4-[(4-HYDROXYPYRIMIDIN-2-YL)AMINO]BENZONITRILE

Endpoint summary

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 1000 mg/kg bodyweight/day (OECD 422; Otterdijk, 2017). The parental and reproduction NOAELs were established as at least 1000 mg/kg body weight/day. The test item is not classified as a 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:

2016-08-22 to 2016-12-14

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:

other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

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:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

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 No.of test material: M16DC1020
- Expiration date of the lot/batch: 2017-04-02
- Purity test date: 08 September 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Solubility and stability of the test substance in the solvent/vehicle: Stability for at least 6 hours at room temperature and 14 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL, Test Facility Study No. 512686


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


OTHER SPECIFICS: correction factor is 1.00

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. 11 weeks (at start pretest) and approx. 13 weeks (at start F0- treatment); Males approx. 10 weeks (at start F0-treatment)
- Weight at study initiation: 227-320 g (males) and 207-249 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 type, 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
- Humidity (%): 40 to 70%
- 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: 2016-10-05 (start pretest, females); 2016-10-19 (start treatment, males); 2016-11-25 to 2016-11-29 (delivery of litters)
To: 2016-11-17 (necropsy males); 2016-12-08/10/12/13/14 (necropsy females); 2016-12-07/09/11-13 (necropsy pups)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% aq. solution

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.
No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. A correction was made for the purity/composition of the test item. A correction factor of 1.00 was used.

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); 22 mg/mL (Group 2); 66 mg/mL (Group 3); 200 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, one female was cohabitated with one male of the same treatment group 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.
- 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 (20 October 2016, Day 2 of treatment) according to a validated method (Test Facility Study No. 512686). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.
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 for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%.

Duration of treatment / exposure:

29 days (males); 50-56 days (females that delivered); 41-44 days (females that failed to deliver). Two females from Group 2, three from Group 3, and two from Group 4 were not dosed on one day as they were littering at the moment of dosing. 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)

Dose / conc.:

110 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

330 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

1 000 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 based on results of a 10-day dose range finding study (Test Facility Study No.514909) and levels of 110, 330 and 1000 mg/kg were selected in consultation
with the Sponsor.
- Rationale for animal assignment (if not random): randomized

Positive control:

no

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. 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.

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 also weighed on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and during lactation on PND 1, 4, 7 and 13. Body weight gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 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 also measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

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: Yes
- 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. 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 CHEMISTRY: Yes
- 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. 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: Yes
- 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). 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 scheduled 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
- 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 in the study report.
- 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 fixed in an identified container containing 70% ethanol as they were not necropsied on the same day.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE:
- Males: following completion of the mating period (a minimum of 28 days of dose administration).
- Females that delivered: PND 14-16
- Females that failed to deliver: on post-coitum day 26 (females that failed to deliver, with evidence of mating)
- Females with total litter loss: within 24 hours of litter loss
- Pups: on PND 4 or PND 13-15
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. On PND 4 pups were killed by decapitation. All remaining pups (PND 7-15) were sacrificed using Euthasol 20% by intraperitoneal (ip) injection. 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.

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.
- 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 (M), 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: Yes
- 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, Epididymides, 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: Epididymides, 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 the selected 5 animals/sex of Groups 1 and 4
- Additional slides of the testes of the selected 5 males and all males that failed to sire.
- All gross lesions of all animals (all dose groups).
- The mammary gland of one female at 110 mg/kg with total litter loss.
- Thyroid gland and kidneys of all selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4 females.

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 ttest) 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 mated) 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, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period.
Incidental findings that were noted included wounds and scabs, alopecia and piloerection.
These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed and in absence of a dose-related incidence across the groups, these findings were considered not to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.
One female at 110 mg/kg was sacrificed on Day 1 of the lactation phase due to total litter loss.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were noted over the treatment period.
On Day 13 of the lactation phase, mean body weight was statistically significantly lower for females dosed at 1000 mg/kg. As this change was only minimal and within the range considered normal for rats of this age and strain, and body weight gain during lactation was similar to control means, this was considered not to be toxicologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Absolute food consumption and relative to body weight was similar between treated and control animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters were considered to be unaffected by treatment.
Any minor statistically significant differences in haematological parameters arising between controls and treated animals were considered not to be related to treatment with test item as they occurred in the absence of a (clear) treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters were considered to be unaffected by treatment.
Any statistically significant changes in clinical biochemistry parameters between controls and treated animals were considered not to be related to treatment as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. One control female showed a notably high bile acid level.
Other individual bile acid values of this control group remained in the range considered normal for rats of this age and strain.
Thyroid hormone analyses
Serum levels of T4 in F0 males were considered to be unaffected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

During the weekly arena observations, no additional treatment-related clinical signs were noted.
Functional observation parameters were considered not affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Mean grip strength and motor activity were similar across the treatment groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the thyroid glands and kidneys of the females of the 1000 mg/kg group.
An increased severity of follicular cell hypertrophy of the thyroid gland up to moderate degree was noted in a female of the 1000 mg/kg group. An increased incidence of mineralization of the kidneys (minimal) was noted in females of the 1000 mg/kg group. In general, this mineralization was focally present at the papilla and in half of the cases unilateral and the other half bilateral.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered to have been unaffected by treatment.
Most females had regular cycles of 4-5 days. An irregular cycle was noted for one control female and two females at 330 mg/kg, which all had normal litters. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic staging profiles were normal for all males examined.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

REPRODUCTION DATA
Mating index, precoital interval, number of implantation sites and fertility index were not affected by treatment. All females showed evidence of mating. One female at 110 mg/kg with a total litter loss had a single implantation site only. As this occurred in the absence of a dose response relationship, this was considered to be unrelated to treatment. One control female, one female at 110 mg/kg and one female at 1000 mg/kg were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was also considered to be unrelated to treatment.

DEVELOPMENTAL DATA
- 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 abortion or premature birth. No deficiencies in maternal care were observed.
Gestation index and duration: Gestation index and duration of gestation were considered to be unaffected by treatment.
Post-implantation survival index: The total number of offspring born compared to the total number of uterine implantations was considered to be unaffected by treatment. For one control female and another treated with 330 mg/kg group, the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation. No toxicological relevance was attached to this finding in the current study.
Litter size: Litter size was considered to be unaffected by treatment.
Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered to be unaffected by treatment.
One pup at 110 mg/kg, five pups at 330 mg/kg and five pups at 1000 mg/kg were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment. No pups were found dead/missing between lactation Days 5 and 13.

Parental results:
There were no adverse effects on parental parameters noted by treatment up to 1000 mg/kg.

Reproduction results:
Fertility, general reproduction parameters and developmental parameters showed no treatment-related changes up to 1000 mg/kg.
Test-item related microscopic findings were noted in the thyroid glands and kidneys of females dosed at 1000 mg/kg. These findings consisted of an increased severity of follicular cell hypertrophy of the thyroid gland (up to moderate degree) and a subtle increase in incidence of mineralization of the kidneys (minimal degree). Since there were no other related findings, and since these findings can incidentally be observed as a background finding in female rats of this age and strain, these subtle exacerbations in severity of follicular cell hypertrophy and of the mineralization of the kidneys were considered as non-adverse.

Analysis of Dose Preparations:
- Accuracy: 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%). No test item was detected in the Group 1 formulation.
- 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:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted in the examined parameters in the study

Remarks on result:

other: Microscopic findings in females treated with the test item at a dose rate of 1000 mg/kg bw/day (i.e., follicular cell hypertrophy in the thyroid and mineralization of the kidneys) were considered to be non-adverse.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted in the examined parameters in the study

Remarks on result:

other: No adverse effects were noted up to 1000 mg/kg

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered to be unaffected by treatment.
One pup of the control group, and one pup at 330 mg/kg were missing on Days 2 or 4, respectively. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered to be unaffected by treatment.
The statistically significantly higher mean pup body weights on Day 1 at 110 mg/kg were attributed to relatively lower litter sizes of a few litters at this dose group. Since mean pup body weights remained within the range considered normal for pups of this age and no doserelated trend was observed, these were considered to be unrelated to treatment.

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 considered to be unaffected by treatment.

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 macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio: Sex ratio was considered to be unaffected by treatment.
Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was considered to be unaffected by treatment.
Areola/nipple retention Treatment up to 1000 mg/kg had no effect on areola/nipple retention. For none of the examined male pups were nipples observed at PND 13.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

DEVELOPMENTAL RESULTS
- No developmental toxicity was observed up to 1000 mg/kg.
- No treatment-related changes were noted in any of the developmetal parameters investigated in this study (i.e., gestation, viability and lactation indices, gestation index and duration, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 for males), T4 thyroid hormone levels (PND 13-15) and macroscopy.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted in any of the parameters examined in this study

Remarks on result:

other: No developmental toxicity was observed up to 1000 mg/kg

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In conclusion, treatment with JNJ-40370226-AAA (T002487) by oral gavage in male and female Wistar Han rats at dose levels of 110, 330 and 1000 mg/kg revealed no adverse effects on parental parameters up to 1000 mg/kg.
Fertility, general reproduction parameters and developmental parameters showed no treatment-related changes up to 1000 mg/kg.
Test-item related microscopic findings were noted in the thyroid glands and kidneys of females dosed at 1000 mg/kg. These findings consisted of an increased severity of follicular cell hypertrophy of the thyroid gland (up to moderate degree) and a subtle increase in incidence of mineralization of the kidneys (minimal degree). Since there were no other related findings, and since these findings can incidentally be observed as a background finding in female rats of this age and strain, these subtle exacerbations in severity of follicular cell hypertrophy and of the mineralization of the kidneys were considered as non-adverse.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 1000 mg/kg
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 mg/kg
Therefore, the substance is not classified as a reproductive toxicant according to the CLP Regulation.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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), 110, 330 and 1000 mg/kg bw/day via gavage (OECD 422; Otterdijk, 2017). The vehicle used was 1% CMC (carboxymethyl cellulose) and the test solutions were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.

No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted included wounds and scabs, alopecia and piloerection. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed and in absence of a dose-related incidence across the groups, these findings were considered not to be related to treatment.

No toxicologically relevant changes in body weights and body weight gain were noted over the treatment period. On Day 13 of the lactation phase, mean body weight was statistically significantly lower for females dosed at 1000 mg/kg. As this change was only minimal and within the range considered normal for rats of this age and strain, and body weight gain during lactation was similar to control means, this was considered not to be toxicologically relevant.

Test item-related microscopic findings were noted in the thyroid glands and kidneys of the females of the 1000 mg/kg group. An increased severity of follicular cell hypertrophy of the thyroid gland up to moderate degree was noted in a female of the 1000 mg/kg group. An increased incidence of mineralization of the kidneys (minimal) was noted in females of the 1000 mg/kg group. In general, this mineralization was focally present at the papilla and in half of the cases unilateral and the other half bilateral.

Length and regularity of the estrous cycle were considered to have been unaffected by treatment. Most females had regular cycles of 4-5 days. An irregular cycle was noted for one control female and two females at 330 mg/kg, which all had normal litters. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Mating index, precoital interval, number of implantation sites and fertility index were not affected by treatment. All females showed evidence of mating. One female at 110 mg/kg with a total litter loss had a single implantation site only. As this occurred in the absence of a dose response relationship, this was considered to be unrelated to treatment. One control female, one female at 110 mg/kg and one female at 1000 mg/kg were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was also considered to be unrelated to treatment.

Based on the above-mentioned considerations, the Parental NOAEL was established as at least 1000 mg/kg and the Reproduction NOAEL as at least 1000 mg/kg.

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; Otterdijk, 2017), the test item was administered daily to rats at dose levels up to 1000 mg/kg body weight/day. The developmental NOAEL was established as being at least 1000 mg/kg body weight/day.

The test substance is considered not classified as a reproductive toxicant. No further testing for fertility and developmental toxicity will be performed, in accordance with the REACH Regulation (Annex IX, section 8.7).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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 study, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: at least 1000 mg/kg

Reproduction NOAEL: at least 1000 mg/kg

Developmental NOAEL: at least 1000 mg/kg

Therefore, the substance is not classified as a reproductive toxicant according to the CLP Regulation.