Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the test substance was administered daily to rats at dose levels up to 500 mg/kg bodyweight/day (OECD 422; Peter, 2017). The parental and reproductive NOAEL were established as 150 mg/kg body weight/day. The substance is not classified as reproductive toxicant according to the CLP Regulation.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-05 to 2016-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/ Developmental Toxicity Screening Test)
Qualifier:
equivalent or similar to guideline
Guideline:
other: OPPTS 870.3050 (Repeated Dose 28–Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: I15FB3131
- Expiration date of the lot/batch: 2017-06-11 (retest date)
- Purity test date: 2015-06-12

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stability for at least 6 hours at room temperature was confirmed over the concentration range 1 to 200 mg/mL, Test Facility Study No. 512034.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Turbid solutions (Groups 2-4).

OTHER SPECIFICS: Correction factor was 1.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: females approx. 10-12 weeks (at start pretest) and approx. 12-14 weeks (at start F0-treatment); males approx. 10-12 weeks (at start F0-treatment)
- Weight at study initiation: 297-325 g (males) and 201-232 g (females)
- Fasting period before study:no
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type,
height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV ty
pe, height 18 cm).
Mating: Females were caged together with males on a one-to-one basis in Macrolon plastic cages (M
III type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm)
with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII
type, height 18 cm).
- Diet (e.g. ad libitum): free access to pelleted rodent diet
- Water (e.g. ad libitum): free access to tap-water
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES:
From: 2016-08-17 (start pretest, females aged approx. 10-12 weeks); 2016-08-31 (start treatment, males aged approx. 10-12 weeks); 2016-10-07/08/09/10/11(delivery of litters)
To: 2016-09-29 (necropsy males); 2016-10-20/21/24/25/26 (necropsy females); 2016-10-19/20/23/24/25 (necropsy pups)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% aqueous CMC
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. A correction was made for the purity/composition of the test item. A correction factor of 1 was used. No adjustment was made for specific gravity/density of the test item, vehicle and/or formulation. Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed
at Charles River Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1); 10 mg/mL (group 2); 30 mg/mL (group 3); 100 mg/ mL
(group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight (Actual dose volumes were calculated according
to the latest body weight)
- Lot/batch no. (if required): no data
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1 animal/sex/cage
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, until detection of mating was confirmed
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating. Detection of mating was not confirmed for animal no. 53 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (2016-09-13; Day 17 of treatment) according to a validated method (Test Facility Study No. 512034). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10.00% compared to those obtained during the method validation. The accuracy of preparation was
considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.
Duration of treatment / exposure:
29 days (males); 50-56 days (females that delivered); 40-43 days (females that failed to deliver). Pups were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control group)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on results of a dose range finding study (Test Facility Study No. 512019) in which animals were dosed for 20 and 7 days at 500 and 1000 mg/kg, respectively. In summary, there were no mortalities for the 500 mg/kg dose group, but 1/3 of the animals in the 1000 mg/kg dose group were found dead on Day 8, while 2/3 were sacrifice d in extremis on Day 8. Piloerection was observed in one animal of the 500 mg/kg group, while hunched posture and piloerection was observed in all animals of the 1000 mg/kg group, with slight salivation in 2/3 animals on Days 6 and/or 7. Body weight loss was observed for both treatment groups. Food consumption was slightly reduced over Days 1-5 for the 500 mg/kg group, but normal thereafter, while food consumption was markedly reduced over Days 1-5 for the 1000 mg/kg group. Macroscopic examination of the 500 mg/kg animals showed no abnormalities, while 1000 mg/kg an imals showed some findings, including reduced size of spleen in one of the animals found dead and reduced size of thymus in two animals sacrificed in extremis. Liver weight was slightly increased in the 500 mg/kg group; organ weights were not recorded for the 1000 mg/kg group. Based on the results of this range finding study, dose levels for the main study were: 50, 150 and 500 mg/kg/day. Since no clear peak effect of occurrence of clinical signs was observed in the range finding study, clinical observations (clinical signs and arena) in the main study were conducted and functional observations were started after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
- Rationale for animal assignment (if not random): randomized
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals after dosing at no specific time point, but within a similar time period after dosing for the respective animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and13. Body weight gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/ kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Relative food consumption was calculated and reported.
- Compound intake calculated as time-weighted averages from the consumption and body weight gaindata: Not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY
- Blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived from food overnight (with a maximum of 24 hours) before blood sampling; but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with K3-EDTA for hematology parameters and with citrate for clotting tests.
- Parameters assessed: white blood cells, differential leukocyte counts, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothombin time, activated thromboplastin time.

CLINICAL BIOCHEMISTRY
- Blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with Li-heparin.
- Parameters assessed: alanine aminotransferase, aspartate aminotrasnferase, alkaline phosphatase , total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.
- thyroid hormone analysis

FUNCTIONAL OBSERVATIONS
- Time schedule: The selected males were tested during Week 4 of treatment and the selected females of Groups 1-3 were tested once during the last week of lactation (e.g. PND 6-13). In Group 4, the selected females were tested during the last week of treatment. These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Parameters: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength recorded as the mean of three measurements per animal, locomotor activity
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generation: additional slides of the testes to examine staging of spermatogenesis; testis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No. All pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink on post-natal day 1 (= day the litter was found completed)
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible) were selected for culling on PND 4; blood samples were collected from two of the surplus pups; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective table.
- Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on PND 1 and 14.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead if possible. Pups found dead during the weekend were necropsied on the same day.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE:
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration).
- Female animals: All surviving animals, on PND 14-16 (females that delivered) or on post-coitum day 25 (females that failed to deliver, with evidence of mating) or approximately 27 days after the last day of the mating period(female without evidence of mating).

GROSS PATHOLOGY: Yes
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. After sacrifice, all animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Necropsy was conducted as soon as possibe after spontaneous death and always within 24 hours.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution):
Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/ F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M) , (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F) (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland ( M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (F), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/ F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F) , Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, and females which failed to deliver, were collected and fixed in 10% buffered formalin:
Cervix (F), Clitoral gland (F), Coagulation gland (M), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levator ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

ORGAN WEIGHTS:
- Absolute organ weights and organ to body weight ratios were reported.
- The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/ group on the scheduled day of necropsy: Adrenal glands, Brain, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix)
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavernosus muscle complex (LABC), Testes, Thyroid

HISTOPATHOLOGY: Yes
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist: The preserved organs and tissues of 5 animals/sex of Groups 1 and 4; The additional slides of the testes of all males of Groups 1, 2, 3 and 4 and all males that failed to sire to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); Mesenteric lymph nodes, epididymides and testes of all selected 5 males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4; Kidneys of all selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in this organ in Group 4; The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups (one couple at 100 mg/kg and 10 couples at 500 mg/kg).
- All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
- A peer review on the histopathology data was performed by a second pathologist.
Postmortem examinations (offspring):
SACRIFICE
- Pups, younger than 7 days were killed by decapitation
- The F1 offspring were sacrificed at PND 4 (by decapitation between 7.00 and 10.30 a.m.), and at PND 7-15 (using Euthasol 20% by intraperitoneal injection).

GROSS NECROPSY
- All pups were sexed by both external and internal examination. Descriptions of all abnormalities were recorded.
- At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin.
- The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means
and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/ Number of females paired) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Survival indices:
Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%) = Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100
Group mean values were calculated from individual litter values.
Sex ratio (percentage males) = (Number of males in litter/Total number of offspring in litter) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related clinical signs of toxicity were noted at 500 mg/kg/day all animals. Frequent findings included hunched posture, piloerection, slightly pale appearance, hypothermia, and slight salivation, and were generally seen from the third week of treatment onwards. Additionally, cramped posture and chromodacryorrhoea (both at a slight degree) were noted in a single male and female at this high dose level. No treatment related clinical signs were observed up to 150 mg/kg/day. During the weekly arena observations, no additional treatment-related clinical signs were noted. Any other clinical signs noted incidentally (alopecia and scabs or scales on the skin) occurred within the range of back ground findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated at 500 mg/kg/day showed slight body weight loss in the first treatment week and severely reduced body weight gain thereafter. At the end of the treatment period, mean body weight of 500 mg/kg/day males was 14% lower than that of controls. At the lower dose levels (50 and 150 mg/kg/ day), males gained slightly less weight than controls during the first two weeks of the treatment period and their mean body weights were up to approximately 5% lower compared with controls at the end of the 4-week treatment period.

Females treated at 500 mg/kg/day showed slight body weight loss in the first treatment week and severely reduced body weight gain or some further weight loss in the second week. Gestational body weights at 500 mg/kg/day were available for only two females (nos. 72 and 74). These females gained less weight than controls, which was consistent with their abnormal pregnancy (low number of implantation sites, no offspring). Body weight development of the one non-mated female and seven non-pregnant females in the 500 mg/kg/day group in post-coitum was in line with that of untreated, nulliparous females of this strain and age.

No clear treatment-related changes in body weight or body weight gain were observed in females treated at 50 or 150 mg/kg/day. It was noted that 6 out of 10 females at 150 mg/kg/day showed a slight body weight loss in the first week of the treatment period. Similar weight loss was noted in 3 out of 10 controls and weight gain of 150 mg/kg/day females was normal in the following weeks. Therefore, the apparent downward trend in the first week was considered not to reflect a toxicologically relevant effect on body weight gain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males, food consumption (before and after allowance for body weight) was reduced at 500 mg/kg/ day, most markedly in the first week of the treatment period.

Females treated at 500 mg/kg/day consumed less food than controls throughout the 2-week pre-mating period. The lower food consumption of the two pregnant females at 500 mg/kg/day during gestation was considered to be due to their abnormal pregnancy (low number of implantation sites, no offs pring). Food consumption of the one non-mated female and seven non-pregnant females in the 500 mg/kg/day group in post-coitum was in line with that of untreated, nulliparous females of this strain and age.

No treatment-related changes in food consumption before or after allowance for body weight were observed in males or females treated up to 150 mg/kg/day. The statistically significantly higher mean food intake (absolute and relative to body weight) noted for females at 50 mg/kg/day during Days 1-4 of lactation was considered to be a chance finding, rather than a treatment-related effect, as no dose-related trend could be established.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in haematology parameters distinguished treated animals from concurrent control animals (differences were statistically significant unless indicated otherwise):
• Lower total white blood cell count (WBC) in both sexes at 500 mg/kg/day (relative difference from controls: approximately 50%), and to a lesser extent at 50 and 150 mg/kg/day in males.
• Lower percentage of monocytes in females at 500 mg/kg/day.
• Lower percentage of eosinophils in females at 150 and 500 mg/kg/day.
• Higher percentage of basophils (not statistically significant) in males at 500 mg/kg/day.
• Lower number of red blood cells in both sexes at 500 mg/kg/day and in males at 150 mg/kg/day.
• Lower haemoglobin and haematocrit in both sexes at 500 mg/kg/day.
• Higher percentage of reticulocytes in males at 500 mg/kg/day, and lower percentage of reticulocytes in females at 500 mg/kg/day.
• Higher red blood cell distribution width (RDW) in both sexes at 500 mg/kg/day, and in males at 150 mg/kg/day.
• Higher mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in both sexes at 150 and 500 mg/kg/day (MCV in 150 mg/kg/day males not statistically significant), and higher MCH in females at 50 mg/kg/day.
• Higher prothrombin time in males at 500 mg/kg/day.
The above changes at 150 and 50 mg/kg/day were slight and/or remained in the normal range. Consequently, these findings were considered not adverse at these dose levels. The increase in prothrombin time in males at 500 mg/kg/day was only slightly above the normal range and therefore, considered to be non-adverse. The changes at 500 mg/kg/day were clearly outside the normal range for rats of this strain and age, except for the changes in WBC in females. Combined with concurrent histopathological findings, these haematological changes at 500 mg/kg/day were regarded adverse.

The higher percentage of lymphocytes and lower percentage of neutrophils in females at 500 mg/kg/day (compared with concurrent controls) were considered to be due to the difference in physiological status.

The higher activated partial thromboplastin time (APTT) in males at 50 and 150 mg/kg/day was considered unrelated to treatment as this change occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in clinical biochemistry parameters distinguished treated animals from concurrent control animals (differences were statistically significant unless indicated otherwise):
• Higher alanine aminotransferase (ALAT) in males at 500 mg/kg/day.
• Lower total protein (not statistically significant) and albumin in females at 150 mg/kg/day.
• Higher total bilirubin in both sexes at 500 mg/kg/day, and in females at 150 mg/kg/day.
• Higher bile acids in both sexes at 500 mg/kg/day (not statistically significant in females), and in females (and a single male) at 150 mg/kg/day and in 3 out of 5 females at 50 mg/kg/day.
Values of the above parameters at 500 mg/kg/day were at the upper limit or above the normal range for rats of this strain and age. The changes in females at 50 and 150 mg/kg/day remained (just) within the normal range, and were consequently regarded as non-adverse.

The following (statistically significant) differences between females treated at 500 mg/kg/day and concurrent controls were considered to be due to the difference in physiological status: higher total protein, albumin, and chloride, and lower aspartate aminotransferase (ASAT) and inorganic phosphate. The slightly lower levels of fasting glucose and cholesterol in 500 mg/kg/day females were not statistically significantly and might be related to the different physiological status. Therefore, these changes were considered not toxicologically significant.

Any other statistically significant variations noted in clinical biochemistry parameters were considered unrelated to treatment due to the absence of a dose-related trend or somewhat high control value.

Thyroid hormone analyses:
The serum T4 level of F0 males was statistically significantly decreased at 500 mg/kg/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Lower mean motor activity (total movements and ambulations) was noted at 500 mg/kg/day in males. This was mainly caused by male nos. 32, 33 and 35. Statistical significance was not achieved, but in view of the general toxicity at this dose level, the decreased activity of 500 mg/kg/day males was considered to be related to treatment and toxicologically relevant. Motor activity habituation profiles in 500 mg/kg/day males showed the normal decreasing trend over the duration of the test period.

No treatment-related changes in motor activity level or habituation profile were noted in males treated up to 150 mg/kg/day and in females treated up to 500 mg/kg/day.

Grip strength was similar across the groups, except for a statistically significantly lower hind limb grip strength in females at 500 mg/kg/day. This finding was not accompanied by a decrease in fore limb grip strength and the values were within the normal range for female rats of this strain and age. Therefore, the change in hind limb grip strength in females at 500 mg/kg/day was considered not to be toxicologically relevant.

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the thyroid gland, thymus, spleen, mesenteric lymph node, testes, epididymides and bone marrow (sternal and femur) in one or both sexes at 150 and/or 500 mg/kg/day.
- Testes: A massive depletion of germ cells was observed in all males treated at 500 mg/kg/day. This mirrored the finding ‘abnormal stages’ recorded for the PAS stained slides of the testes of this group. In general, all round spermatids were affected while elongating spermatids and spermatogonia were still detectable. Tubular degeneration/atrophy and vacuolation of the testes were observed at increased incidence and severity (up to slight) in males treated at 500 mg/kg/day compared with controls (one control male showed these lesions at a minimal degree). This was considered as nonspecific change of the germ cells.
- Epididymides: Cellular luminal debris was observed at increased incidence and severity (up to slight) and reduced luminal sperm was observed up to moderate degree in males treated at 500 mg/kg/day.
- Thyroid gland: Follicular cell hypertrophy was observed at increased severity in males (up to moderate) and females (up to moderate) treated at 500 mg/kg/day.
- Spleen: At 500 mg/kg/day, extramedullary hematopoiesis was observed at increased severity (up to marked) in males and females and pigmentation was observed at increased mean severity in males (majority up to slight) and females (up to moderate).
- Bone marrow (sternum): Increased cellularity (of mainly erythroid cells) was observed in 2 out of 5 males (slight) and all females (up to slight) at 500 mg/kg/day. Decreased cellularity (diffuse, all cell types) was observed in one male (minimal) at 500 mg/kg/day. Increased adipocytes was observed in 2 out of 5 females (minimal) at 500 mg/kg/day.
- Bone marrow (femur): Increased cellularity at the mid part of the metaphysis of the femur (mainly erythroid cells) was observed in one male (minimal) and all females (up to slight) at 500 mg/kg/day. Decreased cellularity at the distal part of the femur was observed in 3 out of 5 males (up to slight) and 2 out of 5 females (slight) at 500 mg/kg/day. Increased adipocytes at the distal part of the femur was observed in the majority of males (up to slight) and one female (minimal) at 500 mg/kg/day.
- Thymus: Lymphoid atrophy was observed at increased incidence and/or severity in males (up to marked) and females (up to moderate) at 150 and 500 mg/kg/day. The single incidence of minimal/slight degree of lymphoid atrophy in one male and one female at 50 mg/kg/day was regarded to be within background findings of rats of this age and strain.
- Mesenteric lymph node: Sinusoidal erythrocytes were observed at increased incidence and severity in males (up to slight) at 500 mg/kg/day. The low incidences with minimal degrees noted in males and females of the control group and in a single male of the 50 mg/kg/day group were considered to be within background findings of rats of this age and strain. Pigmentation (macrophages with brown pigment) was observed in 2 out of 5 males (up to slight) at 500 mg/kg/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles (mostly of 4 days).
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Spermatogenic staging profiles were normal for all examined males of the control group and the 50 and 150 mg/kg/day groups.

Adverse morphological changes noted in male reproductive organs at 500 mg/kg/day consisted of abnormal spermatogenesis with depletion of round spermatids while elongating spermatids and spermatogonia were still present, degeneration/atrophy and vacuolation of the testes, and increased cellular debris and reduced sperm in the epididymides.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
REPRODUCTION DATA
- Mating index: Mating index was not affected by treatment up to 500 mg/kg/day. Except for one female at 500 mg/kg/day (no. 78), all females showed evidence of mating. In this type of study it can be seen occasionally that a female is not mated. As male no. 38 used for pairing of female no. 78 did not have more serious abnormalities in his reproductive organs than the other males that did mate successful, the finding of this single non-mated female is considered unrelated to treatment.
- Fertility index: Fertility index was reduced to 22% at 500 mg/kg/day. Only two of the nine mated females in this group were pregnant (nos. 72 and 74). Fertility index was unaffected by treatment at 50 and 150 mg/kg/day. One female at 50 mg/kg/day (no. 52, mated with male no. 12) was not pregnant (as confirmed by Salewski staining). No abnormalities were seen in the reproductive organs which could account for this non-pregnancy. Therefore, the non-pregnancy of a single low dose female was considered not to be related to treatment.
- Number of implantation sites: At 500 mg/kg/day, there were two females (nos. 72 and 74) with one or two implantations only. All remaining high dose females were not pregnant. The number of implantation sites was unaffected by treatment up to 150 mg/kg/day. For female nos. 47 (control) and 60 (50 mg/kg/day), the number of pups was (slightly) higher than the number of implantations. This was considered to be due to normal resorption of these areas as these enumerations were performed on respectively Days 14 and 16 of lactation.

DEVELOPMENTAL DATA
Note: There were only two pregnant females (nos. 72 and 74) at 500 mg/kg, both had no live offspring. The developmental results described below relate to the groups treated up to 150 mg/kg/day.
- Gestation index and duration: Gestation index and duration of gestation were unaffected by treatment up to 150 mg/kg/day.
- Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of premature birth. No deficiencies in maternal care were observed.
- Post-implantation survival index: Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered not to be affected by treatment up to 150 mg/kg/day. The indices were 93, 96 and 87% in the control, 50 and 150 mg/kg/day groups, respectively. The index at 150 mg/kg/day appeared slightly lower than in controls. As the index at 150 mg/kg/day was within normal limits and individual animals showed no abnormal post-implantation loss, this finding was considered not to be related to treatment.
- Live birth index: Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment up to 150 mg/kg/day. The indices were 97, 100 and 98% in the control, 50 and 150 mg/kg/day groups, respectively. Three females had dead pups at first litter check (control females no. 45 and 47, and female no. 69 at 150 mg/kg/day which had 1 or 2 dead pups). This incidental pup mortality was within normal limits and unrelated to treatment with the test item.
- Viability index: Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment up to 150 mg/kg/day. The viability indices across the groups were 99 or 100%. Post-natal loss up to PND 4 was limited to one pup at 150 mg/kg/day (litter no. 66) which went missing on PND 3. The missing pup was most likely cannibalised. This incidental postnatal loss was considered to be unrelated to treatment with the test item.
- Lactation index No pups were lost between PND 5 and PND 13. The lactation index (number of live offspring on PND 13 as percentage of number of live offspring on PND 4 after culling) was 100% in all groups with litters (control, 50 and 150 mg/kg/day).
Parental results:
- Significant general toxicity was observed in parental males and females treated at 500 mg/kg/day. Clinical signs of toxicity appeared in the third week of the treatment period and included hunched posture, piloerection, slightly pale appearance, hypothermia, slight salivation, and, incidentally cramped posture and chromodacryorrhoea (both at a slight degree). In males this was associated with decreased motor activity. Food consumption and body weight gain were markedly reduced with slight weight loss in the first week of the study. During the post-coitum period, food consumption and body weight gain of 500 mg/kg/day females, most of which were not pregnant, were comparable to that of nulliparous females of similar age.

The following adverse combinations of treatment-related microscopic, macroscopic and/or clinical pathological findings were observed in rats treated at 500 mg/kg/day:
- Increased severity of follicular cell hypertrophy (up to moderate) was observed in the thyroid gland of males and females treated at 500 mg/kg/day. This finding correlated with increased weight and size of the thyroid and decreased serum level of T4 (thyroxine, measured in males only). Based on the somewhat higher severity of follicular cell hypertrophy and significant change in hormone level, and the fact that the finding occurred in the absence of microscopic hepatocellular hypertrophy or organ weight changes of the liver, the thyroid findings were considered adverse. The relevance of the macroscopically observed black-brown discolouration of the thyroid, which had no microscopic correlate, remained unclear.
- Increased severity of extramedullary hematopoiesis (up to marked) and increased mean severity of pigmentation (up to slight in males, up to moderate in females) was observed in the spleen at 500 mg/kg/day in both sexes. This correlated with increased weight and size of the spleen (males only) and changes in blood parameters, including decreases in number of red blood cells, haemoglobin and haematocrit, and increases in reticulocytes (in males only), red blood cell distribution width (RDW), mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH), and total bilirubin.
- Microscopic changes in bone marrow cell population were noted at 500 mg/kg/day in both sexes. In some animals, these changes consisted particularly of decreased cellularity up to slight degree, especially obvious at the distal metaphysis of the femur and most times with increased adipocytes up to slight degree (fatty replacement). While in other 500 mg/kg/day rats increased cellularity of especially the erythroid lineage was noted, which together with the increased severity of extramedullary hematopoiesis of the spleen might be interpreted as a regenerative response to the alterations of the red blood cells. Therefore the combination of changes was considered as adverse.
- Lymphoid atrophy was observed in the thymus at 500 and 150 mg/kg/day in both sexes. This correlated with decreased weight and size of the thymus. Based on the degree of atrophy and significant reduction of thymus weights (over 70 % for absolute and relative to body weight), the thymus atrophy in males at 500 mg/kg/day might be regarded as adverse, whereas that in females at 500 mg/kg/day and in males and females at 150 mg/kg/day might be regarded as non-adverse.
- Adverse, treatment-related decreases in the number of circulating total white blood cells (WBC) were observed at 500 mg/kg/day in both sexes.
- Abnormal spermatogenesis with depletion of round spermatids (while elongating spermatids and spermatogonia were still present), degeneration/atrophy and vacuolation of the testes, and increased cellular debris and reduced sperm in the epididymides were observed at 500 mg/kg/day. Associated changes consisted of macroscopic changes (testes flaccid and reduced in size) and lower weights of the testes and, to a lesser extent, epididymides.
- No parental toxicity was observed in rats treated at 50 or 150 mg/kg/day.

Reproductive results:
- At 500 mg/kg/day, 9 out of 10 females showed evidence of mating but only two of these females were pregnant (implantation sites only), both of which had very few implantation sites (only one or two). This adverse reproductive outcome was related with the adverse effects on the testes and epididymides. There were no test-item related findings in the reproductive organs of 500 mg/kg/day females.
- No reproduction toxicity was observed at 50 and 150 mg/kg/day.

Developmental results:
- No developmental toxicity was observed at 50 and 150 mg/kg/day.

Analysis of dose preparations:
- The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85 .00% and 115.00%). A small response at the retention time of the test item was observed in the chro matograms of the Group 1 formulation. The maximum contribution to the formulation samples at the lo west concentration level was 0.01%. The presence of such minor responses at the retention time of t he test item in Group 1 chromatograms was considered to be acceptable and did not adversely affect the integrity of the study.
- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).
Key result
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
haematology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
Significant general toxicity was observed in parental males and females treated at 500 mg/kg/day. Treatment-related microscopic, macroscopic and/or clinical pathological findings were observed in rats treated at 500 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction toxicity
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance
Remarks on result:
other: based on complete lack of offspring at 500 mg/kg/day due to abnormal spermatogenesis
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
other: Multiple Systems: Endocrine system & Hematopoiesis (Males & Females); and Immune system & Reproductive system (Males only)
Organ:
bone marrow
spleen
testes
thymus
thyroid gland
other: epididymides
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Note: There were only two pregnant females (nos. 72 and 74) at 500 mg/kg, both had no live offspring. The developmental results described in the "Results: F1 generation" section relate to the groups treated up to 150 mg/kg/day.

No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs that were observed remained within the range considered normal for pups of this age.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment up to 150 mg/kg/day. The viability indices across the groups were 99 or 100%.

Post-natal loss up to PND 4 was limited to one pup at 150 mg/kg/day (litter no. 66) which went missing on PND 3. The missing pup was most likely cannibalised. This incidental postnatal loss was considered to be unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights of pups were not affected by treatment up to 150 mg/kg/day.

The slightly, but statistically significantly higher mean weight of male pups in the 50 mg/kg/day group as compared with the concurrent control group was considered to be related to a relative low control value and therefore not caused by treatment with the test item.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were not affected by treatment up to 150 mg/kg/day.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.

The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment up to 150 mg/kg/day.
The slight, but statistically significant, differences in median absolute anogenital distance noted in male pups at 50 and 150 mg/kg/day occurred in the absence of a dose-related trend, and were therefore not attributed to treatment.

Areola/nipple retention: Treatment up to 150 mg/kg/day had no effect on areola/nipple retention, nipples were not observed at PND 13 for any of the examined male pups.

DEVELOPMENTAL DATA
- There were only two pregnant females (nos. 72 and 74) at 500 mg/kg, both had no live offspring.
- Gestation index and duration: Gestation index and duration of gestation were unaffected by treatment up to 150 mg/kg/day.
- Post-implantation survival index: Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered not to be affected by treatment up to 150 mg/kg/day. The indices were 93, 96 and 87% in the control, 50 and 150 mg/kg/day groups, respectively. The index at 150 mg/kg/day appeared slightly lower than in controls. As the index at 150 mg/kg/day was within normal limits and individual animals showed no abnormal post-implantation loss, this finding was considered not to be related to treatment.
- Live birth index: Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment up to 150 mg/kg/day. The indices were 97, 100 and 98% in the control, 50 and 150 mg/kg/day groups, respectively. Three females had dead pups at first litter check (control females no. 45 and 47, and female no. 69 at 150 mg/kg/day which had 1 or 2 dead pups). This incidental pup mortality was within normal limits and unrelated to treatment with the test item.
- Viability index: Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment up to 150 mg/kg/day. The viability indices across the groups were 99 or 100%. Post-natal loss up to PND 4 was limited to one pup at 150 mg/kg/day (litter no. 66) which went missing on PND 3. The missing pup was most likely cannibalised. This incidental post-natal loss was considered to be unrelated to treatment with the test item.
- Lactation index: No pups were lost between PND 5 and PND 13. The lactation index (number of live offspring on PND 13 as percentage of number of live offspring on PND 4 after culling) was 100% in all groups with litters (control, 50 and 150 mg/kg/day).


Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
No developmental toxicity was observed at 50 and 150 mg/kg/day.

Possible developmental toxicity at 500 mg/kg/day could not be evaluated. None of the two pregnant females treated at 500 mg/kg/day had offspring.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental toxicity was observed at 50 and 150 mg/kg/day. No litters were available for developmental toxicity assessment at 500 mg/kg/day.
Remarks on result:
other:
Remarks:
No developmental toxicity was observed at 50 and 150 mg/kg/day. No litters were available for developmental toxicity assessment at 500 mg/kg/day.
Critical effects observed:
no
Reproductive effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
In conclusion, treatment with JNJ-4754724-AAA (T001141) by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg revealed no reproduction and developmental toxicity up to 50 and 150 mg/kg/day. Possible developmental toxicity at 500 mg/kg/day could not be evaluated. None of the two pregnant females treated at 500 mg/kg/day had offspring. Significant general toxicity was observed in parental males and females treated at 500 mg/kg/day. Treatment-related microscopic, macroscopic and/or clinical pathological findings were observed in rats treated at 500 mg/kg/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 150 mg/kg/day (based on clinical signs of toxicity, reduced body weight gain and food consumption, adverse morphological changes in the spleen with associated changes in blood parameters, decreased number of circulating total white blood cells, and adverse morphological changes in the thyroid gland and bone marrow in both sexes, and adverse morphological findings in the thymus, testes and epididymides in males only).
Reproduction NOAEL: 150 mg/kg/day (based on complete lack of offspring at 500 mg/kg/day due to abnormal spermatogenesis).
Developmental NOAEL: 150 mg/kg/day (no litters were available for developmental toxicity assessment at 500 mg/kg/day)

The test item was not classified as a Reproductive Toxicant according to the CLP Regulation.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats were exposed to 0 (vehicle), 50, 150, 500 mg/kg body weight/day via gavage (OECD 422; Peter, 2017).

The vehicle used was 1% aqueous CMC (carboxymethyl cellulose) and the test solutions were prepared daily and administered within 6 hours after preparation.

The test substance did not cause mortality (treatment-related) during the study; however, male and female rats exposed to 500 mg/kg/day presented clinical signs of toxicity, reduced body weight gain and food consumption, adverse morphological changes in the spleen with associated changes in blood parameters, decreased number of circulating total white blood cells, and adverse morphological changes in the thyroid gland and bone marrow. Adverse morphological findings in the thymus, testes and epididymides were found in males only.

At 500 mg/kg/day, 9 out of 10 females showed evidence of mating but only two of these females were pregnant (implantation sites only), both of which had very few implantation sites (only one or two). This adverse reproductive outcome was related with the adverse effects on the testes and epididymides. There were no test-item related findings in the reproductive organs of 500 mg/kg/day females. No reproduction toxicity was observed at 50 and 150 mg/kg/day.

Based on the abovementioned considerations, the Parental and Reproduction NOAEL were considered to be 150 mg/kg bw/day (nominal dose

received).

Effects on developmental toxicity

Description of key information

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422; Peter, 2017), a developmental NOAEL of 150 mg/kg body weight/day was established. Possible developmental toxicity at 500 mg/kg/day could not be evaluated, since none of the two pregnant females treated at 500 mg/kg body weight/day had offspring. The test substance is known to have an adverse effect on fertility. Therefore, no further testing for fertility and developmental toxicity will be necessary, in accordance with the REACH Regulation (Annex IX, section 8.7).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the OECD 422 test, the following No Observed Adverse Effect Levels (NOAEL) were

derived:

Parental NOAEL: 150 mg/kg

Reproduction NOAEL: 150 mg/kg (based on complete lack of offspring at 500 mg/kg/day due to abnormal spermatogenesis)

Developmental NOAEL: 150 mg/kg (Note: No litters were available for developmental toxicity assessment at 500 mg/kg/day)

At 500 mg/kg/day, 9 out of 10 females showed evidence of mating but only two of these females were pregnant (implantation sites only), both of which had very few implantation sites (only one or two). This adverse reproductive outcome was related with the adverse effects on the testes and epididymides. There were no test-item related findings in the reproductive organs of 500 mg/kg/day females.

Although adverse effects on reproductive outcome were observed at the highest dose level, the substance is not classified as reproductive toxicant as these effects were observed together with parental toxicity at the highest dose level.

Additional information