Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity - Oral: In a combined repeated dose toxicity study with reproduction/developmental toxicity screening, the test substance was administered daily to rats at the dose level of 50, 150, and 500 mg/kg bw/day for 29 days for the males and 50-56 days for the females (OECD 422; Pels Rijcken, WR, 2018). A parental NOAEL of at least 500 mg/kg bw/day was established.

 

Repeated dose toxicity - inhalation: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

 

Repeated dose toxicity - dermal: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-20 to 2016-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/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: EPA 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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16AB0081
- Expiration date of the lot/batch: 11 January 2018 (retest date)
- Purity test date: 2016-01-12


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 and 14 days in the refrigerator in the dark is confirmed over the concentration range 1 to 200 mg/mL, Project 513703.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Colorless to white suspensions.

OTHER SPECIFICS: Correction factor is 1
Species:
rat
Strain:
Wistar
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment); Males approx. 11 weeks (at start F0-treatment).
- 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: 5 days prior to start of pretest (females) or treatment (males)


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


IN-LIFE DATES:
From: 2016-10-13 (start pretest, females); 2016-10-27 (start treatment, males)
To: 2016-11-25 (necropsy males); 2016-12-16 and 2016-12-19/20/21/22 (necropsy females)
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:
propylene glycol
Remarks:
specific gravity 1.036
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. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity/composition of the test item. A correction factor of 1 was used.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1); 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 (01 November 2016, Day 6 of treatment) according to a validated method (Test Facility Study No. 513703). 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. Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study (Test Facility Study No. 513703).
Duration of treatment / exposure:
29 days (males); 50-56 days (females that delivered); 41 days (females that failed to deliver). Routinely, females that are littering are left undisturbed. In this study, female nos. 48, 50 (Group 1), 51 (Group 2), 66, 69 (Group 3), 73 and 79 (Group 4) were left out from treatment for one day as they were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.

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 uring/faeces.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control group)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3
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. 513699) in which animals were dosed for 10 days at 500 and 1000 mg/kg. In summary, a dose related increase in liver and kidney weights were observed at 500 and 1000 mg/kg/day. In comparison with laboratory background data, liver weights were increased at both dose levels and kidney weights at 1000 mg/kg/day only. Based on the magnitude of increase in liver weight at 1000 mg/kg/day, it was doubted if this dose level should be tolerated in animals treated for a longer duration. Since signs of (mild) toxicity were observed at 500 mg/kg/day, this dose level was selected to be the highest for the main study. Therefore, 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 clinical signs were observed in the range finding study, clinical observations in the main study were conducted immediately after dosing.

- Rationale for animal assignment (if not random): randomized
Positive control:
no
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. Clinical observations were conducted after dosing at no specific time point, but within a similar time period after dosing on each day. On the day of start of treatment and at weekly intervals thereafter this was also performed outside the home cage in a standard arena.

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 fo od/ 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 aminotransferase, 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: Between 1 and 3 hours after dosing on the selected 5 animals/sex/group. Selected males were tested during week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs (incl. arena observation if applicable)
- Parameters: hearing ability, pupillary reflex, static righting reflex, fore- and hindlimb 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). Female with total litter loss, within 24 hours of litter loss.

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.
- The number of former implantation sites were recorded for all paired females.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution):
Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/ F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord - cervical, midthoracic, lumbar (M/F), Spleen (M/ F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, and females which failed to deliver, were collected and fixed in 10% buffered formalin:
Cervix (F), Clitoral gland (F), Coagulation gland (M), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levator ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

HISTOPATHOLOGY: Yes
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist: The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4; The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); Liver of all selected 5 animals of Groups 2 and 3 (females and males), thymus of all selected 5 males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4; The reproductive organs and mammary gland of female no. 65 (Group 3) that had a total litter loss.
- All abnormalities were described and included in the study 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, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid (including parathyroid if detectable), Uterus (including cervix).
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Epididymides, Prostate, Seminal vesicles including coagulating glands, Testes, Thyroid.
Statistics:
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (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.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.

Salivation seen after dosing among animals of all test groups during the treatment period 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).

Incidental findings that were noted included rales, alopecia, scabs on the cheek or in the neck and red discolouration of the ear. 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.

Female no.65 (at 150 mg/kg/day) was early terminated on PND 1, because of total litter loss.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain of treated males remained in the same range as controls over the treatment period.

In females, body weights and body weight gain over the premating period were similar in all groups. During the post-coitum phase, slightly lower body weights and body weight gain were observed in pregnant females in all three test groups when compared to controls, reaching levels of statistical significance for body weights on Day 14 at 150 mg/kg/day and on Day 17 at 150 and 500 mg/kg/day, and for body weight gain on Days 7, 11, 14 and 17 post-coitum at 500 mg/kg/day. In comparison with historical background data for body weight gain during the post-coitum period, the growth of the control animals was slightly higher than expected. Since body weight gain was similar in all three test groups and also comparable with the normal growth of pregnant rats of this strain and age, the statistically significant differences apparent during the post-coitum phase were considered not indicative of toxicological significant changes.

The difference in body weights on Day 20 post-coitum and Day 1 of lactation, indicative of the weight and size of the litter, showed a mean body weight loss of 85-90 grams in controls and high dose females and of approximately 75 grams in the low and mid dose females. As a result, the difference in mean body weights between controls and females at 50 and 150 mg/kg/day had diminished (to less than 3%), whereas that between controls and females at 500 mg/kg/day was maintained (at approx. 7%) and reaching a level of statistically significance. Levels of statistical significance were also observed for the mean body weights of these high dose females on Days 7 and 13 of lactation in comparison with controls. Body weight gain during lactation, however, showed that growth in controls and high dose females was normal and ran parallel.

In summary, the differences in body weights and body weight gain between treated females and controls were considered to have occurred as a result of a slightly higher than normal body weight gain in controls over the post coitum period, rather than a treatment-related effect. Therefore, no toxicological significance was attached to the changes in growth between the dose groups in this study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption, absolute and relative to body weight, was similar between treated and control males.

In females, food consumption over the pre-mating period was similar in all groups. During the post-coitum phase, slightly higher than normal food consumption was observed in control females, in line with the slightly higher body weight gains in these animals over the same period. Therefore, the statistically significant differences apparent during the post-coitum phase between treated females, i.e. over Days 4-7 in females at 150 and 500 mg/kg/day and over Days 11-14 in females at 500 mg/kg/day, were considered not indicative of changes in food consumption in treated females. During the lactation phase, the food consumption was similar in all groups, except for a slightly lower food consumption (not statistically significant) in the females at 500 mg/kg/day over Days 7-13.

In summary, the food consumption in females was considered not to have been affected by treatment in this study.
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.

No dose related response was observed for any haematology parameter in males and all values were within normal limits and the test group mean values were close to that of the historical control values.

In a single female at 500 mg/kg/day an increase of neutrophil counts with concurrently reduced lymphocyte counts was noted. In addition, a relatively low value for red blood cell count, and relatively high values for red cells distribution width (RDW) and mean corpuscular volume (MCV) were observed in this female. The changes in these parameters might be indicative of a secondary non-specific response to stress and were possibly related to the reddish discolouration of the thymus observed at macroscopic examination and the congestion and lymphoid atrophy of the thymus of this female. Therefore, the findings in female no.78 were considered to have occurred by chance and of no toxicological significance. The statistical significance for the RDW in high dose females was considered to be the result of the deviating value apparent in the single female, rather than an indication of a treatment related effect.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.

Statistically significant decreases in ASAT and bile acids were observed in males at 500 mg/kg/day. The changes in these parameters might be related to the histopathological (non-adverse) alterations observed in the liver, but since decreases in these parameters are considered of limited toxicological significance, these findings were considered to be non-adverse.

Plasma levels of chloride were dose-dependently increased in treated animals, achieving levels of statistical significance in males at 150 and 500 mg/kg/day. Chloride was measured using an Ion Selective Electrode (ISE). It is known that bromide, a constituent of the test substance, can interfere with this measurement, resulting in falsely increased chloride results. Therefore, the apparent dose-related increase in chloride in males and females was considered to be an artifact resulting from interference by test substance-derived bromide with the ISE and of no toxicological relevance.

Any statistically significant changes apparent for calcium in males at 500 mg/kg/day, for potassium and calcium in females at 150 mg/kg/day and for inorganic phosphate in females at 50 and 150 mg/kg/day were considered not to be toxicologically significant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Thyroid hormone analyses: Serum levels of T4 in F0 males were considered not to be affected by treatment. The statistical significances found in all three test groups were considered to the result of a fortuitous difference in T4 values between controls and test groups, rather than an indication of a treatment related effect. In the absence of a clear dose response relationship and since the T4 values in all males were within the normal limits for rats of this strain and age, no toxicological significance was attached to this finding.
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.

The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant higher liver weights were observed in males and females at 500 mg/kg/day (absolute as well as relative to body weight). The liver weights relative to body weight in high dose animals were approximately 40% higher than in controls.

Slightly lower thymic weights were observed in females at 500 mg/kg/day, not reaching a level of statistical significance.

Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

The statistically significant difference apparent for the relative weight of the thyroid gland in males at 150 mg/kg/day was considered to have occurred by chance and of no toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Six out of ten males showed an enlarged liver at macroscopic examination at necropsy, achieving a level of statistical significance in comparison with controls.

Additionally (red-brown) discolouration of the liver was observed at a high incidence in all three test groups. No explanation for the discolouration could be given and in the absence of any corroborative finding in these animals, the discolouration was considered of no toxicological relevance. The incidence of the discolouration of the liver was 1, 5, 5 and 6 (out of ten males per group) in the vehicle and 50, 150 and 500 mg/kg/day groups, respectively.

No treatment related findings were observed in the test item treated female rats.

The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered not to be toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
THYMUS: At 500 mg/kg/day, lymphoid atrophy of the thymus was recorded in 3 females at a minimal degree.

LIVER: At 500 mg/kg/day, centrilobular hepatocellular hypertrophy was recorded in the liver of all males and females at minimal or slight degree.

KIDNEYS: An increased incidence and severity of hyaline droplet accumulation was recorded in males at 500 mg/kg/day. This was recorded in all males at minimal-moderate degree. In the remaining dose groups this was seen in 3/5 males at minimal or slight degree.

There were no other test item-related histologic changes. The remainder of the recorded microscopic findings, including the follicular cell hypertrophy in the male thyroid gland of all dose groups, were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
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%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse changes were noted in any of the parameters examined in this study.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Treatment related changes in the thymus in females, in the kidneys in males and in the liver in both sexes at 500 mg/kg/day were considered non-adverse.
Key result
Critical effects observed:
no
Conclusions:
In conclusion, treatment with JNJ-1597622-AAA (T000625) by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg/day revealed non-adverse, treatment related changes in the thymus in females, in the kidneys in males and in the liver in both sexes at 500 mg/kg/day. Based on these results, the Parental NOAEL was established as at least 500 mg/kg/day.

Therefore, the substance is not classified as a repeated dose toxicant (STOT RE) according to the CLP Regulation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated toxicity: oral


In a combined repeated dose toxicity study with reproduction/developmental toxicity screening, the test substance was administered daily to male and female rats (10 rats/sex/dose level) at the dose level of 50, 150, and 500 mg/kg bw/day (OECD 422; Pels Rijcken, WR, 2018). The vehicle used was propylene glycol. Accuracy and homogeneity of formulations were demonstrated to be acceptable by chemical analyses.


In high dose females at 500 mg/kg/day, (statistically significantly) lower body weights were observed during the lactation phase, in comparison with controls. If this difference had arisen during the post-coitum phase could not be established, because effects on maternal body weight were obscured by the increase due to their pregnancy. Since the difference in mean body weights between the high dose females and controls was less than 10% and body weight gain and food consumption were similar during the lactation period, these lower body weights were considered not indicative of an adverse effect.


No treatment related effects on body weights and food consumption were observed in the parental mid dose females at 50 and 150 mg/kg/day and in males treated up to 500 mg/kg/day. In high dose animals at 500 mg/kg/day, other treatment related changes were observed in the liver (both sexes), thymus (females) and kidneys (males. Hepatocellular hypertrophy was recorded in males and females at 500 mg/kg/day and the severity varied from minimal to light and without evidence of any degenerative, inflammatory or proliferative finding of the liver. The lymphoid atrophy of the thymus in females at 500 mg/kg/day correlated the lower thymic weights. Although lymphoid atrophy can be seen in (non-treated) lactating females, this finding was considered to be test item-related because it was not recorded in control females in the current study. The hyaline droplet accumulation recorded in the kidneys of males at 500 mg/kg/day likely represents alpha2uglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. Mainly based on the incidences and severities observed at histopathology these findings in liver, thymus and kidney were considered to be non-adverse.


No treatment-related were noted after treatment at 500 mg/kg/day in the other parental parameters investigated in this study (i.e. clinical appearance, functional observations and clinical laboratory investigations). Any changes in the various study parameters after treatment at 50 and 150 mg/kg/day were considered no signs of parental toxicity.


Based on these results, the parental No Observed Adverse Effect Level (NOAEL) was derived to be at least 500 mg/kg/day.


 


Repeated toxicity: inhalation


A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.


 


Repeated toxicity: dermal


A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure.

Justification for classification or non-classification

For T000625, the parental NOAEL was established at least 500 mg/kg bw/day in an OECD 422 study.

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