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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)
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

Constituent 1
Chemical structure
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 sterilized by irradiation and analyzed for a predefined list of chemical and bacteriological contaminants.
- Water (e.g. ad libitum): Free access to softened and filtered (0.2 μm) mains drinking water.
- Acclimation period: At least 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
Details on route of administration:
Oral gavage, using a plastic cannula
Vehicle:
propylene glycol
Details on oral 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. Formulations were placed on a magnetic stirrer for at least 15 minutes before and throughout the dosing procedure (except on some occasions).

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.
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 / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control group)
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 3
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

Observations and examinations performed and frequency:
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.


Sacrifice and pathology:
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 PATHOLOGY: Yes
- 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), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, 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: Yes
- 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.
Other examinations:
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.
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 non-parametric 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.

Results and discussion

Results of examinations

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.
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:
no effects observed
Description (incidence and severity):
When compared with controls, there were no test item-related changes in organ weights associated with the administration of JNJ-7820254-AAA (Fluorobenzene).

Occasional organ weight differences were considered to be result of the terminal body weight differences or only to reflect normal individual variation and unrelated to test item.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no observations that were considered to be associated with administration of the test item. Occasional gross lesions were noted with variable incidence in controls and treated animals and were considered to be incidental. Most of these findings were histologically correlated and considered to be part of the spontaneous background observations normally encountered in the rodents.
Neuropathological findings:
not examined
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
Details on results:
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.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Remarks on result:
other: No adverse changes were noted in any of these parameters

Target system / organ toxicity

Critical 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 adverse toxicity up to 200 mg/kg. Based on these results, the No Observed Adverse Effect Level (NOAEL) was concluded to be at least 200 mg/kg.

Therefore, the substance is not classified as a repeated dose toxicant (STOT RE) according to the CLP Regulation.