4-nitrobenzenesulphonyl chloride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-10 to 2018-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

July 2000

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in rodents)

Version / remarks:

October 2008

Deviations:

no

Test guideline 6

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.7 (Repeated dose (28 Days) toxicity (Oral))

Version / remarks:

May 2008

Deviations:

no

Test guideline 7

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

July 2000

Deviations:

no

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

Test material

Test material information

Constituent 1

Reference substance name:

4-nitrobenzenesulphonyl chloride

EC Number:

202-697-9

EC Name:

4-nitrobenzenesulphonyl chloride

Cas Number:

98-74-8

Molecular formula:

C6H4ClNO4S

IUPAC Name:

4-nitrobenzenesulphonyl chloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-39722280-AAA (4-Nitrobenzene-1-sulfonyl chloride)
- Physical state: solid (powder)
- Appearance: Slightly yellow to yellowish brown crystalline powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16LB4523
- Expiration date of the batch: 2018-04-18
- Purity : 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability in vehicle: Stability of formulations bracketing the concentration range used in this study were determined as part of the analytical method development and validation study (Test Facility St udy No. 520060)
- Stability under test conditions: Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 520060)

FORM AS APPLIED IN THE TEST : yellow suspension (groups 2-4)

OTHER SPECIFICS: Correction factor: 1.00 based on purity

Test animals

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 nulliparous and non-pregnant: yes
- Age at study initiation: Males approx. 10 weeks (at start F0-treatment); Females 11weeks (at start pretest) and approx. 13 weeks (at start F0-treatment) - Weight at study initiation: 253-303 g (males); 203-244 g (females)
- Fasting period before study: no
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, heig ht 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV t ype, height 18 cm).
Mating: Males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII 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. During motor activity measurements, animals did not have access to food for a maximum of 2 hours. - Water (e.g. ad libitum): Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutr ients was performed according to facility standard procedures. There were no findings that could in terfere with the study.

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. The light/dark cycle was interrupted for study related activities.

IN-LIFE DATES: From: 2017-10-10 To: 2018-01-24

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 3 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. 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 and on results of the analytical Method Development and Validation performed at the test facility.
- Concentration in vehicle: 0 mg/mL (Group1), 10 mg/mL (Group2), 30 mg/mL (Group3), 20 mg/mL (Group4)
- Amount of vehicle (if gavage): 5 mL/kg (Group1), 1 mL/kg (Group2), 1 mL/kg (Group3), 5 mL/kg (Group4)
- 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, the animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating.
- 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 (2017-11-23, Day 1 of treatment) according to a validated method (Test Facility Study No. 520060). 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 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 va riation was ≤ 10.00%. Stability of formulations bracketing the concentration range used in this study were determined as part of the analytical method development and validation study (Test Facility No. 520060). Formulations were considered stable if the relative difference before and after storage was maximally 10.00%.

Duration of treatment / exposure:

Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were treated for 54-63 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were treated for 38-41 days.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control, Group 1

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 animals/sex/dose level

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. 520059). During the DRF phase, oral (gavage) administration of the test substance to female Wistar rats was given for 10 days at 500 or 200 mg/kg bw/day. At 500 mg/kg bw/ day, all 3 animals were sacrificed in extremis on Day 3. All animals had shown flat/hunched posture, uncoordinated movements, rales, pallor, piloerection, and hypothermia on Day 3 (rales also observed for one animals on day 2). No abnormalities were noted at macroscopic examination of the 500 mg/ kg bw/day group. At 200 mg/kg bw/day, no mortality was observed. All animals showed occasion al hunched posture, rales and/or piloerection on 2 or 3 days across the treatment period. These clinical signs were not persistently noted with continuing treatment. No effects were observed in body weight or food consumption. At macroscopic examination, irregular surface of the stomach was noted for one female, but no abnormalities in the other 2 animals. A higher liver weight, exceeding the range considered normal for rats of this strain and similar age was observed. Kidney weights were considered to be normal. Based on the results of the range finding study, dose levels for the main study were 10, 30 and 100 mg/kg bw/day.
- Rationale for animal assignment (if not random): randomized

Positive control:

No

Examinations

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, at least after dosing at no specific time point, but within a similar time period after dosing for the respective animals. Once prior to start of tr eatment 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.

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 and 13. Body weight gain was calculated and reported.

FOOD CONSUMPTION
- Time schedule for examinations: 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 m easured 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.

WATER CONSUMPTION: No
- Time schedule for examinations: Subjective appraisal was maintained during the study. For the purp ose of collecting historical control data, water consumption was measured daily in control animals only from 01 December 2017 onwards during the entire treatment period. water consumption was measur ed for males and females which were housed together for the mating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. Blood samples were drawn from the retro-orbital sin us and collected into tubes prepared with K3-EDTA for haematological parameters, with citrate for clotting tests and tubes treated with Li-heparin for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Animals were not fasted overnight
- How many animals: 5 animals/sex/group
- Parameters examined : white blood cells, differential leukocyte counts (Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corp uscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood : Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. Blood samples were drawn from the retro-orbital sin us and collected into tubes prepared with K3-EDTA for haematological parameters, with citrate for clotting tests and tubes treated with Li-heparin for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- Animals fasted: Animals were not fasted overnight
- How many animals: 5 animals/sex/group
- Parameters examined: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), Gamma glutamyl transpeptidase (GGT), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, i norganic phosphate

OTHER:
FUNCTIONAL OBSERVATIONS
- functional observations tests were performed on each individual animal of the selected 5 animals/sex/group.
- The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs - Parameters: hearing ability, pupillary reflex, static righting reflex, fore and hind-limb grip strength recorded as mean of three measurements per animal, locomotor activity.

THYROID HORMONE ANALYSIS
-Time schedule: End of study
- How many animals: all animals at planned necropsy (including: females on Day 14-16 of lactation, all females that failed to deliver healthy pups and all males after at least 4 weeks of treatment)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males: Yes, overnight with a maximum of 24 hours before blood sampling but water was available) Males sacrified in extremis: No Females: No
- Blood samples (0.9 mL) were drawn from the retro-orbital sinus After clotting and centrifugation, serum was used as: Males: 1 aliquot of 150 µL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroidstimulating hormone (TSH).
Females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroidstimulating hormone (TSH). Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months.

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 of the selected 5 males and all males that failed to sire for detailed qualitative examination, taking into account the tubular stages of the spermatogenic cycle. The weight of the testes was recorded.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On PND 1, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Mortality/viability: The numbers of live and dead pups were determined on PND1 and daily there after. If possible, defects or cause of death were evaluated. Animals showing pain, distress or dis comfort, which was considered not transient in nature or was likely to become more severe, were sac rificed for humane reasons.
- 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 tables 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 4.
- 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:
- Descriptions of all abnormalities were recorded. If possible, defects or cause of death were evaluated.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE:
Animals were not fasted overnight. All animals surviving to the end of the observation period and all moribund animals were deeply a naesthetized using isoflurane and subsequently exsanguinated.
Necropsy was conducted on the following days:
- Males: Following completion of the mating period (a minimum of 28 days of dose administration).
- Females which delivered: PND 14-16.
- Females which failed to deliver: Post-coitum Days 25 (females with evidence of mating) or a pproximately 26 days after the last day of the mating period (females without evidence of mating).
- Female with total litter loss: Within 24 hours of litter loss.
- Euthanized in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

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.
- 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), C ervix (F), Colon (M/F), Coagulation gland (M), Duodenum (M/F), Epididymides 3 (M), Eyes (with optic nerve (if detectable) and Harderian gland)4 (M/F), Mammary gland area, inguinal region with skin ( M/F), Femur with bone marrow, including joint (M/F), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidne ys (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), 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, mid-thoracic, 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 was not examined by the pathologist, since no signs of toxicity were noted at mac roscopic examination.

- All remaining animals, males that failed to sire, females which failed to deliver and females with total litter loss : Cervix (F), Coagulation gland (M), Epididymides4 (M), Mammary gland area, inguinal region with skin (M/F), Ovaries (F), Prostate gland (M), Seminal vesicles (M), Testes 6 (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F), Identification marks: not processed (M/F).

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 for detailed qualitative examination, taking into account the tubular stages of the spermatogenic cycle,
- The preserved organs and tissues of the animals of all dose groups which were euthanized in ex tremis,
- The mammary gland of the female with total litter loss,
- All gross lesions of all animals (all dose groups),
- Stomach of all selected 5 animals of Groups 2 and 3 (males and females), based on (possible) treatment-related changes in this organ in Group 4,
- Nasal cavity level 3, 4 and caudal to 4 Larynx of intercurrently sacrificed male and female, based on (possible) treatment-related changes in these organs,
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
- A peer review on the histopathology data was performed by a second pathologist

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios are 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, Liver, Ovaries Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyr oid including parathyroid if detectable, Uterus including cervix.
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Epididymides, Prostate, Seminal vesicles, including coagulation glands, Testes, Thyroid, including parathyroid if detectable.

Postmortem examinations (offspring):

SACRIFICE
- Pups, younger than 7 days were euthanized by decapitation.
- All remaining pups (PND 7-16) were sacrificed using Euthasol® 20% by intraperitoneal (ip) injection.

GROSS NECROPSY
- All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
- At terminal sacrifice (PND 14-16), 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 analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-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 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
Precoital time : Number of days between initiation of cohabitation and confirmation of mating.
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
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.
Percentage live males at First Litter Check (%) = (Number of males at first litter check/ Number of live pups at First Litter Check ) x 100
Percentage live females at First Litter Check (%) =(Number of females at first litter check/ Number of live pups at First Litter Check ) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- Animals surviving until scheduled necropsy: Rales were observed in males at 30 and 100 mg/kg and in females across all groups (including the control group). This finding generally occurred in a few animals and on a few days of treatment only. Among these high dose animals, one female (no. 78) presented with gasping on a single day and one male (no. 38) showed laboured respiration on 5 consecutive days.
- Salivation seen among all animals across all groups (including the control group) was considered to be a physiological response rather than a sign of systemic toxicity considering the occurrence of th is finding in the control groups at similar frequency to test item treated groups, the nature and minor severity of the effect and its time of occurrence (after dosing).
- Any other clinical signs noted during the treatment period occurred within the range of backgroun d findings to be expected for rats of this age and strain which are housed and treated under the co nditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment. - No findings were noted during the weekly arena observations in this study

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- No mortality occurred during the study that was considered to represent a systemic test item effect.
- A total of two females at 100 mg/kg (nos. 72 and 80) and two males at 30 mg/kg (nos. 21 and 26) were sacrificed in extremis between Days 16 and 38 of the study period. Respiratory distress was the main clinical sign for these 4 animals resulting in early sacrifice.
- Female no. 72 (100 mg/kg) was sacrificed in extremis on Day 38 (Day 21 post-coitum). This animal presented with respiratory distress (squeaking and gasping), piloerection, pale skin, and hypothermia. Necropsy showed gastrointestinal tract distended with gas. Microscopic exami nation showed marked erosion/ulceration of the trachea and moderate erosion/ulceration of the larynx and nasopharynx together with a marked obstructive granulocytic inflammation of the larynx. Other microscopic findings, such as lymphoid depletion in the spleen and decreased cellularity and incre ased adipocytes in the bone marrow, were considered to be related to the poor condition of the animal. The inflammatory and degenerative lesions in the respiratory tract were considered to be the cause of moribundity for this animal.
- Female no. 80 (100 mg/kg) was sacrificed in extremis on Day 16. This animal showed respiratory di stress (laboured respiration and gasping), squeaking, hunched posture and weight loss of 6% over the last week of premating. Necropsy showed cecum distended with gas, accentuated lobular pa ttern of the liver and enlarged adrenal glands. Microscopic examination revealed massive erosion/ul ceration of the trachea and nasopharynx, and moderate erosion/ulceration and marked obstructive granulocytic inflammation of the larynx. The nasal cavity was noted with erosion/necrosis of the respiratory epithelium and a marked degree of luminal exudate consisting of mucous with granulocytic inf lammatory cells. The inflammatory and degenerative lesions in the respiratory tract were considered to be the cause of moribundity for this animal.
- Male no. 21 (Group 3) was sacrificed in extremis on Day 18. This animal presented with breathing problems (laboured respiration and rales), hunched posture, piloerection, ptosis and a 15% weight loss over the treatment period. Necropsy showed cecum distended with gas, and reduced size of the spleen and thymus. Microscopic examination showed slight lymphogranulocytic inflammation of the forestomach and glandular stomach and slight erosion/ulceration of the forestomach. Other microscopic findings considered to be related to the poor condition of the animal were lymphoid depletion in the thymus and mesenteric lymph nodes together with a decreased cellularity and increased adipocytes in the bone marrow. Although histopathologically no clear cause of moribundity could be established, the distended intestinal tract and inflammatory lesions in the stomach were considered to have contributed to the moribund state of this animal.
- Male no. 26 (Group 3) was sacrificed in extremis on Day 29. This animal showed respiratory d istress (rales and gasping), squeaking and a 20% weight loss over Days 8-15 of the mating period. Necropsy revealed gastrointestinal tract distended with gas, non-collapsed lungs and reduced size of the spleen. Main findings at microscopic examination consisted of slight erosion/ulceration of the larynx. Other microscopic findings considered to be related to the poor condition of the animal were lymphoid depletion in the thymus and decreased cellularity and increased adipocytes in the bone marrow. No clear cause of moribundity could be established from the sections examined, but the distended intestinal tract and the slight erosion/ulceration of the larynx were regarded to have contributed to the moribund state of this animal.
- Respiratory tract lesions recorded for these four animals (nos. 21, 26, 72 and 80) were indicative of a dosing procedure-related occurrence and/or gavage-related reflux.
- One control female (no. 44) was sacrificed on Day 2 of lactation due to total litter loss. One female at 100 mg/kg (no. 74) was found dead on Day 3 (this animal was replaced by a reserve female). No relevant clinical signs were noted for female no. 74 prior to death.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- At 100 mg/kg, mean absolute body weight and body weight gain of females was slightly lower than controls from Day 17 post-coitum onwards (not statistically significant).
- Mean body weight and body weight gain during lactation was not considered to be affected by treatment. The statistically significantly higher mean body weight gain of females at 30 mg/kg on Day 4 of lactation was not considered to be related to treatment as this occurred in the absence of a doserelated trend and was attributed to a higher weight gain of one female (no. 66). One female at 30 mg/kg (no. 69) showed weight loss of 16% over Days 4-7 of lactation (accompanied by a lower food intake over Days 4-7 of lactation). These individual variations in body weight development were not considered to be related to treatment as they occurred in the absence of a dose-related trend.
- Body weights and body weight gain of other treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was not considered affected by treatment. Any statistically significant changes in food consumption were considered to be unrelated to treatment since no dose-related trend was apparent.

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 of treated rats were not considered to be affected by treatment. Any statistically significant changes in haematology parameters were not considered to be related to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Clinical biochemistry parameters of treated animals were not considered to be affected by treatment. Any statistically significant changes in clinical biochemistry parameters were not considered to be related to treatment as they occurred in the absence of a treatment-related distribution.
- Thyroid hormone analyses: Serum levels of T4 in F0 males were not considered to be affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- At 30 and 100 mg/kg, mean forelimb grip strength of selected males was 14 and 20% lower than control values, respectively (statistically significant at 100 mg/kg). Means remained within the range of the historical control data, and hindlimb grip strength of males remained similar to control levels.
- Grip strength in females was considered unaffected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals.
- The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with higher activity in the first interval that decreased over the duration of the test period. The habituation profile of males deviated from that normally encountered, and total movements of all groups (including the control group) were lower than expected for rats of this age and strain. This was attributed to conducted procedures, which did not adversely affect evaluation of the study results.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with the test substance for at least 28-days were noted in the stomach of males. There were no test item-related microscopic observations in fem ale rats after the scheduled study period. In male rats at 100 mg/kg a combination of microscopic findings was recorded for the forestomach, which were considered to be related to the treatment with the test item. These findings included min imal to slight inflammation, slight squamous hyperplasia, slight vacuolation and moderate hyperkera tosis. The inflammation of the glandular stomach of male rats did not show a dose-related trend and was co nsidered to be within background pathology for rats of this age and strain.

Findings of note in the surviving animals were recorded for male no. 32 (100 mg/kg) in the trachea (marked erosion/ulceration) and lung (slight acute alveolar inflammation) and for female no. 41 (control) in the lung (marked ulceration of the bronchi and lipophilic alveolar contents in one lobe, moderate acute bronchioalveolar inflammation and slight alveolar macrophage aggregation). These findings in the respiratory tract in one high dose male and one control female were considered to be gavage procedure related and not a test item effect.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered to be not affected by treatment. Most females had regular cycles of 4-5 days. Extended di-estrus occurred in 5 females: 4 with regular cycle (nos. 54 and 56 at 10 mg/kg and nos. 61 and 68 at 30 mg/kg) and 1 with irregular cycle (no. 74 at 100 mg/kg, with normal litter). Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, the occurrence of extended di-estrus and irregular estrous cycle did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

REPRODUCTIVE DATA:
- Performance:
There was 1 out of 10 females of the control group with total litter loss (couple 44/4) and there were 1 out of 10 females at 10 mg/kg (couple 54/14), 1 out of 10 females at 30 mg/kg (couple 61/21) and 3 out of 10 females at 100 mg/kg (couple 73/33, 77/37 and 80/40) that did not deliver offspring. Male no. 21 (paired with female no. 61) was euthanized moribund before mating on Day 4 of the mating period and female 80 (paired with male no. 40) was euthanized moribund before mating on Day 2 of the mating period. In addition, 1 out of 10 females at 100 mg/kg (couple 72/32) was euthanized moribund in pregnant state on postcoitum day 21.
No abnormalities were seen in the reproductive organs of these couples, which could account for their lack of healthy offspring.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Mating and fertility indices:
- Mating index was not considered to be affected by treatment. The mating indices were 100% for the control group and 90% for the other groups. A total of 3 females (nos. 54, 61 and 80) did not show evidence of mating (one each at 10, 30 and 100 mg/kg). One high dose female (no. 80) was sacrificed before evidence of mating occurred, and for female no. 61, the paired male was sacrificed before mating occurred.
- Fertility index: At 100 mg/kg, fertility index was lower than controls (78% vs. 100% in the control group), and was slightly lower than the range expected for rats of this age and strain8. Two mated females at 100 mg/kg (nos. 73 and 77) were non-pregnant. At 10 and 30 mg/kg, fertility indices (100%) were not affected by treatment.

Precoital time and implantation sites:
- Precoital time was not considered affected by treatment. The higher mean precoital time for females at 100 mg/kg (3.6 days vs. 2.4 days in the control, and 2.8 and 2.6 days at 10 and 30 mg/kg, respectively) was due to one female (no. 74) which was mated after 14 days. Since all other mated females at this dose showed evidence of mating within 4 days, this was not considered to be related to treatment.
- At 100 mg/kg, the mean number of implantation sites was lower than controls (10.6 vs 13.2 in control females). The mean remained within the range expected for rats of this age and strain. At 10 and 30 mg/kg, the number of implantation sites was not considered affected by treatment.


DEVELOPMENTAL DATA:
- Gestation index and duration: Gestation index and duration of gestation were not considered affected by treatment. At 100 mg/kg, gestation index was slightly lower than controls (86%, vs 100% in the other groups). This was attributed to female no. 72 that was sacrificed in extremis in a pregnant state on post-coitum Day 21. Necropsy revealed a total of 13 dead fetuses (most likely they died due to the euthanasia procedure of the dam). This dam showed inflammatory and degenerative lesions in the respiratory tract indicative of a dosing procedure-related occurrence and/or gavage-related reflux which were considered to be the cause of moribundity for this animal. Given that none of the other females that survived until scheduled necropsy at this dose showed fetal deaths, it was considered that fetal mortality for this dam had occurred secondarily to the dam’s moribund state. As such, the lower gestation index at 100 mg/kg should not be considered a primary effect of the test item.

- Parturation/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.

- Post-implantation survival index: Post-implantation survival index was not considered affected 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 not affected by treatment. The live birth indices were 99
or 100% for all groups. One pup of the 10 mg/kg group (from dam no. 60) was found dead on PND 1. No toxicological relevance was attributed to this dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

- viability index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. The viability indices were 87, 97, 100 and
96% for the control, 10, 30 and 100 mg/kg group, respectively. One control female (no. 44) had total litter loss on PND 2 (litter consisted of 15 pups), resulting in a statistically significantly lower postnatal loss at 10 and 30 mg/kg. One pup of the control group (dam no. 50), 3 pups at 10 mg/kg (dam nos. 51 and 60) and 2 pups at 100 mg/kg (dam nos. 76 and 78) were found dead or missing on PND 3-4.
Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/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.
- 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 considered to be not affected by treatment. The lactation indices were 98% at 30 mg/kg and 100% for the other groups. One pup at 30 mg/kg (dam no. 68) had to be sacrificed in extremis between lactation Days 5 and 13 (PND 8). At last litter check and necropsy, the pup presented with swollen and hyperextended right hindleg. No toxicological relevance was attributed to this sacrificed pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Details on results (P0)

Analysis of dose preparations:
- Accuracy of preparation: The concentrations analysed in the formulations of Groups 2, 3 and 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 3 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Parental results:
No adverse parental effects were observed up to the highest dose level tested (100 mg/kg). A total of four animals (two females at 100 mg/kg and two males at 30 mg/kg) were
sacrificed in extremis between Days 16 and 38 due to respiratory distress. These premature decedents were considered to be not due to a direct and systemic effect of the test item.
Instead, microscopic examination of these animals indicated that a dosing procedure-related occurrence and/or gavage-related reflux were the underlying cause as evidenced by
degenerative and inflammatory alterations in the trachea, larynx, nasopharynx and/or nasal cavity. For one male at 30 mg/kg, no clear cause of moribundity could be established histopathologically, but this animal showed clinical signs of respiratory distress comparable to the other premature decedents. In addition to the gavage and/or reflux related findings in the premature decedents, a few surviving animals showed findings in the respiratory system, which were also regarded gavage and/or reflux-related: Erosion/ulceration in the trachea and acute alveolar inflammation in the lung (one male at 100 mg//kg/day) and ulceration of the bronchi, lipophilic alveolar contents, acute bronchioalveolar inflammation and alveolar macrophage aggregation in the lung (one control female). Overall, these effects in the respiratory system were indicative of irritating properties of the test item. Respiratory symptoms were also noted among surviving animals across dose groups (primarily rales, and more occasionally (and at 100 mg/kg only) gasping and laboured respiration). These may also be attributed to the gavage-procedure/reflux. Also, given their low frequency of occurrence they were considered not adverse. At 30 and 100 mg/kg, a non-adverse lower foreleg grip strength was recorded for males (14 and 20% lower than control values, respectively). Since means remained within the range of the historical control data, and no further changes were noted that indicated a deficit in neurobehaviour (including normal hindlimb grip strength of males), this was considered to be not an adverse finding.
At 100 mg/kg, lower body weight and body weight gain was recorded for females from Day 17 post-coitum onwards, which was considered to be reflective of the lower litter size
observed at this dose. As this was considered to be a secondary finding, and given the slight magnitude of this change, this was considered to be not an adverse finding. Test-item related, but non-adverse, forestomach findings were recorded in males at 100 mg/kg, which consisted of minimal to slight inflammation, slight squamous hyperplasia, slight vacuolation and moderate hyperkeratosis. These findings were considered to be local test item-related effects related to the irritating properties of the test item.
Given the generally slight degree of these findings and absence of eg. changes in food consumption this was considered not to represent an adverse effect of the test item.
No treatment-related changes were noted in any of the other parental parameters investigated in this study (i.e. clinical biochemistry investigations including male T4 thyroid hormone
levels, macroscopic examination and organ weights).

Reproductive results:
At 100 mg/kg, a lower number of implantation sites (10.6 vs 13.2 in control females) and lower fertility index (78% vs. 100% in the control group) were recorded. No reproduction toxicity was observed at 10 and 30 mg/kg. No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating index, precoital time, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

other: not specified

Organ:

not specified

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

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 clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Litter size: At 100 mg/kg, a statistically significantly lower mean number of living pups was recorded. This was related to the lower number of implantation sites in this group. At 10 and 30 mg/kg, the mean number of living pups was similar to the control mean.

Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. The live birth indices were 99 or 100% for all groups. One pup of the 10 mg/kg group was found dead on PND 1. No toxicological relevance was attributed to this dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. The viability indices were 87, 97, 100 and 96% for the control, 10, 30 and 100 mg/kg group, respectively.
One control female (no. 44) had total litter loss on PND 2 (litter consisted of 15 pups), resulting in a statistically significantly lower postnatal loss at 10 and 30 mg/kg One pup of the control group (dam no. 50), 3 pups at 10 mg/kg (dam nos. 51 and 60) and 2 pups at 100 mg/kg (dam nos. 76 and 78) were found dead or missing on PND 3-4.
Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/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.

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 considered to be affected by treatment. The lactation indices were 98% at 30 mg/kg and 100% for the other groups. One pup at 30 mg/kg (dam no. 68) had to be sacrificed in extremis between lactation Days 5 and 13 (PND 8). At last litter check and necropsy, the pup presented with swollen and hyperextended right hindleg. No toxicological relevance was attributed to this sacrificed pup 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 not considered to be affected by treatment. Pup weight gain at 100 mg/kg was marginally higher than controls on Days 1 and 4 of lactation (not statistically significant), which was attributed to the lower litter size at this dose. Given the direction of change and secondary occurrence to lower litter size, this finding was not considered to be of toxicological significance.

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 14-16 pups were 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:

effects observed, non-treatment-related

Description (incidence and severity):

ANOGENITAL DISTANCE
Anogenital distance (absolute and normalized for body weight) was not considered to be affected by treatment. The statistically significantly lower mean normalized anogenital distance of female pups at 100 mg/kg was not considered to be an effect of treatment, since the opposite effect would be expected in case of anti-estrogenic/pro-androgenic activity of the test item, and since the change was slight in magnitude. Anogenital distance (absolute and normalized for body weight) in male pups was not considered to be affected by treatment.

AREOLA/NIPPLE RETENTION
Treatment up to 100 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

MACROSCOPY
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.

SEX RATIO
Sex ratio was not considered to be affected by treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Developmental results:
At 100 mg/kg, a lower mean number of living pups (8.8 vs 11.9 in the control group) was recorded, which was related to the lower number of implantation sites in this group. The statistically significantly lower mean normalized anogenital distance of female pups at 100 mg/kg was considered to be not an effect of treatment, since the opposite effect would be expected in case of anti-estrogenic/pro-androgenic activity of the test item, and since the change was slight in magnitude. Anogenital distance (absolute and normalized for body weight) in male pups was considered to be not affected by treatment. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, 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 males), T4 thyroid hormone levels (PND 14-16) and macroscopy.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Target system / organ toxicity (F1)

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

other: not specified

Organ:

not specified

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

JNJ-39722280-AAA was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg. Formulation analysis showed that the formulations were prepared accurately and homogenously. Based on the results, the parental No Observed Adverse Effect Level (NOAEL) was derived to be at least 100 mg/kg, the reproduction NOAEL at 30 mg/kg (based on lower number of implantation sites and lower fertility index at 100 mg/kg), and the Developmental NOAEL at 30 mg/kg (based on lower mean number of living pups at 100 mg/kg).