1-acetyl-4-(4-hydroxyphenyl)piperazine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-05 to 2016-10-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (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

Test material information

Constituent 1

Reference substance name:

1-acetyl-4-(4-hydroxyphenyl)piperazine

EC Number:

267-744-8

EC Name:

1-acetyl-4-(4-hydroxyphenyl)piperazine

Cas Number:

67914-60-7

Molecular formula:

C12H16N2O2

IUPAC Name:

1-acetyl-4-(4-hydroxyphenyl)piperazine

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: JNJ-119379-AAA (T001141)
- Physical state: solid (powder)
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: I15FB3131
- Expiration date of the lot/batch: 2017-06-11 (retest date)
- Purity test date: 2015-06-12

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

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

OTHER SPECIFICS: Correction factor was 1.

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 (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: females approx. 10-12 weeks (at start pretest) and approx. 12-14 weeks (at start F0-treatment); males approx. 10-12 weeks (at start F0-treatment)
- Weight at study initiation: 297-325 g (males) and 201-232 g (females)
- Fasting period before study:no
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one 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
- Water (e.g. ad libitum): free access to tap-water
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES:
From: 2016-08-17 (start pretest, females aged approx. 10-12 weeks); 2016-08-31 (start treatment, males aged approx. 10-12 weeks); 2016-10-07/08/09/10/11(delivery of litters)
To: 2016-09-29 (necropsy males); 2016-10-20/21/24/25/26 (necropsy females); 2016-10-19/20/23/24/25 (necropsy pups)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Method: Oral gavage, using a plastic feeding tube
Frequency: 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.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% aqueous CMC

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. A correction was made for the purity/composition of the test item. A correction factor of 1 was used. No adjustment was made for specific gravity/density of the test item, vehicle and/or formulation. Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1); 10 mg/mL (group 2); 30 mg/mL (group 3); 100 mg/ mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight (Actual dose volumes were calculated according to the latest body weight)
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (2016-09-13; Day 17 of treatment) according to a validated method (Test Facility Study No. 512034). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10.00% compared to those obtained during the method validation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.

Duration of treatment / exposure:

29 days (males); 50-56 days (females that delivered); 40-43 days (females that failed to deliver).

Pups were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (control group)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on results of a dose range finding study (Test Facility Study No. 512019) in which animals were dosed for 20 and 7 days at 500 and 1000 mg/kg, respectively. In summary, there were no mortalities for the 500 mg/kg dose group, but 1/3 of the animals in the 1000 mg/kg dose group were found dead on Day 8, while 2/3 were sacrificed in extremis on Day 8. Piloerection was observed in one animal of the 500 mg/kg group, while hunched posture and piloerection was observed in all animals of the 1000 mg/kg group, with slight salivation in 2/3 animals on Days 6 and/or 7. Body weight loss was observed for both treatment groups. Food consumption was slightly reduced over Days 1-5 for the 500 mg/kg group, but normal thereafter, while food consumption was markedly reduced over Days 1-5 for the 1000 mg/kg group. Macroscopic examination of the 500 mg/kg animals showed no abnormalities, while 1000 mg/kg animals showed some findings, including reduced size of spleen in one of the animals found dead and reduced size of thymus in two animals sacrificed in extremis. Liver weight was slightly increased in the 500 mg/kg group; organ weights were not recorded for the 1000 mg/kg group. Based on the results of this range finding study, dose levels for the main study were: 50, 150 and 500 mg/kg/day. Since no clear peak effect of occurrence of clinical signs was observed in the range finding study, clinical observations (clinical signs and arena) in the main study were conducted and functional observations were started after dosing at no specific time point, but within a similar time period after dosing for the respective animals
- Rationale for animal assignment (if not random): randomized

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

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

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females
were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Body weight gain was calculated and reported.

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

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

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

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

FUNCTIONAL OBSERVATIONS
- Time schedule: The selected males were tested during Week 4 of treatment and the selected females of Groups 1-3 were tested once during the
last week of lactation (e.g. PND 6-13). In Group 4, the selected females were tested during the last week of treatment. These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Parameters: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength recorded as the mean of three measurements per animal, locomotor activity

Sacrifice and pathology:

SACRIFICE:
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration).
- Female animals: All surviving animals, on PND 14-16 (females that delivered) or on post-coitum day 25 (females that failed to deliver, with evidence of mating) or
approximately 27 days after the last day of the mating period(female without evidence of mating).

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

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

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, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix)
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavernosus muscle complex (LABC), Testes, Thyroid

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical signs of toxicity were noted at 500 mg/kg/day all animals. Frequent findings included hunched posture, piloerection, slightly pale appearance, hypothermia, and slight salivation, and were generally seen from the third week of treatment onwards. Additionally, cramped posture and chromodacryorrhoea (both at a slight degree) were noted in a single male and female at this high dose level. No treatment related clinical signs were observed up to 150 mg/kg/day. During the weekly arena observations, no additional treatment-related clinical signs were noted. Any other clinical signs noted incidentally (alopecia and scabs or scales on the skin) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated at 500 mg/kg/day showed slight body weight loss in the first treatment week and severely reduced body weight gain thereafter. At the end of the treatment period, mean body weight of 500 mg/kg/day males was 14% lower than that of controls. At the lower dose levels (50 and 150 mg/kg/day), males gained slightly less weight than controls during the first two weeks of the treatment period and their mean body weights were up to approximately 5% lower compared with controls at the end of the 4-week treatment period.

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

No clear treatment-related changes in body weight or body weight gain were observed in females treated at 50 or 150 mg/kg/day. It was noted that 6 out of 10 females at 150 mg/kg/day showed a slight body weight loss in the first week of the treatment period. Similar weight loss was noted in 3 out of 10 controls and weight gain of 150 mg/kg/day females was normal in the following weeks. Therefore, the apparent downward trend in the first week was considered not to reflect a toxicologically relevant effect on body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, food consumption (before and after allowance for body weight) was reduced at 500 mg/kg/day, most markedly in the first week of the treatment period.

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

No treatment-related changes in food consumption before or after allowance for body weight were observed in males or females treated up to 150 mg/kg/day. The statistically significantly higher mean food intake (absolute and relative to body weight) noted for females at 50 mg/kg/day during Days 1-4 of lactation was considered to be a chance finding, rather than a treatment-related effect, as no dose-related trend could be established.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following changes in haematology parameters distinguished treated animals from concurrent control animals (differences were statistically significant unless indicated otherwise):
• Lower total white blood cell count (WBC) in both sexes at 500 mg/kg/day (relative difference from controls: approximately 50%), and to a lesser extent at 50 and 150 mg/kg/day in males.
• Lower percentage of monocytes in females at 500 mg/kg/day.
• Lower percentage of eosinophils in females at 150 and 500 mg/kg/day.
• Higher percentage of basophils (not statistically significant) in males at 500 mg/kg/day.
• Lower number of red blood cells in both sexes at 500 mg/kg/day and in males at 150 mg/kg/day.
• Lower haemoglobin and haematocrit in both sexes at 500 mg/kg/day.
• Higher percentage of reticulocytes in males at 500 mg/kg/day, and lower percentage of reticulocytes in females at 500 mg/kg/day.
• Higher red blood cell distribution width (RDW) in both sexes at 500 mg/kg/day, and in males at 150 mg/kg/day.
• Higher mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in both sexes at 150 and 500 mg/kg/day (MCV in 150 mg/kg/day males not statistically significant), and higher MCH in females at 50 mg/kg/day.
• Higher prothrombin time in males at 500 mg/kg/day.
The above changes at 150 and 50 mg/kg/day were slight and/or remained in the normal range. Consequently, these findings were considered not adverse at these dose levels. The increase in prothrombin time in males at 500 mg/kg/day was only slightly above the normal range and therefore, considered to be non-adverse. The changes at 500 mg/kg/day were clearly outside the normal range for rats of this strain and age, except for the changes in WBC in females. Combined with concurrent histopathological findings, these haematological changes at 500 mg/kg/day were regarded adverse.

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

The higher activated partial thromboplastin time (APTT) in males at 50 and 150 mg/kg/day was considered unrelated to treatment as this change occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following changes in clinical biochemistry parameters distinguished treated animals from concurrent control animals (differences were statistically significant unless indicated otherwise):
• Higher alanine aminotransferase (ALAT) in males at 500 mg/kg/day.
• Lower total protein (not statistically significant) and albumin in females at 150 mg/kg/day.
• Higher total bilirubin in both sexes at 500 mg/kg/day, and in females at 150 mg/kg/day.
• Higher bile acids in both sexes at 500 mg/kg/day (not statistically significant in females), and in females (and a single male) at 150 mg/kg/day and in 3 out of 5 females at 50 mg/kg/day.
Values of the above parameters at 500 mg/kg/day were at the upper limit or above the normal range for rats of this strain and age. The changes in females at 50 and 150 mg/kg/day remained (just) within the normal range, and were consequently regarded as non-adverse.

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

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

Thyroid hormone analyses:
The serum T4 level of F0 males was statistically significantly decreased at 500 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Lower mean motor activity (total movements and ambulations) was noted at 500 mg/kg/day in males. This was mainly caused by male nos. 32, 33 and 35. Statistical significance was not achieved, but in view of the general toxicity at this dose level, the decreased activity of 500 mg/kg/day males was considered to be related to treatment and toxicologically relevant. Motor activity habituation profiles in 500 mg/kg/day males showed the normal decreasing trend over the duration of the test period.

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

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

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant test item-related organ weights changes (absolute and/or relative to body weight) were noted in the thymus (both sexes) starting at 150 mg/kg/day, and in the thyroid gland (both sexes), spleen (males), testes and epididymides at 500 mg/kg/day, as described below:
- Testes: statistically significantly lower weight (absolute and relative to body weight) at 500 mg/kg/day. The microscopic correlate was germ cell depletion.
- Epididymides: statistically significantly lower weight (absolute) at 500 mg/kg/day. The microscopic correlate was reduced sperm.
- Thyroid: statistically significantly higher weight (absolute and relative to body weight) at 500 mg/kg/day in both sexes.
- Spleen: statistically significantly higher weight (relative to body weight) at 500 mg/kg/day in males only.
- Thymus: statistically significantly lower weight (absolute and relative to body weight) at 150 and 500 mg/kg/day in both sexes.

Other organ weight differences (liver, prostate gland, kidneys) were statistically significant when compared with the control group but were considered to be the result of a test item-related effect on final body weight or difference in physiologic status (former pregnancy or no pregnancy).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were observed starting at 150 mg/kg/day in the thyroid gland of females and at 500 mg/kg/day in thyroid gland of males, the thymus of both sexes, and spleen and testes of males of the 500 mg/kg /day group:
Findings at 150 mg/kg/day and 500 mg/kg/day:
• Thyroid gland, black-brown discolouration in 4 out of 10 females at 150 mg/kg/day and in 10 out of 10 males and 9 out of 10 females at 500 mg/kg/day (no microscopic correlate) and enlarged in 1 out of 10 females at 150 mg/kg/day and in 6 out of 10 males at 500 mg/kg/day (microscopic correlate follicular cell hypertrophy).
Findings only at 500 mg/kg/day:
• Testes, flaccid in 9 out of 10 males (microscopic correlate germ cell depletion) and reduced in size in 4 out of 10 males, with microscopic correlate germ cell depletion
• Spleen, enlarged in 3 out of 10 males, (microscopic correlate extramedullary hematopoiesis).
• Thymus, reduced in size in 9 out of 10 males and 1 out of 10 females, (microscopic correlate atrophy).
• Mesenteric lymph node, discolouration in 2 out of 10 males (microscopic correlate intrasinusoidal erythrocytes).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the thyroid gland, thymus, spleen, mesenteric lymph node, testes, epididymides and bone marrow (sternal and femur) in one or both sexes at 150 and/or 500 mg/kg/day.
- Testes: A massive depletion of germ cells was observed in all males treated at 500 mg/kg/day. This mirrored the finding ‘abnormal stages’ recorded for the PAS stained slides of the testes of this group. In general, all round spermatids were affected while elongating spermatids and spermatogonia were still detectable. Tubular degeneration/atrophy and vacuolation of the testes were observed at increased incidence and severity (up to slight) in males treated at 500 mg/kg/day compared with controls (one control male showed these lesions at a minimal degree). This was considered as nonspecific change of the germ cells.
- Epididymides: Cellular luminal debris was observed at increased incidence and severity (up to slight) and reduced luminal sperm was observed up to moderate degree in males treated at 500 mg/kg/day.
- Thyroid gland: Follicular cell hypertrophy was observed at increased severity in males (up to moderate) and females (up to moderate) treated at 500 mg/kg/day.
- Spleen: At 500 mg/kg/day, extramedullary hematopoiesis was observed at increased severity (up to marked) in males and females and pigmentation was observed at increased mean severity in males (majority up to slight) and females (up to moderate).
- Bone marrow (sternum): Increased cellularity (of mainly erythroid cells) was observed in 2 out of 5 males (slight) and all females (up to slight) at 500 mg/kg/day.
Decreased cellularity (diffuse, all cell types) was observed in one male (minimal) at 500 mg/kg/day. Increased adipocytes was observed in 2 out of 5 females (minimal) at 500 mg/kg/day.
- Bone marrow (femur): Increased cellularity at the mid part of the metaphysis of the femur (mainly erythroid cells) was observed in one male (minimal) and all females (up to slight) at 500 mg/kg/day. Decreased cellularity at the distal part of the femur was observed in 3 out of 5 males (up to slight) and 2 out of 5 females (slight) at 500 mg/kg/day. Increased adipocytes at the distal part of the femur was observed in the majority of males (up to slight) and one female (minimal) at 500 mg/kg/day.
- Thymus: Lymphoid atrophy was observed at increased incidence and/or severity in males (up to marked) and females (up to moderate) at 150 and 500 mg/kg/day. The single incidence of minimal/slight degree of lymphoid atrophy in one male and one female at 50 mg/kg/day was regarded to be within background findings of rats of this age and strain.
- Mesenteric lymph node: Sinusoidal erythrocytes were observed at increased incidence and severity in males (up to slight) at 500 mg/kg/day. The low incidences with minimal degrees noted in males and females of the control group and in a single male of the 50 mg/kg/day group were considered to be within background findings of rats of this age and strain. Pigmentation (macrophages with brown pigment) was observed in 2 out of 5 males (up to slight) at 500 mg/kg/day.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

- Analysis of dose preparations: The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%). A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation. The maximum contribution to the formulation samples at the lowest concentration level was 0.01%. The presence of such minor responses at the retention time of the test item in Group 1 chromatograms was considered to be acceptable and did not
adversely affect the integrity of the study.
- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

other: Multiple Systems: Endocrine system and Hematopoiesis (Males & Females); Immune system, Reproductive system (Males only)

Organ:

bone marrow

spleen

testes

thymus

thyroid gland

other: epididymides

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with JNJ-119379-AAA (T001141) by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg revealed clinical signs of toxicity, reduced body weight gain and food consumption, adverse morphological changes in the spleen with associated changes in blood parameters, decreased number of circulating total white blood cells, and adverse morphological changes in the thyroid gland and bone marrow in both sexes; and adverse morphological findings in the thymus, testes and epididymides in males only. Based on the results, the following NOAEL was derived: 150 mg/kg/day.

Therefore, the test item is not classified for STOT RE according to the CLP Regulation.