/day). This gastric change was judged to be in connection with the local effect of high and medium doses of test item.
One or both sided pyelectasia occurred in each group of male and female animals as follows:
- male 5/24 control, 1/1 at 51 mg/kg bw/day, 3/3 at 102/mg/kg bw/day, 5/15 at 180/225 mg/kg bw/day:
- female: 2/24 control, 2/2 at 51 mg/kg bw/day, 1/1 at 102/mg/kg bw/day, 3/19 at 180/225 mg/kg bw/day:
Pyelectasia without significant histological lesions (inflammation, degeneration, fibrosis etc.) is common in laboratory rats of this strain and is considered to be an individual disorder without pathological significance.
Signs of inflammatory processes were detected in some organs, which are considered as species specific alterations i.e., these occurs commonly in non-treated experimental rats of this strain:
- purulent necrotic foci in the spleen – 1/1 male at 51 mg/kg bw/day;
- encapsulated inflammatory focus in the abdominal cavity – 1/24 female in control group;
- acute inflammation on the skin of tail – 1/19 female at 180/225 mg/kg bw/day;
Hemorrhage in the thymus is considered to be in connection with the hypoxia and circulatory disturbance developing during the exsanguination procedure and was seen in single control male animal (1/24, surviving) in this study.
Dilatation of uterine horn was observed in some animals in each group independently from doses: 3/24 in control group, 2/2 at 51 mg/kg bw/day; 4/4 at 102 mg/kg bw/day; 3/19 at 180/225 mg/kg bw/day. Dilatation of uterine horns – without inflammatory or other pathological lesions – is a slight neurohormonal phenomenon and is in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
There was no morphological evidence of test item-related acute or subacute injury (degeneration, inflammation, necrosis etc.) in the small and large intestines, liver, pancreas, cardiovascular system, respiratory system, immune system, hematopoietic system, skeleton, muscular system, central, or peripheral nervous system, eyes, integumentary system.
The cytomorphology of endocrine glands were the same in the control and treated animals.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The estrous cycle was not adversely influenced by the test item at 51, 102 or 180/225 mg/kg bw/day.
The examined parameters of the estrous cycle were comparable in the control and 51, 102 and 180/225 mg/kg bw/day groups.
Statistically or biologically significances were not detected at the percentage of animals with regular or irregular cycle, mean number of cycles, length of cycles, mean number of days in pro-estrous, estrous or diestrous as well as in the percentage of animals in prolonged estrous or diestrous at 51, 102 or 180/225 mg/kg bw/day.
The percentage of female animals with irregular cycle (38 – 48 %) was higher than the mean historical control (12 %) in each group (control, 51, 102 and 180/225 mg/kg bw/day) during the two last weeks of an overall of 10 weeks pre-mating period. However, they met well the historical control data range of 0 – 58 %. This finding is not considered test-item-related as it was observed in all groups without dose-relation.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm examinations did not reveal any test item-related influence on the sperm cells at 102 or 180/225 mg/kg bw/day.
Statistical significances were detected at the mean percentage of immotile sperm cells and cells with separated head in parental male animals at 180/225 mg/kg bw/day (both less than control).
The mean percentage of sperms with normal morphology was similar in the control, 102 and 180/225 mg/kg bw/day groups.
Differences in the percentage of immotile sperm cells and cells with separated head were considered to be indicative of biological variation and normal function of male genital organs.
Reproductive performance:
no effects observed
Description (incidence and severity):
The reproductive performance of male and female animals was not adversely affected by the treatment at 51, 102 or 180/225 mg/kg bw/day with respect to their control.
The copulatory index was similar in all groups (control, 51, 102 and 180/225 mg/kg bw/day, male and female) and the fertility index was higher than in the control group in male animals at 102 and 180/225 mg/kg bw/day and in female animals at 102 mg/kg bw/day. The gestation index exceeded the control value at 51, 102 and 180/225 mg/kg bw/day.
In female animals, statistical significance was detected at the lower mean percentage of non-pregnant females and higher mean percentage of pregnant females at 102 mg/kg bw/day. The percentage of delivered dams was higher than in the control group at 51 and 102 mg/kg bw/day.
The differences between the control and test item- treated groups are without any biological relevance and lay within the biological range of variation.
Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
51 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other: Systemic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
102 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Offspring
There were no adverse clinical signs in the F1 offspring from post-natal day 0 to 21.
The percentage of offspring showing signs (no milk in the stomach, cold, smaller than normal, pale, hemorrhage, found dead, missing) was higher in test item-treated groups than in the control. Most of these observations were detected on the day of delivery and corresponded with the findings of inadequate nursing behavior of the respective dams.
The percentage of offspring, which were cold were higher than in the control group (2 %) at 51 mg/kg bw/day (15 %), at 102 mg/kg bw/day (14 %) and at 180/225 mg/kg bw/day (24 %). Percentage of not suckled pups was higher than in the control group at 51 and 102 mg/kg bw/day (10 and 14 % versus 1 %).
Some other sporadic clinical signs were detected with similar incidence (smaller than normal, pale, hemorrhage, found dead or missing) in the control and 51, 102 or 180/225 mg/kg bw/day dose groups.
F1 Cohort 1A Generation
Daily Observations
There were no test item-related clinical signs referring to systemic toxicity in male or female animals in F1 Cohort 1A generation in 51, 102 or 180 mg/kg bw/day groups.
In dead animals, dyspnea (1/1 male and 1/3 female at 102 mg/kg bw/day, for one day; 1/6 male at 180 mg/kg bw/day, for two weeks; 2/4 female at 180 mg/kg bw/day, for one day) and noisy breathing (1/6 at 180 mg/kg bw/day, for five day) were observed before death. All other dead animals were symptom free during the days preceding their death: 2/3 female at 102 mg/kg bw/day, 5/6 male and 2/4 female at 180 mg/kg bw/day.
In surviving animals in the control, 51 and 102 and 180 mg/kg bw/day groups, the behaviour and physical condition of animals were normal during the entire treatment period (PND22-PND114) except for three animals as follows:
- Skin: Scar behind right ear – 1/22 male at 180 mg/kg bw/day; between PND91-97;
- Dyspnea – 1/24 female at 180 mg/kg bw/day, on PND33;
- Noisy breathing: 1/24 female at 180 mg/kg bw/day, on PND 40 and 41;
Transient breathing disturbance in two female animals was judged to be a local effect related to the treatment with the test item, which however did not result in death of animals. Scar on the skin is a species-specific sign of this strain of experimental rats.
F1 Cohort 1B Generation
Daily clinical observations revealed clinical signs (disturbance in breathing) related to the test item in F1 Cohort 1B animals at 180 mg/kg bw/day.
Dead animals
In the dead female animal at 51 mg/kg bw/day, dyspnea was detected immediately before death. The physical state and behavior, body weight development was normal up to post-natal day 36 and animal died on post-natal day 37.
Dead male animal at 102 mg/kg bw/day showed normal behavior and body weight development before the death and was found dead one day after the treatment (PND50).
At 180 mg/kg bw/day, dyspnea was detected (2/4 male and 1/3 female) before the death. There were no preceding clinical signs or body weight change in 2/4 male and 2/3 female animals.
Surviving animals
In the control group, scar on the skin of the neck (1/20) and reddish colored hairs around the right eye (1/20) were detected in male animals.
There were no clinical signs in female animals in the control group and in male and female animals at 51 and 102 mg/kg bw/day.
At 180 mg/kg bw/day, dyspnea (1/21 male, 1/22 female), noisy breathing (1/21 male), scar on the skin behind the right ear or on the scapula (2/22 females) and alopecia on the chest, hindlimbs and right side fore-limb (1/22 females) were detected.
Dyspnea and noisy breathing were considered to be a consequence of the reflux of the test item. Alopecia and scars on the skin are species-specific findings detectable in non-treated experimental rats of this strain. Brownish hairs around eye are indicative of enhanced porphyrin production of Harderian gland – observed in one male animal in control group in this study – is a common observation in this strain of experimental rats.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There was no test item-related effect on offspring’s extra uterine mortality. The extrauterine mortality was low and comparable in the control, 51, 102 and 180/225 mg/kg bw/day group on post-natal day 0 and from birth to post-natal day 21.
Test item-related mortality was observed in F1 Cohort 1A animals at 102 mg/kg bw/day (1/20 M, 3/20 F) and 180/225 mg/kg bw/day (6/28 M, 4/28 F).
Test item-related mortality was observed in F1 Cohort 1B animals at 51 mg/kg bw/day (1/20 M), 102 mg/kg bw/day (1/20 M) and 180/225 mg/kg bw/day (4/25 M, 3/25 F).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Offspring
The body weight development of the F1 offspring was not adversely affected at 51, 102 and 180/225 mg/kg bw/day.
Slight reduction in the mean body weight (between -2 and -4 %) and mean body weight gain (between -3 and – 7 %) was considered to be toxicologically not relevant. The mean litter weight and litter weight gain were comparable with control and 51, 102 and 180/225 mg/kg bw/day groups during the 21-day observation period. Statistical significance with respect to the control was detected at the lower mean body weight of pups at 51 mg/kg bw/day on post-natal days 0, 4, 7, at 102 mg/kg bw/day on post-natal days 0 and 21 and at 180/225 mg/kg bw/day on post-natal days 0, 4, 7, 14 and 21. The mean body weight gain of pups was below the control value at 51 mg/kg bw/day between post-natal days 4-7, 7-14, 14-21 and at 102 mg/kg bw/day between postnatal day 14-21 and 0-21. Similarly, the mean body weight gain of offspring was below the control at 180/225 mg/kg bw/day from birth up to post-natal day 21. The mean body weight of male pups was slightly lower than in the control at 102 and 180/225 mg/kg bw/day and in female animals at 51 mg/kg bw/day on post-natal day4 (evaluated separately by gender).
F1 Cohort 1A Generation
The body weight development was undisturbed in F1 Cohort 1A (male and female) at 51, 102 and 180 mg/kg bw/day during the entire treatment period.
The mean body weight was comparable to their control in F1 Cohort 1A male and female animals at 51, 102 and 180 mg/kg bw/day during the entire observation period.
Sporadic statistically significant difference with respect to the control were detected in the body weight gain as follows:
Male animals
- 51 mg/kg bw/day: lower mean body weight gain between post-natal days 77-84;
- 102 mg/kg bw/day: lower mean body weight gain between post-natal days 22-25 and 49-56;
- 180 mg/kg bw/day: higher mean body weight gain between post-natal days 70-77, lower mean body weight gain between post-natal days 77- 84;
Female animals:
- 51 mg/kg bw/day: lower mean body weight gain between post-natal days 56-63;
- 102 mg/kg bw/day: lower mean body weight gain between post-natal days 25-29;
These minor changes in the body weight gain did not result in significant changes in the body weight of male or female animals, were not consistent, and therefore, were considered to be toxicologically not relevant.
F1 Cohort 1B Generation
There were no adverse test item-related changes in the body weight or body weight gain in F1 Cohort 1B animals administered with 51, 102 or 180 mg/kg bw/day (male and female).
The mean body weight was comparable in male animals in the control and 51, 102 and 180 mg/kg bw/day body during the entire observation period.
The mean body weight gain was lower than in the control group in male animals at 51 mg/kg bw/day between post-natal day 98 and 105, at 102 mg/kg bw/day between post-natal day 49-56 and between 98-105 and at 180mg/kg bw/day between post-natal day 49-56.
In the female animals, the following statistically significant differences with respect to the control were detected:
- 51 mg/kg bw/day: lower mean body weight on PND98 and PND112 (-5 %); higher mean body weight gain between PND 29-32 and lower mean body weight gain between PND 22-112;
- 102 mg/kg bw/day: lower mean body weight on PND29 and from PND36 up to 112 at each occasion (up to -7 %); lower mean body weight gain between PND 25-29 and PND 32-36, between PND 22-112;
- 180 mg/kg bw/day: the mean body weight was comparable with the control during the entire observation period; lower mean body weight gain between PND 29-32;
These minor changes in the body weight gain did not result in significant changes in the body weight of male or female animals and were not related to doses. Therefore, the slightly reduced body weight at 102 mg/kg bw/day was considered to be independent from treatment and were caused by the reduced feed intake in this dose group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
F1 Cohort 1A Generation
The mean daily food consumption of F1 Cohort A male and female animals was not adversely affected at 51, 102 or 180 mg/kg bw/day during the observation period.
The mean daily food consumption was comparable with the control in all treated female animals and in male animals at 51 and 180 mg/kg bw/day during the entire observation period. Statistically significant difference with respect to the control was detected at the lower mean daily food consumption in male animals at 102 mg/kg bw/day between post-natal days 22-29 (-7 %). These minor difference to the control was judge to be toxicologically not relevant because of inconsistent occurrence and low degree.
F1 Cohort 1B Generation
The mean daily food consumption of male and female animals in F1 Cohort 1B animals was not adversely affected at 51, 102 or 180 mg/kg bw/day during the observation period.
The mean daily food consumption was similar to their control in male animals of F1 Cohort 1B at 51 mg/kg bw/day during the observation period (between PND22 and PND112). A lowered mean daily food consumption was observed in male animals at 102 mg/kg bw/day between PND49-56 and PND56-63 and at 180 mg/kg bw/day between PND49-56, when compared to the control. In the female animals at 51 mg/kg bw/day, the mean daily food consumption was comparable with the control during the course of the entire study. At 102 mg/kg bw/day, lower food consumption was observed during the course of the study being statistically significant from PND 22 up to and including PND 63 consistently, between PND70-77 and 105-112. At 180 mg/kg bw/day, the mean daily food consumption of female animals was lower than in the control group between PND42-49 and 56-63.
The difference with respect to the control reached statistical significance especially in female animals at 102 mg/kg bw/day, however these changes were not dose-dependent. Therefore, the changes were considered to be independent from the treatment and toxicologically not relevant.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related adverse changes in the examined hematological and blood coagulation parameters in male or female animals at 51, 102 or 180 mg/kg bw/day in F1 Cohort 1A.
An elevated percentage of reticulocytes in male animals at 180 mg/kg bw/day and in female animals at 51, 102 or 180 mg/kg bw/day (not dose dependent) was considered to have little or no toxicological relevance.
In the absence of further statistically significant changes in the related red-blood cell parameters or signs of blood loss, hemorrhage or anemia, increased reticulocyte count was regarded as incidental and not treatment- related.
In male animals, statistical significances with respect to the control were detected at the slightly shorter mean prothrombin time (PT) at 51 and 180 mg/kg bw/day, at higher mean corpuscular volume (MCV) at 180 mg/kg bw/day.
In the female animals, the mean hemoglobin concentration (HGB) was below the control at 180 mg/kg bw/day.
The minor changes noted in MCV, PT and HGB were considered to have little or no toxicological relevance. Values were either close to or within the historical control range.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Pathologic alterations were not detected at the evaluation of clinical chemistry parameters in F1 Cohort 1A male or female animals at 51, 102 or 180 mg/kg bw/day.
In the male animals, reduced mean activity of alanine aminotransferase (ALT) was detected in all treatment groups being statistically significant at 51 and 102 mg/kg bw/day,
In the female animals, statistical significance with respect to the control was detected at the higher mean concentration of glucose (GLUC) at 51 and 180 mg/kg bw/day and higher mean urea concentration at 180 mg/kg bw/day.
Changes in enzyme activity of ALT has no toxicological relevance in the absence of histological changes.
Elevated mean concentration of urea in female animals at 180 mg/kg bw/day may refer to enhanced renal function. Clinical pathology, necropsy or histopathological investigations did not reveal any related changes in the examined parameters or organs.
Higher mean glucose concentration may be related to the over-night fasting of animals. Therefore, these minor changes were judged to be of little or no toxicological relevance.

The thyroid hormone (FT3, FT4 and TSH) levels were elevated dose-dependently in female animals at 102 and 180 mg/kg bw/day in the F1 Cohort 1A.
There were no statistically significant differences with respect to their control in the FT3, FT4 and TSH concentrations in male animals at 51, 102 or 180 mg/kg bw/day levels.
The mean FT3 and TSH levels were statistically significantly higher than in the control group in female animals at 102 and 180 mg/kg bw/day groups. Statistically significantly higher mean FT4 levels were detected in females at 180 mg/kg bw/day.
Although statistically significant differences were detected in females, a relation to treatment is unlikely since values are within the historical control data range and there were no collaborative findings (no macroscopic or microscopic changes in relevant organs (pituitary, thyroid), no organ weight impairment, no changes on estrous cycle, development of ovarian follicles not affected). Moreover, increase of FT3 and FT4 in the presence of increased TSH level is biologically not consistent.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in the examined urine parameters in F1 Cohort 1A animals (male or female) at 51, 102 or 180 mg/kg bw/day.
The examined urine parameters were comparable in the control and 51, 102 and 180 mg/kg bw/day groups (male and female).
Sexual maturation:
no effects observed
Description (incidence and severity):
The sexual maturity was not adversely affected in male or female animals at 51, 102 or 180 mg/kg bw/day in F1 Cohort 1A and Cohort 1B.
The balano-preputial separation was completed in all animals in F1 Cohort 1A and Cohort 1B on post-natal day 25, and the mean body weight on this day was comparable in the control and test item groups.
In the F1 Cohort 1A and Cohort 1B female animals, post-natal days of vaginal patency and post-natal days of appearance of the first cornified vaginal smear and the interval between days of vaginal patency and first cornified smear were similar in control, 51 and 180 mg/kg bw/day groups.
The onset of vaginal patency and appearance of first cornified smear were slightly later than in the control group in female animals at 102 mg/kg bw/day. Values met well the historical control (i.e., were well within or near to the historical control range) and similar finding was not detected in animals of the high dose group. Therefore, this minor change was considered to be toxicologically not relevant.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The F1 offspring’s development (surface righting reflex, pinna detachment, eye opening, anogenital distance) was not affected at 51, 102 or 180/225 mg/kg bw/day.
Statistical significance was detected for the minimally longer mean normalized anogenital distance of male pups at 51 mg/kg bw/day and for the minimally longer mean absolute and normalized anogenital distances in male pups at 102 mg/kg bw/day when compared to the control.
The absolute and normalized anogenital distances were similar in female offspring at 51, 102 and 181/225 mg/kg bw/day.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Nipples/areoles were not visible in any of the examined male offspring in the control or 51, 102 or 180/225 mg/kg bw/day groups on post-natal day 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes in the weights of examined organs (absolute and relative to body and brain weights) in male and female F1 offspring at the necropsy at weaning.
Statistical significance with respect to the control were detected at the lower mean absolute spleen weight at 102 and 180/225 mg/kg bw/day (-16 and -25% respectively) and at the slightly lower mean spleen weight relative to brain weight at 180/225 mg/kg bw/day (-25 %). These changes in the spleen weight were judged to be toxicologically not relevant because of the minor degree.
The weights of the examined organs were not adversely affected in male and female animals at 51, 102 or 180 mg/kg bw/day in F1 Cohort 1A and 1B.
F1 Cohort 1A Generation
At 51 mg/kg bw/day, statistically significant difference with respect to the control was observed at the higher mean weights of thyroid glands (absolute and relative to body and brain weights), at the lower mean weight of submandibular lymph node (absolute and relative to body weight) and at the higher mean weight of popliteal lymph node relative to brain weight in male animals.
In the male animals at 102 mg/kg bw/day, the mean weight of submandibular lymph node (absolute and relative to body and brain weights) was lowered and the mean thyroid weight relative to body and brain weights was higher with respect to their control. In the male animals at 180 mg/kg bw/day, the mean weight of pituitary (absolute and relative to body and brain weights) and popliteal lymph nodes relative to brain weight exceeded the control and the weight of submandibular lymph node (relative to body weight) were below the control.
In the female animals at 51 mg/kg bw/day, the thymus weights (absolute and relative to body and brain weights) and the weight of ovaries (absolute and relative to brain weight) were below the control.
At 102 mg/kg bw/day, the weight of examined organs were comparable with their control.
At 180 mg/kg bw/day, lower mean weight of thymus was observed when compared to the control.
The sporadic, statistically significant differences with respect to the control were of minor degree and independent from the doses. Morphological changes were not detected at the histopathological examination and hematology investigations as well as clinical chemistry parameters did not reveal test item-related abnormalities. Therefore, changes in organ weights were judged to have little or no toxicological relevance due to the minor degree and in the lack of dose dependency or associated histopathological alterations.
F1 Cohort 1B Generation
In the male animals, the mean testes weight (absolute) was below the control value at 180 mg/kg bw/day and the weights of all other organs were comparable with the control at 51, 102 and 180 mg/kg bw/day.
In the female animals at 51 mg/kg bw/day, higher mean weight of uterus (relative to body and brain weight) was observed when compared to the control.
In the female animals at 102 mg/kg bw/day group, the mean fasted body weight (absolute and relative to brain weight) was below the control and the brain weight relative to body weight exceeded the control value. The weights of ovaries (absolute and relative to body and brain weights) were also lower than in the control group in female animals in the mid dose group.
The weight of all examined organs (absolute and relative to body and brain weights) was comparable with their control in female animals at 180 mg/kg bw/day.
The statistically significant differences with respect to control were considered to be of little or no toxicologically significance. There were no supporting macroscopic or histological findings and no dose-dependency could be observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Offspring
Specific macroscopic alterations were not found in F1 offspring subjected to gross pathological examination before the weaning or at the weaning.
Before the weaning pups were subjected to necropsy because of death (stillborn and pups found dead) or after euthanasia on PND 4 (surplus pups). Pups in one litter were subjected to early euthanasia because of maternal death at 102 mg/kg bw/day on PND3:
- stillborn: 1/12 control, 1/4 at 51 mg/kg bw/day, 3/25 at 102 mg/kg bw/day
- found dead: 1/12 control, 1/25 at 102 mg/kg bw/day;
- early euthanasia: 11/25 at 102 mg/kg bw/day
The following common necropsy findings were detected in pups subjected to necropsy before the weaning:
- no milk in the stomach: 12/25 at 102 mg/kg bw/day;
- cold body temperature: 11/25 at 102 mg/kg bw/day;
- pale skin: 1/12 control, 4/25 at 102 mg/kg bw/day;
- smaller than normal: 1/12 control, 1/25 at 102 mg/kg bw/day;
Necropsy observations revealed some sporadic macroscopic findings at weaning as follows:
- brownish red thymus: 1/57 in control group;
- right or both sided pyelectasia: 3/57 control, 3/58 at 102 mg/kg bw/day

F1 Cohort 1A Generation
Macroscopic alterations related to the effect of the test item were detected in the stomach of surviving animals at 102 mg/kg bw/day (male) and at 180 mg/kg bw/day (male and female) at the necropsy.
In dead animals, the main macroscopic observations revealed changes in the stomach at 102 and 180 mg/kg bw/day (male and female) and lungs at 102 and 180 mg/kg bw/day (female).
Dead animals
In dead animals, necropsy observation revealed the following findings:
Male
- control (1/20): thymic hemorrhage, mottled lung lobes, collapse of lung lobes after incision;
- 102 mg/kg bw/day (1/20): brown-reddish colored lungs;
- 180 mg/kg bw/day (6/28): thymic hemorrhage (2/6), brown-reddish colored lungs (4/6), collapse of lung lobes after incision (1/6), granular (1/6) or erythematous (1/6) mucous membrane at cardia, bited nose and left eye (1/6, cannibalism), right side pyelectasia (1/6);
Female
- 102 mg/kg bw/day (3/20): brown reddish colored lungs (1/3), gas filled stomach (1/3), autolyzed brain (1/3), autolyzed hypophysis (1/3), cannibalism (2/3);
- 180 mg/kg bw/day (4/28): brown reddish colored lungs (3/4), greyish-pink colored lungs (1/4), oily liquid content in the thoracic cavity (1/4), cannibalism (1/4);
Necropsy findings of dead animals refer to local effects of the test item in lungs or gastrointestinal tract, which led to systemic changes, acute stress and agony.
Surviving animals
Male
- control: one or both sided pyelectasia (7/19), smaller than normal (empty, one side) seminal vesicle (1/19);
- 51 mg/kg bw/day: one or both sided pyelectasia (9/20);
- 102 mg/kg bw/day: one or both sided pyelectasia (5/19); thickened mucous membrane at cardia (6/19), Hernia diaphragmatica including liver (1/19);
- 180 mg/kg bw/day: one or both sided pyelectasia (2/22); thickened mucous membrane at cardia (21/22), Hernia diaphragmatica including liver (1/22);
Female
- control: one or both sided pyelectasia (9/20), slight, moderate or marked hydrometra (9/20);
- 51 mg/kg bw/day: one or both sided pyelectasia (2/20), slight, moderate or marked hydrometra (8/20);
- 102 mg/kg bw/day: one or both sided pyelectasia (3/17), slight, moderate or marked hydrometra (5/17);
- 180 mg/kg bw/day: right side pyelectasia (2/24), thickened mucous membrane at cardia (4/24), slight or moderate hydrometra (8/24), missing right side uterine horn (1/24), ovarian cyst filled with brownish-red liquid (1/24, 0.5 cm, right side)
Alterations in the stomach were supported by the results of histological investigations in surviving animals. These were considered to be related to the local effect of the test item at 102 and 180 mg/kg bw/day.
Pyelectasia and hydrometra are common macroscopic findings in experimental rats of this strain with similar age. Histological examination did not reveal degeneration, inflammation or fibrosis. Therefore, these findings were considered as individual lesion without toxicological significance. Smaller than normal seminal vesicle, missing right side uterine horn, ovarian cyst and Hernia diaphragmatica were individual alterations and are considered to be also species-specific changes and not treatment-related.

F1 Cohort 1B Generation
Necropsy observations revealed test item-related local alterations in the stomach at 180 mg/kg bw/day in male animals.
In dead animals, macroscopic observations revealed main findings in the lungs at 180 mg/kg bw/day (male and female).
Dead animals
At 51 mg/kg bw/day, brownish-red colored lungs were observed in dead female animal (1/20).
In dead male animal at 102 mg/kg, bw/day (1/20), oily liquid content in the thoracic cavity, cannibalized abdominal wall, mucous coat on the left lung lobes (right side), pale spleen and right side pyelectasia were detected at the necropsy.
At 180 mg/kg bw/day, four male animals (4/25) were subjected to early necropsy because of death. Brownish-red/ reddish mottled lungs (2/4), collapse of lungs after incision (2/4), pink color and foamy liquid content in the lungs (1/4), granular mucous membrane at cardia of the stomach (1/4), greyish colored mesenteric lymph nodes (1/4), sanguineous discharge around nose and mouth (1/4) were observed.
In dead female animals at 180 mg/kg bw/day (3/25), brownish-red (3/3) and mottled lungs (1/3), brownish-green (1/3) and greyish colored (1/3) mesenteric lymph nodes, moderate hydrometra (1/3) and cannibalized head and cervical organs (1/3) were detected at the necropsy.
Surviving animals
In the male animals, necropsy observations revealed the following findings:
- control group: right or both sided pyelectasia in the kidneys (9/20) and thymic hemorrhage (1/20);
- 51 mg/kg bw/day: right or left sided pyelectasia in the kidneys (5/20);
- 102 mg/kg bw/day: right or both sided pyelectasia in the kidneys (7/19);
- 180 mg/kg bw/day: right or both sided pyelectasia in the kidneys (2/21); and thickened mucous membrane at cardia (14/21);
In the female animals, necropsy observations revealed the following findings:
- control group: one or both sided pyelectasia in kidneys, (9/20), thymic hemorrhage (2/20), moderate or marked hydrometra (9/20);
- 51 mg/kg bw/day: right or both sided pyelectasia in the kidneys (3/19), slight, moderate or marked hydrometra (8/19), dilated uterine horns with yellowish liquid content (1/19), mucous yellowish liquid filled urinary bladder (1/19), smaller than normal and pale ovaries (1/19);
- 102 mg/kg bw/day: one or both sided pyelectasia in the kidneys (5/20); thymic hemorrhage (1/20), slight, moderate or marked hydrometra (6/20);
- 180 mg/kg bw/day: right side pyelectasia in the kidneys (2/22); thickened mucous membrane at cardia of the stomach (1/22), slight, moderate or marked hydrometra (6/22), alopecia on the skin of forelimb, right side (1/22), scar on the skin of right-side scapula (1/22).
Alterations in the stomach were supported by the results of histological investigations in surviving animals. These were considered to be related to the local effect of the test item at 102 and 180 mg/kg bw/day.
Pyelectasia, hydrometra and dermal changes (alopecia and scar) are common findings in experimental rats of this strain and age. Hydrometra (i.e., dilatation of uterine horns with clear liquid content) related to the female sexual cycle, is a physiological phenomenon. In the lack of related inflammatory or other pathological signs, these findings were judged to be toxicologically not relevant and not test item-related as no dose response was noted. Thymic hemorrhage is also frequently observed in experimental rats in connection with the exsanguination procedure. Dilated uterine horns with yellowish liquid content, mucous yellowish liquid filled urinary bladder, smaller than normal and pale ovaries were considered to be individual macroscopic alterations and not related to the test item.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxic or other test item-related lesions detectable by histological examination of investigated genital organs of the experimental male and female animals at 51, 102 or 180 mg/kg bw/day doses in Cohort 1A.
Squamous cell hyperplasia in the mucous membrane of non-glandular stomach was detected in a dose dependent manner in surviving animals a 102 mg/kg bw/day (male) and at 180 mg/kg bw/day (male and female). This gastric change was judged to be in connection with the local effect of high and medium doses of test item.
In dead animals at 102 mg/kg bw/day (male) and 180 mg/kg bw/day (male and female), histological lesions revealed serious congestion in the lungs. Squamous cell hyperplasia in the mucous membrane of non-glandular stomach was observed in male animals at 180 mg/kg bw/day.
The investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic for the sexually mature organism in most of examined male animals. Decreased amount of the secreta in the seminal vesicle (1/24 in control group) and severely decreased intensity of spermatogenesis in the testes and lack of mature spermatozoa in the epididymides (1/1 dead at 102 mg/kg bw/day, 1/6 dead at 180 mg/kg bw/day) were detected in single animals. These findings could be in connection with the unscheduled death of affected animals.
In the examined female animals (control, 180 mg/kg bw/day, dead animals at 102 mg/kg bw/groups), the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle except for one dead animal. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation except for one dead animal. The epithelial capsule and ovarian stroma were normal in most cases. Quantitative analysis of ovaries in animals of F1 Cohort 1A verified normal structure and function of the organ (primordial, primary, secondary and tertiary follicles, follicular atresia and corpora lutea) in the control and high dose treated animals. The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
Dead animals
In dead male animal in the control group (1/1), thymic hemorrhage and purulent necrotic bronchopneumonia was detected, which is considered as an individual disease.
Severe congestion in the lungs (1/1, 102 mg/kg bw/day, 4/6 at 180 mg/kg bw/day), decreased intensity of the spermatogenesis in the testes and lack of mature spermatozoa in the epididymides (1/1, 102 mg/kg bw/day, 1/6, 180 mg/kg bw/day), thymic hemorrhage (2/6, 180 mg/kg bw/day), squamous cell hyperplasia in the stomach (5/6, 180 mg/kg bw/day), both sided pyelectasia (1/6, 180 mg/kg bw/day) were observed.
In dead female animals, severe or mild pulmonary congestion (2/2, 102 mg/kg bw/day, 3/4, at 180 mg/kg bw/day) and lack of corpora lutea (1/2, 102 mg/kg bw/day) were observed by microscopic examination.
Surviving animals
In the control group, one or both sided pyelectasia (7/9 male and 9/20 female) and decreased amount of the secreta (1/19 male) was detected in the seminal vesicle in accordance with the necropsy findings (smaller than normal, no seminal fluid, left side). Dilatation of uterine horn was seen in some female animal (9/20).
In the low and mid dose group, squamous cell hyperplasia (5/5 male at 102 mg/kg bw/day), one or both sided pyelectasia was detected: 9/9 male and 2/2 female at 51 mg/kg bw/day; 5/5 male and 3/3 female at 102 mg/kg bw/day. Focal fibrosis in the Glisson’s capsule (1/1 male at 102 mg/kg bw/day) in connection with diaphragm hernia is an individual disorder.
At 180 mg/kg bw/day, one or both sided pyelectasia (2/22 male and 2/25 female), focal fibrosis in the Glisson’s capsule (1/22 male), mild congestion in the lungs (1/25 female), squamous cell hyperplasia (22/22 male and 4/25 female) and dilatation of the uterine horns (8/25) were observed.
Quantitative examinations of ovaries did not reveal test item-related changes in the number of developing follicles, corpora lutea or in the number of follicular atresia at the examined level of section in female animals at 180 mg/kg bw/day of F1 Cohort 1A. The mean number of primordial and primary follicles was similar in the control and at 180 mg/kg bw/day. The values met well the historical control ranges thus indicating normal morphology and function of female genital organ in the control and high dose groups.
There was no morphological evidence of test item-related acute or subacute injury (degeneration, inflammation, necrosis etc.) in the small and large intestines, liver, pancreas, cardiovascular system, respiratory system, immune system, hematopoietic system, skeleton, muscular system, central, or peripheral nervous system, eyes, integumentary system. The cytomorphology of endocrine glands were the same in the control and treated animals.
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle
The estrous cycle was not affected in the F1 Cohort 1A and 1B female animals at 51, 102 or 180 mg/kg bw/day.
The examined parameters of the estrous cycle – percentage of animals with regular or irregular cycles, in prolonged estrous or in prolonged diestrous, number of cycles, length of cycles, days in proestrous estrous diestrous –were comparable in the control and 51, 102 or 180 mg/kg bw/day groups of Cohort 1A.
In Cohort 1B, statistical significances were observed at the lower mean number of days in pro-estrous in female animals 51 mg/kg bw/day and higher mean number of days in estrous at 51 and 102 mg/kg bw/day group when compared to control. The examined parameters of the estrous cycle were comparable in the control and 180 mg/kg bw/day groups.
These minor differences with respect to the control in the low and mid dose groups of Cohort 1B were independent from doses and were considered to be indicative of biological variation and have no toxicological relevance regarding the short (observable) period of pro-estrous.

Sperm analysis
Sperm examinations did not point out any test item-related influence on the sperm cells at 180 mg/kg bw/day.
Statistical significances were detected at the lower mean percentage of immotile sperm cells in male animals at 180 mg/kg bw/day. This minor difference was considered to be indicative of biological variation and normal function of male genital organs.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
sexual maturation
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
51 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other: Systemic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
102 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes
Key result
Reproductive effects observed:
no
Conclusions:
Under the conditions of the present study, administration of the test item via oral gavage caused signs of systemic toxicity in parental male and female animals of the high dose group manifested in mortality, clinical signs and changes in the lungs and the gastro-intestinal tract accompanied by histological findings in these organs. The copulatory and fertility ability of male and female animal were not impaired at 180/225 mg/kg bw/day.
At 102 mg/kg bw/day of P generation, mortality, macroscopic and histopathological findings (stomach, lungs) were observed. The reproductive performance of male and female animals was not affected.
No signs of systemic toxicity or influence on the reproductive performance were observed in parental male and female animals at 51 mg/kg bw/day.
In F1 offspring, undisturbed development (sex distribution, clinical signs, surface righting reflex, pinna detachment, eye opening, anogenital distance, body weight development, thyroid hormone levels, necropsy findings and organ weight) were observed at 51, 102 and 180 mg/kg bw/day.
F1 adult (F1 Cohort 1A, Cohort 1B)
102 and 180 mg/kg bw/day caused mortality in F1 adult animals (male and female) accompanied by clinical signs mainly in dead animals.
At 51 mg/kg bw/day of F1 adults, no test item- related effects were detected.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for toxicity (P/ F1 adult male and female): 51 mg/kg bw/day
NOAEL for reproductive performance (male and female): 180 mg/kg bw/day
NOAEL for F1 Offspring: 180 mg/kg bw/day
Executive summary:

The subject of this study was to investigate the reproductive toxicity of the test item by conducting the Extended One-Generation Reproduction Toxicity Study in the rat according to OECD 443 as requested by the European Chemical Agency (ECHA). The guideline is designed for using the rat, which is the preferred rodent species for reproduction toxicity testing.


The aim of the extended one-generation reproduction toxicity study was to provide an evaluation of the pre- and post-natal effects of the test item on development as well as a thorough evaluation of systemic toxicity in male and female animals, in pregnant and lactating females and in young and adult offspring when repeatedly administered via oral gavage to animals at doses of 51, 102 and 180/225 mg/kg bw/day compared to control animals.


The effect of the test item was examined on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation, lactation and weaning (P generation) and on the offspring viability, neonatal health and mortality, growth and development of the offspring (F1 generation; Cohort 1A and Cohort 1B) to adulthood following oral (by gavage) administration.


Four groups of Han:WIST rats (n= 24/sex/group) were administered with the test item via oral gavage once a day at 0 (vehicle), 51, 102 and 180/225 mg/kg bw/day doses corresponding to concentrations of 0, 17, 34 and 60/75 mg/mL in parental generation in a volume of 3 mL. Control animals received the vehicle, sunflower oil, only in an identical manner. Due to the observed mortality in the mid and high dose parental animals caused by the irritating properties of the test substance, F1 animals were administered with doses of 51, 102 and 180 mg/kg bw/day at a dosing volume of 4 mL/kg bw/day corresponding to concentrations of 0, 12.75, 25.5 and 45 mg/mL.


The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). Analyses of formulations used for administration were performed five times during the study. Test item concentrations in the samples varied within the range of 92 % and 101 % in comparison to the nominal values.


All animals of the parent (P) generation were dosed for 10 weeks prior to mating and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 124, 125 days). Dams were exposed through the mating and gestation periods and at least up to lactation day 21 (altogether for 115-123 days). Not delivered, not mated and non-pregnant females were administered for 103 days.


Clinical observations (clinical signs, body weight, food consumption, estrous cycle) and pathology (clinical and organ pathology) examinations were performed on parental (P) animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems. Clinical pathology examinations - urinalysis, hematology, blood coagulation, clinical chemistry - were conducted in randomly selected ten male and ten female animals from each group (parental animals and Cohort 1A). Estrous cycle was monitored in parental animals by examining vaginal smears before the mating for two weeks and during the mating period until evidence of mating and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 post-partum.


All F1 offspring were observed individually for health, growth, development and function up to and including post-natal day 21 (clinical signs, body weight, surface righting reflex, pinna detachment, eye opening, anogenital distance, nipple retention).


Twenty F1 animals/sex/group were randomly selected for Cohort 1A and twenty F1 animals/sex/group were selected for Cohort 1B in the control, low and mid dose group on post-natal day 21 for follow-up examinations. Twenty-eight animals/ sex were selected for Cohort 1A and twenty-five animals/ sex were selected for Cohort 1B in high dose group to ensure the appropriate number of animals due to possible mortality.


Dosing of F1 offspring selected for follow-up examinations (Cohort 1A and Cohort 1B) begun on post-natal day 22 and treatment was continued up to the day before the necropsy.


F1 adult animals (Cohort 1A and Cohort 1B) were observed identically to parental animals – clinical signs, body weight, food consumption, estrous cycle, clinical pathology (Cohort 1A only) and organ pathology. Sexual maturity of offspring was investigated by observation of balano-preputial separation, vaginal patency (Cohort 1A and Cohort 1B) and appearance of first cornified vaginal smear (Cohort 1A). Cohort 1A animals were subjected to necropsy, organ weighing and sperm analysis – one day after the termination of the exposure – on PND 98-110. Cohort 1B animals were observed identically to parental (P) animals and were subjected to necropsy and organ weighing on PND 106-116.


Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 10 parent (P) animals/sex/ group at termination, in surplus pups at PND 4 (pooled by litters), from F1 pups not selected for cohorts on PND 22 and from 10 adult F1 Cohort 1A male and female animals/group at termination. Thyroid hormone levels were determined in adult animals (P, F1 Cohort 1A) and in PND22 F1 offspring.


All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue preservation one day after the last treatment. Brain, spleen, thymus and mammary tissues were preserved for 10 male and 10 female pups per group – where feasible – in F1 offspring not selected for Cohorts on PND22 or shortly thereafter.


Special attention was paid to the organs and tissues of the reproductive system for P or F1 animals.


Selected organ weights were determined in adult animals (P, F1) and in offspring (PND22 or shortly thereafter).


Sperm parameters were determined in all control, mid and high dose male animals in P generation and in all control and high dose male animals F1 generation (Cohort 1A).


At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female Cohort 1A animals from each group.


Full histopathology examinations were performed on the organs and tissues of adult animals (P, F1 Cohort 1A) in control and high dose groups with special emphasis on sexual organs and tissues. The stomach of all parental male animals (control, low, mid and high dose group) was also examined histologically on the basis of necropsy observation. In addition, organs showing macroscopic changes were also processed and examined histologically in adult animals in low or mid dose groups (P, F1 Cohort 1A). Reproductive organs were also processed and examined histologically in non-mated and non-pregnant female animals and their mating partners in the low and mid dose groups.


A quantitative evaluation of follicles (primordial, primary, secondary and tertiary follicles, follicular atresia) as well as corpora lutea was performed in F1 Cohort 1A female animals in the control and 180 mg/kg bw/day groups.


The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.


Results


Analytical control of dosing formulations


Concentration of the test item in the dosing formulations varied in the acceptable range between 92 % and 101 % of the nominal values and formulations were homogenous thereby confirming the proper dosing of parental animals (P) and F1 generations.


Parental (P) generation


Mortality


Test item-related mortality was detected in male and female animals (P) at 102 mg/kg bw/day (1/24 male and 2/24 female) and at 180/225 mg/kg bw/day (9/24 male and 5/24 female). Clinical signs, necropsy and histopathological findings refer to reflux in the laryngopharyngeal area during the oral gavage of test item and collapse of fluid balance, which caused the death of these animals.


One female animal at 51 mg/kg bw/day group (1/24) in parental generation (1/24) was early euthanized in moribund state due to para-gastric administration.


Clinical observation


Test item-related clinical signs were observed in parental male and female animals at 102 and 180/225 mg/kg bw/day in a dose related manner and incidence.


Dyspnea and noisy breathing were observed in dead and surviving animals at 102 and 180/225 mg/kg bw/day (male and female). As consequences, decreased activity, piloerection and sanguineous nose were detected in dead female animals at 102 or 180/225 mg/kg bw/day.


Nuzzling up the bedding material and salivation were detected shortly after the administration at 102 and 180/225 mg/kg bw/day and ceased within a short time period and was likely caused by the irritant character of the test item. The incidence of signs was similar during pre-mating, gestation and lactation period.


Some animal showed these signs of elaborated breathing at the detailed weekly observations.


Body weight and body weight gain


The body weight development was comparable in parental (male and female) animals in the control and 51, 102 and 180/225 mg/kg bw/day groups during the entire treatment period.


Food consumption


The mean daily food consumption was not adversely affected in parental male or female animals at 51, 102 or 180/225 mg/kg bw/day during the pre-mating, gestation or lactation period.


Estrous cycle


The estrous cycle was similar in the control and 51, 102 and 180/225 mg/kg bw/day groups during the two weeks observation period.


Delivery data of dams


There were no differences between the control and 51, 102 or 180/225 mg/kg bw/day groups in the delivery data of dams.


Reproductive performance


The reproductive performance of male and female animals was normal (the same as in not treated animals of this strain of experimental animals) in control, 51, 102 and 180/225 mg/kg bw/day groups.


Urinalysis


The examined urine parameters were not adversely affected in male and female animals at 51, 102 or 180/225 mg/kg bw/day.


Clinical pathology


There were no adverse changes in the examined clinical pathology parameters in parental male or female animals at 51, 102 or 180/225 mg/kg bw/day.


Thyroid hormones


The serum thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals or in PND22 F1 offspring at any dose levels.


Necropsy


Macroscopic alterations related to the effect of the test item were detected in the stomach of surviving male animals at 102 and 180/225 mg/kg bw/day at the necropsy.


In dead animals at 102 and 180/225 mg/kg bw/day, necropsy observations revealed changes in the lungs and gastro-intestinal tract (stomach, intestines).


Organ weight


The weights of examined organs were not adversely affected in male or female parental animals at 51, 102 and 180/225 mg/kg bw/day.


Sperm examinations


Sperm examinations did not reveal any test item-related influence on the sperm cell morphology and motility, and total sperm count at 102 or 180/225 mg/kg bw/day (parental (P) male animals).


Histopathology


Histological examinations revealed changes in gastrointestinal tract referring to local effects of high and medium doses of the test item (squamous cell hyperplasia in the mucous membrane of non-glandular stomach) in dead and surviving animals and systemic effects in dead animals (dilatation of stomach, small and large intestines along with large amount of fluid accumulation in the lumen, collapse of fluid balance; circulatory disturbance, serious congestion in the lungs)


The investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviduct, uterus cervix, vagina) were histologically normal and characteristic of the sexually mature organism in all parental (P) animals at 180/225 mg/kg bw/day (including not mated and non-pregnant female animals and their mating partners).


Offspring


The F1 offspring’s development was not affected at 51, 102 or 180/225 mg/kg bw/day. The mortality, sex distribution, clinical signs, surface righting reflex, pinna detachment, eye opening, anogenital distance, body weigh development, necropsy findings and organ weight were comparable to control.


F1 generation (Cohort 1A and Cohort 1B)


Mortality


102 or 180 mg/kg bw/day doses of the test item caused mortality in F1 animals (Cohort 1A and Cohort 1B) with higher incidence in males than in females.


Clinical observation


Clinical signs related to the test item were detected with low incidence in male and female animals in F1 Cohort 1A or Cohort 1B in 102 and 180 mg/kg bw/day groups (dead and surviving animals) at the daily clinical observations. Dyspnea and noisy breathing were considered to be a consequence of reflux of the test item in the laryngopharyngeal area during administration. Disturbances in breathing also appeared at the weekly clinical observations in some animals at 180 mg/kg bw/day.


Body weight and body weight gain


The body weight development was undisturbed in male and female animals of F1 Cohorts 1A and 1B at 51, 102 and 180 mg/kg bw/day during the entire treatment period.


Food consumption


The mean daily food consumption was not adversely affected in males and females of F1 Cohort 1A and Cohort 1B at 51, 102 and 180 mg/kg bw/day during the observation period.


Sexual maturity


The sexual maturity was similar to control in male or female animals of F1 Cohort 1A and 1B at 51, 102 and 180 mg/kg bw/day.


Estrous cycle


The estrous cycle was comparable with their control in the F1 Cohort 1A and Cohort 1B female animals at 51, 102 and 180 mg/kg bw/day.


Clinical pathology


There were no test item-related pathologic changes among the examined clinical pathology parameters (urine, hematology and blood coagulation, clinical chemistry) in male or female animals at 51, 102 or 180 mg/kg bw/day in F1 Cohort 1A.


Thyroid hormones


The thyroid hormone (FT3, FT4 and TSH) levels were elevated dose-dependently in female animals at 102 and 180 mg/kg bw/day in the F1 Cohort 1A. There were no statistically significant differences with respect to their control in the FT3, FT4 and TSH concentrations in male animals at 51, 102 or 180 mg/kg bw/day levels.


Although statistically significant differences were detected in females, a relation to treatment is unlikely since values are within the historical control data range and there were no collaborative findings. Moreover, increase of FT3 and FT4 in the presence of increased TSH level is biologically not consistent.


Necropsy


Necropsy observations revealed test item-related local alterations in the stomach at 102 mg/kg bw/day (male, Cohort 1 A) and at 180 mg/kg bw/day (male and female Cohort 1 A and Cohort 1B).


In dead animals, additional changes were detected in the lungs at 102 mg/kg bw/day (male and female, Cohort 1A) and at 180 mg/kg bw/day (male and female, Cohorts 1A and 1B).


Organ weight


The weights of the examined organs were not adversely affected in male and female animals of F1 Cohort 1A and F1 Cohort 1B at 51, 102 or 180 mg/kg bw/day.


Sperm examinations


Sperm morphology and motility, and total sperm count were similar to the control in male animals at 180 mg/kg bw/day in F1 Cohort 1A.


Splenic Lymphocyte Subpopulation Analysis


There were no test item-related changes in the splenic lymphocyte subpopulation in male or female animals in F1 Cohort 1A animals at 51, 102 or 180 mg/kg bw.


Histopathology


Squamous cell hyperplasia in the mucous membrane of non-glandular stomach was detected in a dose- related manner at 102 mg/kg bw/day (male) and at 180 mg/kg bw/day (male and female) in surviving animals due to the high local concentration of the test item.


In dead animals at 102 and 180 mg/kg bw/day (male and female, F1 Cohort 1A), histological lesions referred to serious congestion in the lungs and local lesions in the stomach (squamous cell hyperplasia in the mucous membrane of non-glandular stomach).


 


Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:


NOAEL for toxicity (P/ F1 adult male and female): 51 mg/kg bw/day


NOAEL for reproductive performance (male and female): 180 mg/kg bw/day


NOAEL for F1 Offspring: 180 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
51 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and OECD Guideline conform 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

Extended One-Generation Reproductive Toxicity Study


The subject of this study was to investigate the reproductive toxicity of the test item by conducting the Extended One-Generation Reproduction Toxicity Study in the rat according to OECD 443 as requested by the European Chemical Agency (ECHA). The guideline is designed for using the rat, which is the preferred rodent species for reproduction toxicity testing.


The aim of the extended one-generation reproduction toxicity study was to provide an evaluation of the pre- and post-natal effects of the test item on development as well as a thorough evaluation of systemic toxicity in male and female animals, in pregnant and lactating females and in young and adult offspring when repeatedly administered via oral gavage to animals at doses of 51, 102 and 180/225 mg/kg bw/day compared to control animals.


The effect of the test item was examined on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation, lactation and weaning (P generation) and on the offspring viability, neonatal health and mortality, growth and development of the offspring (F1 generation; Cohort 1A and Cohort 1B) to adulthood following oral (by gavage) administration.


Four groups of Han:WIST rats (n= 24/sex/group) were administered with the test item via oral gavage once a day at 0 (vehicle), 51, 102 and 180/225 mg/kg bw/day doses corresponding to concentrations of 0, 17, 34 and 60/75 mg/mL in parental generation in a volume of 3 mL. Control animals received the vehicle, sunflower oil, only in an identical manner. Due to the observed mortality in the mid and high dose parental animals caused by the irritating properties of the test substance, F1 animals were administered with doses of 51, 102 and 180 mg/kg bw/day at a dosing volume of 4 mL/kg bw/day corresponding to concentrations of 0, 12.75, 25.5 and 45 mg/mL.


The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). Analyses of formulations used for administration were performed five times during the study. Test item concentrations in the samples varied within the range of 92 % and 101 % in comparison to the nominal values.


All animals of the parent (P) generation were dosed for 10 weeks prior to mating and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 124, 125 days). Dams were exposed through the mating and gestation periods and at least up to lactation day 21 (altogether for 115-123 days). Not delivered, not mated and non-pregnant females were administered for 103 days.


Clinical observations (clinical signs, body weight, food consumption, estrous cycle) and pathology (clinical and organ pathology) examinations were performed on parental (P) animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems. Clinical pathology examinations - urinalysis, hematology, blood coagulation, clinical chemistry - were conducted in randomly selected ten male and ten female animals from each group (parental animals and Cohort 1A). Estrous cycle was monitored in parental animals by examining vaginal smears before the mating for two weeks and during the mating period until evidence of mating and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 post-partum.


All F1 offspring were observed individually for health, growth, development and function up to and including post-natal day 21 (clinical signs, body weight, surface righting reflex, pinna detachment, eye opening, anogenital distance, nipple retention).


Twenty F1 animals/sex/group were randomly selected for Cohort 1A and twenty F1 animals/sex/group were selected for Cohort 1B in the control, low and mid dose group on post-natal day 21 for follow-up examinations. Twenty-eight animals/ sex were selected for Cohort 1A and twenty-five animals/ sex were selected for Cohort 1B in high dose group to ensure the appropriate number of animals due to possible mortality.


Dosing of F1 offspring selected for follow-up examinations (Cohort 1A and Cohort 1B) begun on post-natal day 22 and treatment was continued up to the day before the necropsy.


F1 adult animals (Cohort 1A and Cohort 1B) were observed identically to parental animals – clinical signs, body weight, food consumption, estrous cycle, clinical pathology (Cohort 1A only) and organ pathology. Sexual maturity of offspring was investigated by observation of balano-preputial separation, vaginal patency (Cohort 1A and Cohort 1B) and appearance of first cornified vaginal smear (Cohort 1A). Cohort 1A animals were subjected to necropsy, organ weighing and sperm analysis – one day after the termination of the exposure – on PND 98-110. Cohort 1B animals were observed identically to parental (P) animals and were subjected to necropsy and organ weighing on PND 106-116.


Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 10 parent (P) animals/sex/ group at termination, in surplus pups at PND 4 (pooled by litters), from F1 pups not selected for cohorts on PND 22 and from 10 adult F1 Cohort 1A male and female animals/group at termination. Thyroid hormone levels were determined in adult animals (P, F1 Cohort 1A) and in PND22 F1 offspring.


All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue preservation one day after the last treatment. Brain, spleen, thymus and mammary tissues were preserved for 10 male and 10 female pups per group – where feasible – in F1 offspring not selected for Cohorts on PND22 or shortly thereafter.


Special attention was paid to the organs and tissues of the reproductive system for P or F1 animals.


Selected organ weights were determined in adult animals (P, F1) and in offspring (PND22 or shortly thereafter).


Sperm parameters were determined in all control, mid and high dose male animals in P generation and in all control and high dose male animals F1 generation (Cohort 1A).


At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female Cohort 1A animals from each group.


Full histopathology examinations were performed on the organs and tissues of adult animals (P, F1 Cohort 1A) in control and high dose groups with special emphasis on sexual organs and tissues. The stomach of all parental male animals (control, low, mid and high dose group) was also examined histologically on the basis of necropsy observation. In addition, organs showing macroscopic changes were also processed and examined histologically in adult animals in low or mid dose groups (P, F1 Cohort 1A). Reproductive organs were also processed and examined histologically in non-mated and non-pregnant female animals and their mating partners in the low and mid dose groups.


A quantitative evaluation of follicles (primordial, primary, secondary and tertiary follicles, follicular atresia) as well as corpora lutea was performed in F1 Cohort 1A female animals in the control and 180 mg/kg bw/day groups.


The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.


Results


Analytical control of dosing formulations


Concentration of the test item in the dosing formulations varied in the acceptable range between 92 % and 101 % of the nominal values and formulations were homogenous thereby confirming the proper dosing of parental animals (P) and F1 generations.


Parental (P) generation


Mortality


Test item-related mortality was detected in male and female animals (P) at 102 mg/kg bw/day (1/24 male and 2/24 female) and at 180/225 mg/kg bw/day (9/24 male and 5/24 female). Clinical signs, necropsy and histopathological findings refer to reflux in the laryngopharyngeal area during the oral gavage of test item and collapse of fluid balance, which caused the death of these animals.


One female animal at 51 mg/kg bw/day group (1/24) in parental generation (1/24) was early euthanized in moribund state due to para-gastric administration.


Clinical observation


Test item-related clinical signs were observed in parental male and female animals at 102 and 180/225 mg/kg bw/day in a dose related manner and incidence.


Dyspnea and noisy breathing were observed in dead and surviving animals at 102 and 180/225 mg/kg bw/day (male and female). As consequences, decreased activity, piloerection and sanguineous nose were detected in dead female animals at 102 or 180/225 mg/kg bw/day.


Nuzzling up the bedding material and salivation were detected shortly after the administration at 102 and 180/225 mg/kg bw/day and ceased within a short time period and was likely caused by the irritant character of the test item. The incidence of signs was similar during pre-mating, gestation and lactation period.


Some animal showed these signs of elaborated breathing at the detailed weekly observations.


Body weight and body weight gain


The body weight development was comparable in parental (male and female) animals in the control and 51, 102 and 180/225 mg/kg bw/day groups during the entire treatment period.


Food consumption


The mean daily food consumption was not adversely affected in parental male or female animals at 51, 102 or 180/225 mg/kg bw/day during the pre-mating, gestation or lactation period.


Estrous cycle


The estrous cycle was similar in the control and 51, 102 and 180/225 mg/kg bw/day groups during the two weeks observation period.


Delivery data of dams


There were no differences between the control and 51, 102 or 180/225 mg/kg bw/day groups in the delivery data of dams.


Reproductive performance


The reproductive performance of male and female animals was normal (the same as in not treated animals of this strain of experimental animals) in control, 51, 102 and 180/225 mg/kg bw/day groups.


Urinalysis


The examined urine parameters were not adversely affected in male and female animals at 51, 102 or 180/225 mg/kg bw/day.


Clinical pathology


There were no adverse changes in the examined clinical pathology parameters in parental male or female animals at 51, 102 or 180/225 mg/kg bw/day.


Thyroid hormones


The serum thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals or in PND22 F1 offspring at any dose levels.


Necropsy


Macroscopic alterations related to the effect of the test item were detected in the stomach of surviving male animals at 102 and 180/225 mg/kg bw/day at the necropsy.


In dead animals at 102 and 180/225 mg/kg bw/day, necropsy observations revealed changes in the lungs and gastro-intestinal tract (stomach, intestines).


Organ weight


The weights of examined organs were not adversely affected in male or female parental animals at 51, 102 and 180/225 mg/kg bw/day.


Sperm examinations


Sperm examinations did not reveal any test item-related influence on the sperm cell morphology and motility, and total sperm count at 102 or 180/225 mg/kg bw/day (parental (P) male animals).


Histopathology


Histological examinations revealed changes in gastrointestinal tract referring to local effects of high and medium doses of the test item (squamous cell hyperplasia in the mucous membrane of non-glandular stomach) in dead and surviving animals and systemic effects in dead animals (dilatation of stomach, small and large intestines along with large amount of fluid accumulation in the lumen, collapse of fluid balance; circulatory disturbance, serious congestion in the lungs)


The investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviduct, uterus cervix, vagina) were histologically normal and characteristic of the sexually mature organism in all parental (P) animals at 180/225 mg/kg bw/day (including not mated and non-pregnant female animals and their mating partners).


Offspring


The F1 offspring’s development was not affected at 51, 102 or 180/225 mg/kg bw/day. The mortality, sex distribution, clinical signs, surface righting reflex, pinna detachment, eye opening, anogenital distance, body weigh development, necropsy findings and organ weight were comparable to control.


F1 generation (Cohort 1A and Cohort 1B)


Mortality


102 or 180 mg/kg bw/day doses of the test item caused mortality in F1 animals (Cohort 1A and Cohort 1B) with higher incidence in males than in females.


Clinical observation


Clinical signs related to the test item were detected with low incidence in male and female animals in F1 Cohort 1A or Cohort 1B in 102 and 180 mg/kg bw/day groups (dead and surviving animals) at the daily clinical observations. Dyspnea and noisy breathing were considered to be a consequence of reflux of the test item in the laryngopharyngeal area during administration. Disturbances in breathing also appeared at the weekly clinical observations in some animals at 180 mg/kg bw/day.


Body weight and body weight gain


The body weight development was undisturbed in male and female animals of F1 Cohorts 1A and 1B at 51, 102 and 180 mg/kg bw/day during the entire treatment period.


Food consumption


The mean daily food consumption was not adversely affected in males and females of F1 Cohort 1A and Cohort 1B at 51, 102 and 180 mg/kg bw/day during the observation period.


Sexual maturity


The sexual maturity was similar to control in male or female animals of F1 Cohort 1A and 1B at 51, 102 and 180 mg/kg bw/day.


Estrous cycle


The estrous cycle was comparable with their control in the F1 Cohort 1A and Cohort 1B female animals at 51, 102 and 180 mg/kg bw/day.


Clinical pathology


There were no test item-related pathologic changes among the examined clinical pathology parameters (urine, hematology and blood coagulation, clinical chemistry) in male or female animals at 51, 102 or 180 mg/kg bw/day in F1 Cohort 1A.


Thyroid hormones


The thyroid hormone (FT3, FT4 and TSH) levels were elevated dose-dependently in female animals at 102 and 180 mg/kg bw/day in the F1 Cohort 1A. There were no statistically significant differences with respect to their control in the FT3, FT4 and TSH concentrations in male animals at 51, 102 or 180 mg/kg bw/day levels.


Although statistically significant differences were detected in females, a relation to treatment is unlikely since values are within the historical control data range and there were no collaborative findings. Moreover, increase of FT3 and FT4 in the presence of increased TSH level is biologically not consistent.


Necropsy


Necropsy observations revealed test item-related local alterations in the stomach at 102 mg/kg bw/day (male, Cohort 1 A) and at 180 mg/kg bw/day (male and female Cohort 1 A and Cohort 1B).


In dead animals, additional changes were detected in the lungs at 102 mg/kg bw/day (male and female, Cohort 1A) and at 180 mg/kg bw/day (male and female, Cohorts 1A and 1B).


Organ weight


The weights of the examined organs were not adversely affected in male and female animals of F1 Cohort 1A and F1 Cohort 1B at 51, 102 or 180 mg/kg bw/day.


Sperm examinations


Sperm morphology and motility, and total sperm count were similar to the control in male animals at 180 mg/kg bw/day in F1 Cohort 1A.


Splenic Lymphocyte Subpopulation Analysis


There were no test item-related changes in the splenic lymphocyte subpopulation in male or female animals in F1 Cohort 1A animals at 51, 102 or 180 mg/kg bw.


Histopathology


Squamous cell hyperplasia in the mucous membrane of non-glandular stomach was detected in a dose- related manner at 102 mg/kg bw/day (male) and at 180 mg/kg bw/day (male and female) in surviving animals due to the high local concentration of the test item.


In dead animals at 102 and 180 mg/kg bw/day (male and female, F1 Cohort 1A), histological lesions referred to serious congestion in the lungs and local lesions in the stomach (squamous cell hyperplasia in the mucous membrane of non-glandular stomach).


 


Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:


NOAEL for toxicity (P/ F1 adult male and female): 51 mg/kg bw/day


NOAEL for reproductive performance (male and female): 180 mg/kg bw/day


NOAEL for F1 Offspring: 180 mg/kg bw/day


 


Reproduction/Developmental Toxicity Screening Test


A reproduction/developmental toxicity screening test was performed with the methyl-ethylketone peroxide in TXIB and DMP according to OECD guideline 421. This study was conducted to provide preliminary information on the potential adverse effects of methyl-ethylketone peroxide on male and female reproduction. This investigation encompassed gonadal function, mating behaviour, conception, parturition and lactation of the F0 generation and the development of offspring from conception through day 4 of postnatal life. Three groups of male and female Crl:CD(SD) rats (12/sex/group) were administered the test article, methyl-ethylketone peroxide, in the vehicle (0.1% polysorbate 80), daily by oral gavage for at least 14 consecutive days prior to mating. Dosage levels for the F0 generation were 25, 50 and 100 mg/kg/day; however, due to the mortality/moribundity of 1 male and 2 females in the 100 mg/kg/day group following 2 days of dose administration, the dosage level was lowered to 75 mg/kg/day.


Based on the results of this study, F0 parental systemic toxicity was observed at 100/75 mg/kg/day as mortality/moribundity, reductions in body weight and food consumption, and macroscopic and microscopic findings in the stomach. No signs of parental systemic toxicity were observed at 25 and 50 mg/kg/day; therefore, the no-observed-adverse-effect level (NOAEL) for parental systemic toxicity was 50 mg/kg/day.


No effects on F0 reproduction were noted in animals administered methyl-ethylketone peroxide at dosage levels of 25, 50 or 100/75 mg/kg/day, therefore the NOAEL for F0 reproductive toxicity was 75 mg/kg/day. F1 growth and survival were unaffected by parental test article administration; however, because mean F1 pup body weights in the 100/75 mg/kg/day group were lower than control values, the NOAEL for F1 neonatal toxicity was 50 mg/kg/day. There were no differences between the vehicle control groups when F0 and F1 parameters were evaluated; therefore, the toxicity observed at the 100/75 mg/kg/day dosage level was due to the methyl-ethylketone peroxide and not to the diluent components.


 


In conclusion, methyl-ethylketone peroxide did not have an impact on the reproductive performance of the parental animals, the delivery data of dams and on the development of the offspring up to and including the dose that cause signs of severe systemic toxicity including mortality.

Effects on developmental toxicity

Description of key information

The No Observed Adverse Effect Level (NOAELs) were determined as follows:
Rat
NOAEL (maternal toxicity): 65 mg/kg bw/day
NOAEL (developmental toxicity): 200 mg/kg bw/day.

Rabbit
NOAEL maternal toxicity: 100 mg/kg bw/day
NOAEL developmental toxicity: 200 mg/kg bw/day

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-06-28 to 2020-01-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance No. 43 on mammalian reproductive toxicity testing and assessment
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: S & K-LAP Kft. Császár út 135 2173 Kartal HUNGARY
- Age at study initiation: young, healthy and breeding mature rabbits Females were nulliparous before first insemination at study initiation
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 15 - 21°C
- Humidity: 29 - 62 %
- Air changes: 8 - 12 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light
Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (sunflower oil). Formulations were prepared in the formulation laboratory of the Test Facility with a frequency and stored according to the results of stability measurements in the frame of the analytical method validation from daily to every three days and stored at room temperature or in the refrigerator.

VEHICLE
- Justification for use and choice of vehicle: the test item is not stable in water or aqueous vehicles. Therefore, sunflower oil was used.
- Concentration in vehicle: 100 mg/mL, 50 mg/mL, 25 mg/mL and 5 mg/mL
- Amount of vehicle: a constant volume of 2 mL/kg bw was administered
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suitability of the chosen vehicle for the test item was analytically proven. Recovery was 95 % of nominal concentrations at 2 mg/mL and 200 mg/mL in sunflower oil respectively. The test item was proved to be stable in the formulations at least for 24 hours at room temperature and at least 3 days in the refrigerator (5 ± 3°C). Analytical control of dosing solutions (control of test item concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility two times during the study.
Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on two occasions. Similarly, five samples were taken from the vehicle (Control, Group 1) and analyzed.
Details on mating procedure:
- Impregnation procedure: artificial insemination
Day of insemination was regarded as day 0 of gestation. Synchronization of the cycle was completed 48 hours prior to insemination by administering PMSG (gonadotropin) hormone subcutaneously into the neck region. The insemination procedure was performed at the test facility by the breeder.
Duration of treatment / exposure:
From gestation day (GD) 6 to 27
Frequency of treatment:
The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Duration of test:
21 days
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
corresponding to a concentration of 100 mg/mL
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
corresponding to a concentration of 50 mg/mL
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
corresponding to a concentration of 25 mg/mL
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
corresponding to a concentration of 5 mg/mL
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
No. of animals per sex per dose:
24 inseminated females per dose group
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale:
Justification of the dose selection:
The dose setting was based on findings obtained in the non-GLP preliminary study. According to this dose range finding study the dose level of 300 mg/kg bw/day caused abortion in two of three females after treating with 3 mL/kg treatment volume of sunflower oil up to gestation day 11 and 2 mL/kg from gestation day 12.
At 100 and 300 mg/kg bw/day dose lower (negative) corrected body weight gain was indicated. In this main study two high doses (100 mg/kg bw/day and 200 mg/kg bw/day) were chosen accordingly with the aim to induce some maternal or/and developmental toxicity but not death or severe suffering. The low dose was selected to induce no toxicity. The intermediate dose level of 50 mg/kg bw/day was interpolated geometrically.

- Rationale for animal assignment: random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after treatment

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded on gestation days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28 (accuracy 1 g).
The corrected body weight was calculated for the 28th day of pregnancy (body weight on day 28 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes; The food consumption was measured between gestation days 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 27 and 27 to 28 by re-weighing the non-consumed diet (accuracy 1 g).

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, visual inspection

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
- Organs examined: The organs of neck, thorax and abdomen of the does were examined macroscopically. Organs pathological changes which could not be diagnosed macroscopically were fixed in 4 % neutral formaldehyde solution. Corresponding organs from control animals were kept for comparison. Histological examination on organs was not performed. The ovaries and uterus were removed and the uterus (including cervix) of the pregnant females was weighed (accuracy 1 gram). Uterus of each female was examined for early, late embryonic and fetal death and for the number of live fetuses.
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
The statistical evaluation of data was performed with the 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 is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to assess the significance of intergroup differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.

Does or litters were excluded from the data evaluation in cases of:
- A disease or death of the doe unrelated to the treatment (total exclusion)

- Non pregnant females i.e. females with no implantation and no corpora lutea (total exclusion)

- Body weight, body weight gain, food consumption, clinical signs and necropsy findings of females with no implantation but corpora lutea i.e. total preimplantation loss (only the intrauterine parameters were evaluated, partial exclusion) - Circumstances unrelated to the test item which are considered to be reason for exclusion, at the discretion of the Study Director Although these animals were excluded from the data evaluation the study report contains all data. A male/female fetus was considered as retarded in body weight/crown-rump length, when its weight/length was below the average minus twofold standard deviation of the control male/female fetuses.
Indices:
Pre-Implantation loss
Number of corpora lutea - Number of implantations / Number of corpora lutea x 100 (%, group mean)

Post-implantation loss
Number of implantation loss – Number of live fetuses / Number of implantations x 100 (%, group mean)


Fetuses

Sex distribution
Number of Male (Female) fetuses / Number of fetuses x 100 (%, group mean)

External abnormalities/litter
Number of fetuses with abnormality / Number of fetuses x 100 (%, group mean)

Visceral abnormalities/litter
Number of fetuses with abnormality / Number of fetuses examined x 100 (%, group mean)

Skeletal abnormalities/litter
Number of fetuses with abnormality / Number of fetuses examined x 100 (%, group mean)
Historical control data:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Few amount of faeces was observed on different days in several animals without a dose response. Diarrhea was recorded for one female on G.D. 28 in the 10 mg/kg bw/day group. Noisy breath was observed in three animals in the 50 mg/kg bw/day group 2 to 4 days long and stopped on G.D. 25 the latest as well as in three animals in the 200 mg/kg bw/day group 3 to 9 days long (in one case stopped on G.D. 23 and in two cases on G.D. 28). Considering the lack of clear dose response this was not attributed to the test item, rather to a technical reason by the treatment with oral gavage. Swelling and wound on the muzzle was recorded for one animal in the 50 mg/kg bw/day dose group. This finding disappeared from G.D. 21 and was not judged to be in relationship with the treatment.
These clinical observations were not attributed to be an effect of the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the animals died before scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant reductions observed in the body weight parameters including corrected body weight/gain. However, between G.D. 6 to 9 and 21 to 24, 24 to 27 as well as 0 to 28 the body weight gain was lower at 200 mg/kg bw/day than in the other groups which cannot be excluded to be a slight effect of the test item. Between G.D. 27 and 28 the body weight gain was statistically significantly (p<0.01) higher in the 100 and 200 mg/kg bw/day and 10 mg/kg bw/day (p<0.05) groups than in the control.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
G.D. 6 to 9 in the 10, 100 and 200 mg/kg bw/day groups (p<0.05) and not at 50 mg/kg bw/day. considering the lack of clear dose response this finding was not associated with the test item. Between G.D. 9 and 12 as well as 24 and 27 the food consumption was lower (-16 and -19%) in the 200 mg/kg bw/day group than in the other groups which cannot completely be ruled out to be due to an effect of the test item.
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
Endocrine 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:
effects observed, treatment-related
Description (incidence and severity):
Observations like pinhead-sized or point-like or pin-prick -sized haemorrhages in the lungs and reddish mottled lungs were seen in the groups. Bloody region of vaginal orifice as well as stomach filled by feed and fetal remains was observed in relationship abortion or early delivery. Anal region was tainted by faeces of one doe with diarrhea in the 10 mg/kg bw/day group. These macroscopic findings were not attributed to the treatment with the test item considering the lack of dose response. Doubled gall bladder was seen in one dam in the control group as a finding unrelated from the treatment. Ca. 10 to 30 mm diameter dark red or/and corrosed-like area was observed in the stomach of three high dose does. In addition one more doe (excluded due to total pre-implantation loss) in this group had similar finding. Considering that this change was seen only in the 200 mg/kg bw/day group, it was attributed to the treatment with the test item and its locally irritating properties.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female aborted on G.D. 26 in the 50 mg/kg bw/day group and was euthanized on G.D. 27. Considering the lack of dose response, abortion was not attributed to an effect of the test item.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Blood on the tray under the cage was found for one doe in the control group on G.D. 28 and blood/tissue was found for another doe (total post-implantation loss was revealed at Caesarean section) in the 10 mg/kg bw/day group.
There were no statistically significant differences observed in the mean number of corpora lutea, implantation, pre-implantation- and post-implantation loss (including early-, late- and fetal death), total intrauterine mortality (pre- and post-implantation loss summarized).
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no statistically significant differences observed in the mean number of viable fetuses.
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
The occurrence of early delivery (GD 27 or 28 (the day before or in the morning of scheduled necropsy) was one in each group including control and excluded 100 mg/kg bw/day. Considering the lack of dose response, early delivery was not attributed to an effect of the test item.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
please refer to details on maternal toxic effects
Other effects:
no effects observed
Details on maternal toxic effects:
The number of inseminated females was 24 in all groups. There were 4 females without any implantation and corpora lutea in the control group and one each in the test item treated groups. The females with corpora lutea but without implantation (total pre-implantation loss) was 1, 0, 2, 1 and 1 in the control, 10, 50 100 and 200 mg/kg bw/day dose groups, respectively. The pregnancy rate was between 79 and 96%, the lowest in the control group. One female in the 10 and two in the 50 mg/kg bw/day dose group had total post-implantation loss. One of these 50 mg/kg bw/day females was excluded from the data evaluation based on a developmental disorder and the other female in this group was excluded due to a disease. One female aborted on G.D. 26 in the 50 mg/kg bw/day dose group and was euthanized. In all groups (except 100 mg/kg bw/day) one female delivered early (on G.D. 27 or to 28 morning).
The number of litters revealed at Caesarean section was 18, 21, 17, 22 and 21 in the control, 10, 50, 100 and 200 mg/kg bw/day dose groups, respectively.
Does or litters listed below were excluded from the data evaluation:
Non pregnant females i.e. females with no implantation:
No implantation and no corpora lutea (total exclusion):
- control: No.: 160, 051, 093, 109
- 10 mg/kg bw/day: 017
- 50 mg/kg bw/day: 012
- 100 mg/kg bw/day: 019
- 200 mg/kg bw/day: 188
No implantation but corpora lutea i.e. total pre-implantation loss (partial exclusion, the intrauterine parameters were evaluated):
- control: No.: 116
- 50 mg/kg bw/day: No.: 130
- 100 mg/kg bw/day: No.: 143
- 200 mg/kg bw/day: No.: 007
Females with total post-implantation loss (partial exclusion, body weight and food consumption parameters excluded)
- 10 mg/kg bw/day: No.: 124
- 50 mg/kg bw/day: No.: 182, 145 (both total excluded, see * and **)
Females with abortion: (excluded: body weight and food consumption data from the day of observed abortion)
- 50 mg/kg bw/day: No.: 115
Females and litters with early delivery: (excluded: body weight and food consumption data of the does from the day of observed early delivery)
- control: No.: 170
- 10 mg/kg bw/day: No.: 095
- 50 mg/kg bw/day: No.: 085
- 200 mg/kg bw/day: No.: 094
A disease of the doe unrelated to the treatment (survived) (total exclusion):
Disease (survived, post-implantation loss):
- 50 mg/kg bw/day: No.: 145 ** (thoracic cavity filled by whitish-yellowish dense phlegma, lungs sticked to pleura)
Developmental disorder of the doe (survived, total post-implantation loss) (total exclusion):
- 50 mg/kg bw/day: No.: 182 * (right kidney absent, left kidney 2-3 fold larger than normal, hypoplastic right uterine horn)
Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
necropsy findings
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no significant differences indicated in the fetal growth (body weight and crown-rump length) as well as placental weight (absolute and relative).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Malformations
The number of affected litters was one in each group including control and except the 10 mg/kg bw/day group.
Spina bifida occulta was observed in one control fetus (No.: 0950055/4). Partial acrania was seen in one fetus (No.:1175196/2) in the 50 mg/kg bw/day group. Exencephaly was found in one fetus (No.: 1456135/3) in the 100 mg/kg bw/day group.
Hyperflexion of one forelimb was seen in one fetus (No.: 1570045/6) in the 200 mg/kg bw/day group. These findings were not attributed to the test item, considering the sporadic distribution in the groups and that failure of neural tube closure occurred also in the control group. Hyperflexion of the forelimbs may occur in control fetuses according to the Historical control data.

Variations
There was no increase in incidence of fetuses and litters with variations (retardation in body weight or crown-rump length). Moreover, the incidence of affected litters with crown-rump length retardation was statistically significantly lower (p<0.005) in the high dose.

Placentas
There were no placental changes found.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There was a statistically significant increase in incidence of fetuses with skeletal abnormalities due to incidence of variations (p<0.05) in the 200 mg/kg bw/day dose group. There were no significant increases indicated investigating the different types of variations observed. There was no increase indicated in the incidence of malformations.

Malformations:
The following skeletal malformations were observed in the fetuses found with malformation at external examination.
The lumbar II arches of fetus No.: 0950055/4 were partially, from lumbar III to sacral I completely unclosed (spina bifida at external examination) in the control group. The skull bones were absent cranial from atlas (brain and facial), the mandible was considered as misshapen and hypoplastic and interrupted in fetus No.:1175196/2 in the 50 mg/kg bw/day group (partial acrania and hypoplastic mandible at external examination).
Interconnecting anterior and posterior fontanelle (dilated suture), hole in parietal; misshapen frontal and parietal; absent interparietal and supraoccipital were found in one fetus (No.: 1456135/3 with exencephaly) in the 100 mg/kg bw/day group. Slight shortness of one side ulna was seen in fetus No.: 1570045/6 (hyperflexion of the forelimb at external examination) in the 200 mg/kg bw/day group.
Sternebral fusions of different degree (fused in a small point, fused by peak, fused) or/and combined with wideness were recorded. Spliss or hole in or between sternebra 5th or 6th or/and xiphoid cartilage were found with low incidences and without a dose response. Multiply malformed ribs or/and vertebra, misshapen cervical vertebra, dumb-bell shaped cartilage and hemicentric ossification of thoracic centrum, multiply malformed vertebrae were found in single cases or/and without a dose response.
When the whole incidence or the single type of skeletal malformations was investigated, there were no significant dose related increases observed, hence the skeletal malformations were not attributed to the treatment.

Variations:
Variations of the skull bones like larger or slightly larger anterior or/and posterior fontanelle or/and slightly dilated suture, slightly incomplete ossification of parietal bone, sternal variations such as less than 5 ossified sternebra, dumb-bell shaped, bipartite or/and asymmetric, misaligned ossification, extra ossification point, fusing tendency, small hole in xiphoid cartilage and slightly bent cartilage, variation of the vertebra, such as bipartite, dumb-bell shaped or asymmetric ossification, ossification delay of pelvic girdle (unossified pubic), talus, pollex, phalanges as well as asymmetric degree of ossification of phalanges of the two sides were recorded.

There was no statistically significant increase indicated in any of these parameters.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the visceral abnormalities/variations/malformations.

Malformations:
There were no malformations found at visceral examinations.

Variations:
Slightly or moderately dilated IIIrd brain ventricle was found in two fetuses in the 100 and two in the 200 mg/kg bw/day groups. This alteration may also occur in control animals according to the Historical data. In one fetus in the 100 mg/kg bw/day group the inter-medial lung lobe was absent. Convoluted- or hydroureter was found in the fetuses with similar incidences in the groups. Acute angled ureter connection to kidney, markedly convoluted and slightly dilated ureter and dilated renal pelvis and doubled gall bladder were found in single cases and not in the high dose group.
The variations found were not attributed to the test item considering the low incidences and/or lack of dose response.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: no adverse effects observed up to the highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the results of this OECD 414 compliant study in rabbits, the NOAEL was determined as follows:
NOAEL (maternal toxicity): 100 mg/kg bw/day
NOAEL (developmental toxicity): 200 mg/kg bw/day
Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of 24 inseminated New Zealand White rabbits were treated with the test item by oral (gavage) administration daily at four dose levels of 10, 50, 100 and 200 mg/kg bw/day respectively from day 6 up to and including day 27 post insemination. A control group of 24 inseminated females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.


The test item was proved to be stable in sunlflower oil formulations at 2 mg/mL and 200 mg/mL concentration levels for 24 hours at room temperature and at least for three days in the refrigerator (5 ± 3°C) according to the analytical method validation (Study no.: (Toxi-Coop study no. 552-100-4500)) at Toxi-Coop Zrt. The suitability of the chosen vehicle for the test item was analytically proven. Analytical control of dosing solutions was performed during the first and last week of treatment. The mean of the test item concentrations of the test item in the dosing formulations varied in the acceptable range between 90 and 96 % of nominal concentrations at both analytical occasions confirming proper dosing.


During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the does were recorded. The day of insemination was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 28. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed, measured and examined for gross external abnormalities. The placentas were weighed and examined externally.


Fresh visceral examination of each fetus was performed including the determination of the gender.


Head of about 50 % of each litter was removed, fixed in modified Sanomiya solution and washed in 90 % isopropanol. Examination of the heads was done by Wilson's free-hand razor blade method. All skeletons were examined by means of a dissecting microscope after cartilage-bone staining.


There was no test item related mortality, moribund state, abortion or early delivery observed.


The number of litters revealed at Caesarean section was 18, 21, 17, 22 and 21 in the control, 10, 50, 100 and 200 mg/kg bw/day dose groups, respectively.


There were no test item related clinical signs observed. Dark red and/or corrosion- like areas in the stomach revealed at necropsy in the 200 mg/kg bw/day group were attributed to the treatment and irritant properties of the test item.  
Between G.D. 6 to 9 and 21 to 24 24 to 27 as well as 0 to 28 the body weight gain was lower at 200 mg/kg bw/day than in the other groups. Between G.D. 9 and 12 as well as 24 and 27 the food consumption was lower (-16 and -19%) in the 200 mg/kg bw/day group than in the other groups. It cannot completely ruled out that both findings are caused by the test item treatment. There were no treatment related effects observed.
The fetal growth and placental weight were not affected by the treatment. The test item was judged not to increase the incidence of fetal malformations and variations. The summarized incidence of skeletal variations increased statistically significantly (p<0.05) in the high dose group but not the different type of variations. In the lack of a dose- response relationship this observation is not attributed to the test item.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-04-03 to 2015-05-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: young adult females, males: 20-21 weeks
- Housing: Before mating: 1-3 females per cage 1-2 males per cage
Mating: 1 male and 1-3 females / cage
During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water. ad libitum
- Acclimation period: 22 days for females, 85 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 10 mg/mL, 32.5 mg/mL and 100 mg/mL. Formulations were prepared in the laboratory of Toxi-Coop Zrt. daily or according to the stability data of the formulations.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Amount of vehicle: A constant treatment volume of 2 mL dose preparation/kg body weight was administered.
- Lot/batch no.: 1410-5159
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
MEKP content was determined in sunflower oil formulations using the previously validated reverse phase HPLC method with UV detection on a Kinetex 2.6u C18 100A column (100x4.6 mm). Detector: 205 nm.
MEKP concentrations in the samples varied in the range from 95% to 100 % in comparison to the nominal values.
Recovery of MEKP from sunflower oil formulations at two concentration levels (~10 and ~200 mg/mL) was 96 and 102 %.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male: one to three females
- Length of cohabitation: in the mornings for two to four hours
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19. The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time.
Frequency of treatment:
daily
Duration of test:
gestational day 5 to 19, gross pathology afterwards
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
65 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
only females were treated, at least 24 per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting is based on findings obtained in a 28-Day Repeated Dose Oral Toxicity Study (OECD 407) in the Rat.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: viscera, uterus with cervix and the ovaries
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
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
The statistical evaluation of data was performed with the 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 is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Indices:
Pre-implanation loss:
((number of corpora lutea)-(number of implantations))/(number of corpora lutea)*100

Post-implanation loss:
((number of implantations)-(number of live fetuses))/(number of implantations)*100

Sex distribution:
(number of male (female)fetuses)/(number of fetuses)*100

External abnormalities/ litter:
(number of fetuses with abnormality/(number of fetuses)*100

Visceral abnormalities/litter:
(number of fetuses with abnormality/(number of fetuses examined)*100

Skeletal abnormalities/litter
(number of fetuses with abnormality/(number of fetuses examined)*100
Historical control data:
Findings were compared to historical control data.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The death of one as well as clinical signs of three further pregnant females (squish breath, dyspnoea and breath with open mouth started in all cases immediately after drawing the gavage out of the esophagus) in the 200 mg/kg bw/day group were suspected to be due to an aspiration or inhalation of the test item hence as a consequence of a technical reason probably due to the properties of the test item at this concentration.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
The death of one as well as clinical signs of three further pregnant females (squish breath, dyspnoea and breath with open mouth started in all cases immediately after drawing the gavage out of the esophagus) in the 200 mg/kg bw/day group were suspected to be due to an aspiration or inhalation of the test item hence as a consequence of a technical reason probably due to the properties of the test item at this concentration. There was no mortality and treatment related clinical signs and necropsy findings in the 65 and 20 mg/kg bw/day dose groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slight, but statistically significant reduction in the body weight gain was observed in the food consumption and body weight gain of the dams in the 200 mg/kg bw/day group between gestation days 11 and 17 which was attributed to an effect of the test item. There were no treatment related differences in the food consumption and body weight of the animals in the 65 and 20 mg/kg bw/day groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A slight, but statistically significant reduction in the body weight gain was observed in the food consumption and body weight gain of the dams in the 200 mg/kg bw/day group between gestation days 11 and 17 which was attributed to an effect of the test item. There were no treatment related differences in the food consumption and body weight of the animals in the 65 and 20 mg/kg bw/day groups.
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:
effects observed, treatment-related
Description (incidence and severity):
The stomach and intestines of the dam which died before scheduled necropsy were empty and filled with gas. The necropsy findings (reddish mottled lungs, dark red liver) of the non-pregnant female in the 200 mg/kg bw/day group that died before scheduled termination might confirm the suspect of aspiration/asphyxia.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
65 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no fetal malformations found at external and skeletal examination. External hydrocephaly was found in one fetus as a malformation at visceral examination in the 200 mg/kg bw/day dose group which was judged to be unrelated from the treatment based on the historical control data
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
The mean number of implantations, intrauterine mortality and sex distribution of the fetuses was not influenced by the treatment. There were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity no adverse effects observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The No Observed Adverse Effect Level (NOAELs) weres determined as follows:
NOAEL (maternal toxicity): 65 mg/kg bw/day
NOAEL (developmental toxicity): 200 mg/kg bw/day
Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of 24 sperm-positive female Hsd. Brl. Han: Wistar rats were treated with the test substance by oral administration daily at three dose levels of 20, 65 and 200 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 24 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test substance in sunflower oil was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 3 days at the concentrations of 10 and 200 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 95 and 100 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, there were 23 evaluated litters each in the control and 20 mg/kg bw/day group, 24 and 21 in the 65 and 200 mg/kg bw/day groups respectively.

The death of one as well as clinical signs of three further pregnant females (squish breath, dyspnoea and breath with open mouth started in all cases immediately after drawing the gavage out of the esophagus) in the 200 mg/kg bw/day group were suspected to be due to an aspiration or inhalation of the test item hence as a consequence of a technical reason probably due to the properties of the test item at this concentration.

The stomach and intestines of the dam which died before scheduled necropsy were empty and filled with gas. The necropsy findings (reddish mottled lungs, dark red liver) of the non-pregnant female in the 200 mg/kg bw/day group that died before scheduled termination might confirm the suspect of aspiration/asphyxia.

There was no mortality and treatment related clinical signs and necropsy findings in the 65 and 20 mg/kg bw/day dose groups.

A slight, but statistically significant reduction in the body weight gain was observed in the food consumption and body weight gain of the dams in the 200 mg/kg bw/day group between gestation days 11 and 17 which was attributed to an effect of the test item. There were no treatment related differences in the food consumption and body weight of the animals in the 65 and 20 mg/kg bw/day groups.

The mean number of implantations, intrauterine mortality and sex distribution of the fetuses was not influenced by the treatment.

There were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations. There were no fetal malformations found at external and skeletal examination. External hydrocephaly was found in one fetus as a malformation at visceral examination in the 200 mg/kg bw/day dose group which was judged to be unrelated from the treatment based on the historical control data.

As a conclusion,based on these observations the No Observed Adverse Effect Level (NOAELs) weres determined as follows:

NOAEL (maternal toxicity): 65 mg/kg bw/day

NOAEL (developmental toxicity): 200 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable GLP and OECD Guideline conform 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

OECD 414, rat


The test item was examined for its possible prenatal developmental toxicity. Groups of 24 sperm-positive female Hsd. Brl. Han: Wistar rats were treated with the test substance by oral administration daily at three dose levels of 20, 65 and 200 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 24 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test substance in sunflower oil was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 3 days at the concentrations of 10 and 200 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 95 and 100 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, there were 23 evaluated litters each in the control and 20 mg/kg bw/day group, 24 and 21 in the 65 and 200 mg/kg bw/day groups respectively.


The death of one as well as clinical signs of three further pregnant females (squish breath, dyspnoea and breath with open mouth started in all cases immediately after drawing the gavage out of the esophagus) in the 200 mg/kg bw/day group were suspected to be due to an aspiration or inhalation of the test item hence as a consequence of a technical reason probably due to the properties of the test item at this concentration.


The stomach and intestines of the dam which died before scheduled necropsy were empty and filled with gas. The necropsy findings (reddish mottled lungs, dark red liver) of the non-pregnant female in the 200 mg/kg bw/day group that died before scheduled termination might confirm the suspect of aspiration/asphyxia. There was no mortality and treatment related clinical signs and necropsy findings in the 65 and 20 mg/kg bw/day dose groups.


A slight, but statistically significant reduction in the body weight gain was observed in the food consumption and body weight gain of the dams in the 200 mg/kg bw/day group between gestation days 11 and 17 which was attributed to an effect of the test item. There were no treatment related differences in the food consumption and body weight of the animals in the 65 and 20 mg/kg bw/day groups.


The mean number of implantations, intrauterine mortality and sex distribution of the fetuses was not influenced by the treatment. There were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations. There were no fetal malformations found at external and skeletal examination. External hydrocephaly was found in one fetus as a malformation at visceral examination in the 200 mg/kg bw/day dose group which was judged to be unrelated from the treatment based on the historical control data.


 


As a conclusion, based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows:


 


NOAEL (maternal toxicity): 65 mg/kg bw/day


NOAEL (developmental toxicity): 200 mg/kg bw/day


 


 


OECD 414, rabbit


The test item was examined for its possible prenatal developmental toxicity. Groups of 24 inseminated New Zealand White rabbits were treated with the test item by oral (gavage) administration daily at four dose levels of 10, 50, 100 and 200 mg/kg bw/day respectively from day 6 up to and including day 27 post insemination. A control group of 24 inseminated females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.


The test item was proved to be stable in sunlflower oil formulations at 2 mg/mL and 200 mg/mL concentration levels for 24 hours at room temperature and at least for three days in the refrigerator (5 ± 3°C) according to the analytical method validation (Study no.: (Toxi-Coop study no. 552-100-4500)) at Toxi-Coop Zrt. The suitability of the chosen vehicle for the test item was analytically proven. Analytical control of dosing solutions was performed during the first and last week of treatment. The mean of the test item concentrations of the test item in the dosing formulations varied in the acceptable range between 90 and 96 % of nominal concentrations at both analytical occasions confirming proper dosing.


During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the does were recorded. The day of insemination was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 28. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed, measured and examined for gross external abnormalities. The placentas were weighed and examined externally.


Fresh visceral examination of each fetus was performed including the determination of the gender.


Head of about 50 % of each litter was removed, fixed in modified Sanomiya solution and washed in 90 % isopropanol. Examination of the heads was done by Wilson's free-hand razor blade method. All skeletons were examined by means of a dissecting microscope after cartilage-bone staining.


There was no test item related mortality, moribund state, abortion or early delivery observed.


The number of litters revealed at Caesarean section was 18, 21, 17, 22 and 21 in the control, 10, 50, 100 and 200 mg/kg bw/day dose groups, respectively.


There were no test item related clinical signs observed. Dark red and/or corrosion- like areas in the stomach revealed at necropsy in the 200 mg/kg bw/day group were attributed to the treatment and irritant properties of the test item.  
Between G.D. 6 to 9 and 21 to 24 24 to 27 as well as 0 to 28 the body weight gain was lower at 200 mg/kg bw/day than in the other groups. Between G.D. 9 and 12 as well as 24 and 27 the food consumption was lower (-16 and -19%) in the 200 mg/kg bw/day group than in the other groups. It cannot completely ruled out that both findings are caused by the test item treatment. There were no treatment related effects observed.
The fetal growth and placental weight were not affected by the treatment. The test item was judged not to increase the incidence of fetal malformations and variations. The summarized incidence of skeletal variations increased statistically significantly (p<0.05) in the high dose group but not the different type of variations. In the lack of a dose- response relationship this observation is not attributed to the test item.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on toxicity to reproduction the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) No 2021/849.

Additional information