Fluorobenzene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-14 to 2017-03-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developm ental Toxicity Screening Test

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 6

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.

Test material

Test material information

Constituent 1

Reference substance name:

Fluorobenzene

EC Number:

207-321-7

EC Name:

Fluorobenzene

Cas Number:

462-06-6

Molecular formula:

C6H5F

IUPAC Name:

fluorobenzene

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15FB2364
- Expiration date of the lot/batch: 2017-08-12
- Purity test date: Date not specified on the Certificate of Analysis

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (between +15 and +25 °C).


OTHER SPECIFICS: Correction factor was 1

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 Lyon 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 Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males approx. 10 weeks (at start F0-treatment); Females approx. 10 weeks (at start pretest) and approx. 12 weeks (at start F0-treatment).
- Weight at study initiation: 281-330 g (males) and 185-246 g (females)
- Fasting period before study: No
- Housing:
- Accommodation: Group housed males and females and individual housed females, including females during mating and with litters, were housed in plastic cages meeting European directive 2010/63/EU requirements.
- From arrival to randomisation: Females were housed in groups of 8 to 9 per cage. Males were housed in groups of 7 to 8 per cage.
- From randomisation and during the pre-mating period: Animals were housed in groups of 5/sex/cage.
- Mating: Males and females were cohabitated on a 1:1 basis.
- Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed.
- Diet (e.g. ad libitum): Free access to pelleted complete rodent diet.
- Water (e.g. ad libitum): Free access to softened and filtered (0.2 μm) mains drinking water.
- Acclimation period: 7 days prior to start of pretest (females) or treatment (males).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): >35 %
- 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: 2017-01-03 (start pretest, females); 2017-01-17 (start treatment, males); 2017-02-23 through 2017-03-09 (delivery of litters)
To: 2017-02-17 (necropsy males); 2017-03-09 through 2017-03-23 (necropsy females and necropsy pups

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/v) were prepared daily within 6 hours prior to dosing (based on stability study no. AB21506) and were homogenized to a visually acceptable level. A correction was made for the purity/composition of the test item. A correction factor of 1.0 was used. No adjustment was made for specific gravity/density of the test item, vehicle and/or formulation.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Lyon.
- Concentration in vehicle: 0 mg/mL (group 1); 3 mg/mL (group 2); 10 mg/mL (group 3); 40 mg/ mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight/day. Actual dose volumes were calculated according to the latest body weight.

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, avoiding sibling mating, 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 plastic cages meeting European directive 2010/63/EU requirements.
- 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 on formulations used on the first day of treatment during the main phase (17 January 2017), according to a validated method (Test Facility Study No. AB21506). One set of duplicate samples (2 x 1 g) was collected. Samples of formulations were analysed for homogeneity (highest and lowest concentrations) and accuracy of preparation (all concentrations). In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10.00 % compared to those obtained during the method validation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤10.00 %.

Duration of treatment / exposure:

31 days (males); 51-65 days (females that delivered); 42 (females which failed to deliver).

Pups were not dosed directly but were potentially 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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control group)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

200 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: During the dose-range finding (DRF) phase, oral (gavage) administration of JNJ-7820254-AAA (Fluorobenzene) to the female Han Wistar rat for 3 days at doses of 500 and 1000 mg/kg/day was associated with a marked reduction in food intake, significant body weight loss, marked clinical signs and the sacrifice of one female due to its poor clinical condition. The dose levels were therefore decreased up to 100 and 200 mg/kg/day and were administrated for up to 10 days. These doses were associated with slightly reduced food consumption, intermittent clinical signs such as whole body twitching, hunched posture, hypersalivation and persistant rapid breathing. Therefore, a high dose of up to 200 mg/kg/day could be employed in the subsequent reproduction/developmental toxicity screening test study in the rat.
- 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 (i.e. at the beginning and at the end of the working day, including weekends and public holidays).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made for all animals before and at least once after dosing between 1 and 3 hours, except for some occasions, from start of treatment onwards up to the day prior to necropsy.


BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed once pretest, 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.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/ kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Relative food consumption was calculated and reported.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
- 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 F0 animals/sex/group under anaesthesia using isoflurane. The animals were deprived of food overnight (less than 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with K2-EDTA for hematology parameters, and with citrate for clotting tests.
- Parameters assessed: white blood cells, differential white blood counts, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial 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 F0 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 taken from the abdominal aorta as part of the necropsy procedure in tubes without anticoagulant for clinical biochemistry parameters (1.5 mL). An additional blood sample was collected into serum tubes for determination of bile acids.
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate
- Thyroid hormone analysis


FUNCTIONAL OBSERVATIONS
- Time schedule: The selected males were tested once during Week 5 of treatment and the selected females were tested once during the last week of lactation (i.e. PND 13). These tests were performed before observation for clinical signs.
- Parameters: The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group: hearing ability, pupillary reflex and static righting reflex; fore- and hind-limb grip strength, recorded as the mean of three measurements; locomotor activity in an open field test.

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. During pretest, this was done for 48 females. On the day of necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generation: additional slides of the testes to examine staging of spermatogenesis; testis weight

Litter observations:

STANDARDISATION OF LITTERS
- The pups were not identified individually. Maximum of 8 pups/litter (4/sex/litter as nearly as possible) were selected for culling on PND4; 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 PND1 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 each weighing day. Sex ratio (% male pups on PND 1) was calculated per group
- 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.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE:
Necropsy was conducted according to the following schedule:
- Males: following completion of the mating period (after 31 days of dose administration).
- Females which delivered: on PND 14.
- Female which failed to deliver: on post-coitum Day 26 (female without evidence of mating).
- Pups: on PND 4 or PND 13.
The 5 selected males and females for clinical pathology blood sampling were deeply anaesthetized using isoflurane and subsequently exsanguinated. Other adult animals surviving to the end of
the observation period and all non-pregnant females were killed by carbon dioxide inhalation and exsanguination then necropsied. On PND 4 and PND 13, pups were sacrificed by intraperitoneal injection of sodium pentobarbitone. Pups that died were necropsied and their stomach examined for the presence of milk, if possible. Defects or cause of death were evaluated, if possible.

GROSS NECROPSY
- 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: 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), (Glanspenis) (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, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin:
Cervix (F), Clitoral gland (F), Coagulation gland (M), Epididymides (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)

HISTOPATHOLOGY
- 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 of Groups 1 and 4 and all males that failed to sire to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina).
- All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
- A pathology peer review was conducted by an appropriately experienced pathologist nominated by Charles River Lyon.

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported.
- The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/ group on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, 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 (including parathyroid if detectable), Prostate, Seminal vesicles including coagulating glands.

Postmortem examinations (offspring):

SACRIFICE
- On PND 4 and PND 13, pups were sacrificed by intraperitoneal injection of sodium pentobarbitone. Pups that died were necropsied and their stomach examined for the presence of milk, if possible. Defects or cause of death were evaluated, if possible.

GROSS NECROPSY
- All pups were sexed by both external as well as internal examination. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external - reproductive genitals to examine signs of altered development.
- At terminal sacrifice (PND 13), the thyroid from 1 male and 1 female pup 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. Defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGHTS
not examined

Statistics:

Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
- The best transformation for the data (none, log or rank) was determined depending upon the kurtosis of the data, the probability of the Bartlett's test for homogeneity of the variances and an assessment of whether the size of the groups were approximately equal or not.
- Non- or log-transformed data were analysed by parametric methods.
- Rank transformed data were analysed using non-parametric methods.
- Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for nonparametric data.
- Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
- If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
- Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
- Functional test (locomotor activity in an open field test), oestrous cycle and pre-coital interval and anogenital distance data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test.
- Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hypersalivation associated or not with abnormal foraging and/or pedaling was observed with a dose-related incidence in all treated male and female groups during the study period from Day 14. Occasionnal hypersalivation was also observed in control groups, associated on occasions with abnormal foraging from Day 21 (males) or from day 9 of gestation (females). In all female groups, the occurrence of these signs tended to decrease during the lactation period. This effect was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test item and/or vehicle.

Incidental findings that were noted included chromodacryorrhea, scabs, limping or localized hairloss. 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, these were considered not to be signs of toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weight and body weight gain were noted in males or females over the treatment period. Although the overall mean body weight and body weight gain was slightly lower in the high dose group for males and in all treated female groups when compared with the control, this was considered to be due to the slightly lower mean body weight of treated female groups and males given 200 mg/kg/day from the start of the dosing period. In addition, the effect was not dose-related and the mean body weight gain was higher in the 50 and 200 mg/kg/day groups than in controls during the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption over the treatment period in males were noted. There was a slightly lower mean food consumption for females given 50 and 200 mg/kg/day during premating and gestation periods compared with the control. This change was considered of no toxicological relevance in view of the low magnitude of the differences. No toxicologically relevant changes in food consumption over the lactation period in females were noted.

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 considered not to be affected by treatment. Any statistically significant changes were considered not to be toxicologically relevant as they occurred in the absence of a treatment-related distribution, were of low magnitude 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 of treated rats were considered not to be affected by treatment. Any statistically significant changes were considered not to be toxicologically relevant as they occurred in the absence of a treatment-related distribution, were of low magnitude and remained within the range considered normal for rats of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.

There was a statistically significant higher mean grip strength (hindlimb) in the male 50 and 200 mg/kg/day groups. Lower mean values in the control and 15 mg/kg/day groups might have been influenced by 2 males in each group (nos. 104, 105, 121 and 122) for which one single limb was tested, due to a high gap between the two hindlimbs. These differences were therefore considered as unrelated to the test item.
The variation in motor activity did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of any test item-related microscopic findings in animals given 200 mg/kg/day. The nature or incidence of histological findings in the organs examined did not indicate any relationship to treatment and they were considered to be incidental and part of the normal background changes normally encountered in reproductive/developmental studies in rodents.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had regular cycles of approximately 4 days.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

REPRODUCTION DATA
No toxicologically relevant effects on reproductive parameters were noted.
- Mating index: There were 10 mated pairs of animals in each group. All mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal oestrus cycle), except control female no. 114 paired with male no. 104 with a pre-coital interval of 14 days. The mating index was therefore not affected by treatment.
- Fertility index: There were no toxicologically relevant effects on fertility. Female nos. 137 and 179 in the 15 and 200 mg/kg/day groups, respectively, were not pregnant. The fertility index was therefore lower in these two groups (90 %) than in the control and 50 mg/kg/day groups (100 %). In the absence of a dose-related incidence of non-pregnancy and given their low incidence, these findings were considered not to be related to treatment.
- Pre-coital interval: Precoital time was considered not to be affected by treatment. There was one control pair (control female no. 114 paired with male no. 104) with a pre- coital interval of 14 days. This was considered incidental.
- Number of implantation sites: Number of implantation sites was considered not to be affected by treatment.

DEVELOPMENTAL DATA
No toxicologically relevant effects on developmental parameters were noted. All pregnant females delivered live offspring.
- Gestation index and duration: There was a marginally longer duration of gestation among the 50 and 200 mg/kg/day groups (22.7 and 22.9 days, respectively) when compared with the control and 15 mg/kg/day groups (22.4 and 22.2 days, respectively). However, there was no adverse effect on pup viability at birth. This effect was therefore considered not to be toxicologically significant. The mean gestation index was not affected by treatment in any group.
- Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. No deficiencies in maternal care were observed.
- Post-implantation survival index: The mean percent pre-birth loss (total number of offspring born compared to the total number of uterine implantations) was considered not to be affected by treatment. There was a slightly higher mean percent pre-birth loss in the 15 and 50 mg/kg/day groups (11.57 and 13.26 %, respectively) when compared with the control and 200 mg/kg/day groups (6.13 and 8.36 %, respectively). The mean value in the 50 mg/kg/day was exacerbated by one female (no. 151) with a pre-birth loss of 54.5 %.
No toxicological relevance was attributed to this change since the mean percent pre-birth loss did not show a dose-related trend and in view of the low magnitude of the differences.
- Live birth index: The number of live offspring at birth after littering compared to the total number of offspring born was considered not to be affected by treatment. Two pups in the control group (female no. 117) and one pup in the 200 mg/kg/day group (female no. 176) were delivered dead or missing. No toxicological relevance was attributed to these isolated dead/missing pups. The mean number of pups delivered per litter was slightly lower in the 15 and 200 mg/kg/day groups (10.3 and 10.4, respectively) than in the control and
50 mg/kg/day groups (11.2 and 11.0, respectively). No toxicological relevance was attributed to these changes since there were not dose-related and mean values remained within the historical control data range.
- Viability index: The number of live offspring on post-natal Day 4 before culling compared to the number of offspring alive at birth was considered not to be affected by treatment. There were 2,1,1 and 3 found dead or missing pups in the control, 15, 50 and 200 mg/kg/day, respectively between post-natal Day 0 and 4 (before culling). No toxicological relevance was attributed to these dead/missing pups since the viability index for all groups remained within the historical control data range.
- 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 not to be affected by treatment. One pup in the 50 mg/kg/day group (female no. 156) was found dead/missing between lactation Days 4 and 7. No toxicological relevance was attributed to this isolated dead/missing pup.

Details on results (P0)

Analysis of Dose Preparations:
All formulations at 3, 10 and 40 mg/mL of fluorobenzene in vehicle (propylene glycol), including the vehicle, used on the first day of treatment of the main study, were in agreement with acceptance criteria. The formulations at 3 and 40 mg/mL were homogenous. The deviations from the nominal concentrations ranged from -4.8 % to -3.4 %, and the RSD was ≤2.8 %. No significant amount of test item was detected in the vehicle sample.

Parental results:
No parental toxicity was observed up to the highest dose level tested (200 mg/kg).

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (200 mg/kg).

Developmental results:
No developmental toxicity was observed up to the highest dose level tested (200 mg/kg).

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: No adverse changes were noted in any of these parameters

2

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other:

Remarks:

No adverse changes were noted in any of the parameters determined in this study.

Target system / organ toxicity (P0)

Critical effects observed:

no

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. Incidental clinical signs of pups consisted of occasional pale pup, incomplete hair growth, scabs, sores and haematoma. No toxicological relevance was attributed to these isolated findings.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on post-natal Day 4 before culling compared to the number of offspring alive at birth was considered not to be affected by treatment. There were 2, 1, 1 and 3 found dead or missing pups in the control, 15, 50 and 200 mg/kg/day, respectively between post-natal Day 0 and 4 (before culling). No toxicological relevance was attributed to these dead/missing pups since the viability index for all groups remained within the historical control data range.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not affected by treatment. Minor differences were not dose-related and remained within the historical control data range.

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 pups were considered not to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment. The statistically significantly slightly higher mean anogenital distance of male and female pups at 200 mg/kg/day occurred was attributed to relatively high intra-group differences. These variations were considered not to be related to treatment.

Areola/nipple retention: Treatment up to 200 mg/kg/day had no effect on areola/nipple retention. No nipples were observed on PND 13 for any of the examined male pups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Developmental results:
- No developmental toxicity was observed up to the highest dose level tested (200 mg/kg).
- No treatment-related changes were noted in any of the 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 (PND1), areola/nipple retention (PND13 males), T4 thyroid hormone levels (PND13-15) and macroscopy.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

Remarks on result:

other: No adverse changes were noted in any of these parameters.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with JNJ-7820254-AAA (Fluorobenzene) by oral gavage in male and female Wistar Han rats at dose levels of 15, 50 and 200 mg/kg revealed no parental, reproduction and developmental toxicity up to 200 mg/kg.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 200 mg/kg
Reproduction NOAEL: at least 200 mg/kg
Developmental NOAEL: at least 200 mg/kg
Therefore, the substance is not classified as a reproductive toxicant according to the CLP Regulation.