2-Butanone, peroxide

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

15 July 2014

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-hydroperoxybutan-2-yl)peroxy]butane-2-peroxol; butane-2,2-diperoxol

EC Number:

700-954-4

Cas Number:

1338-23-4

Molecular formula:

Mixture of C4H10O4 and C8H18O6

IUPAC Name:

2-[(2-hydroperoxybutan-2-yl)peroxy]butane-2-peroxol; butane-2,2-diperoxol

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

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

2

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

Target system / organ toxicity (F1)

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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