α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

Endpoint summary

Description of key information

Repeated dose toxicity - oral: In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the test substance was administered daily to rats up to a dose level of 330 mg/kg body weight/day (OECD 422; Meijer, 2018). The NOAEL is established to be 30 mg/kg body weight/day based on clinical signs of toxicity (piloerection of the fur in both males and females) and inflammation of the endometrium of the uterus (related to reproductive failure in females). The substance is therefore classified as a repeated dose toxicant (STOT RE 2) according to the CLP Regulation.

 

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

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:

2017-06-14 to 2017-11-07

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)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

Minor deviations occurred which were determined not to have adversely affected the integrity of the study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2000

Deviations:

yes

Remarks:

Minor deviations occurred which were determined not to have adversely affected the integrity of the study.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

October 2008

Deviations:

yes

Remarks:

Minor deviations occurred which were determined not to have adversely affected the integrity of the study.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

yes

Remarks:

Minor deviations occurred which were determined not to have adversely affected the integrity of the study.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

July 2000

Deviations:

yes

Remarks:

Minor deviations occurred which were determined not to have adversely affected the integrity of the study.

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: Sponsor; M15KB5305
- Expiration date of the lot/batch: 2019-11-24
- Purity test date: 2016-10-21

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability in vehicle: Stable for at least 6 hours at room temperature under normal laboratory light conditions and for at least 14 days in refrigerator (2-8°C) is confirmed over the concentration range 1 to 200 mg/mL (Project 517993).
- Stability under test conditions: Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Project 517993).

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Solution (Group 2-3) and suspension (Group 4).

OTHER SPECIFICS: Correction factor was 1.

Species:

rat

Strain:

Wistar

Remarks:

rat

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: Males approx. 10 weeks (at starat F0-treatment); Females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: 269-313 g (males); 203-236 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 in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Females were housed in Macrolon plastic cages (MIII type,height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet. During motor activity measurements, animals did not have access to food for a maximum of 2 hours
- Water (e.g. ad libitum): Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES
From: 2017-08-23 (start pretest, females); 2017-09-06 (start treatment, males); 2017-10-13 through 2017-10-17 and 2017-10-24 through 2017-10-25 (delivery of litters)
To: 2017-10-05 (necropsy males); 2017-10-18/23/26/27/30/31 and 2017-11-1/6/7 (necropsy females);
through (necropsy pups)

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Vehicle:

propylene glycol

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); 6 mg/mL (group 2); 20 mg/mL (group 3); 66 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): not reported
- Purity: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (2017-09-06, Day 1 of treatment) according to a validated method (Test Facility Study No. 517993). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.
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%.
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. 517993).

Duration of treatment / exposure:

Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were treated for 50-62 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were treated for 41-47 days.

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:

Control, group 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

group 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

group 3

Dose / conc.:

330 mg/kg bw/day (nominal)

Remarks:

group 4

No. of animals per sex per dose:

10 animals/sex/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 10-day dose range finding study (Test Facility Study No. 517995). During the DRF phase, oral (gavage) administration of T00824 to female Wistar rats for 10 days (500 mg/kg) or 4 days (600 mg/kg). Dose limiting effects occurred at 600 mg/kg (clinical signs of toxicity, body weight loss and reduced food consumption, leading to early sacrifice at Day 4). Based on these results, dose levels of 0, 30, 100, and 330 mg/kg/day were selected for the main study.
- 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 in all animals at 0-15 minutes and 1 hour (±15 minutes) after dosing. 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. Arena observations were conducted at least 1 hour (± 15 min) after dosing. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Females of Group 4 were also weighed on Day 41 in order to aid in confirmation of pregnancy. Body weight gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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.

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: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. Blood samples were drawn from the retroorbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters, with citrate for clotting tests, and tubes treated with Li-heparin for clinical biochemistry parameters.
- Animals fasted: F0-Female animals were not fasted prior to blood sampling. F0-Male animals were fasted prior to blood sampling, but water was available.
- Parameters examined: White blood cells (WBC); Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width (RDW), Haemoglobin, Haematocrit Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration, Platelets, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. Blood samples were drawn from the retroorbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters, with citrate for clotting tests, and tubes treated with Li-heparin for clinical biochemistry parameters.
- Animals fasted: F0-Female animals were not fasted prior to blood sampling. F0-Male animals were fasted prior to blood sampling, but water was available.
- How many animals: 5 animals/sex/group
- Parameters examined: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP) ,Gamma glutamyl transferase (GGT), Total protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate (Inorg. Phos).

FUNCTIONAL OBSERVATIONS: Yes
- Time schedule for examinations: The selected males were tested once during Week 4 of treatment. The selected females of Groups 1 and 2 were tested once during the last week of lactation (PND 6-13) and selected females of Group 3 were tested once at PND 5-8. Selected females of Group 4 (no offspring) were tested once at Day 23-26 post-coitum. These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R); fore- and hind-limb grip strength, recorded as the mean of three measurements per animal; locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system. Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

Sacrifice and pathology:

SACRIFICE
F0-Female animals were not fasted prior to necropsy. F0-Male animals (except for male no.32 which was sacrificed in extremis) were deprived of food overnight (with a maximum of 24 hours) prior to necropsy, but water was available. Necropsy was conducted on the following days:
- Males: Following completion of the mating period (a minimum of 28 days of doseadministration).
- Females which delivered: PND 14 or 16.
- Females which failed to deliver: Post-coitum Days 25-27 (females with evidence of mating).
- Females with total litter loss: Within 24 hours of litter loss.
- Euthanized in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

GROSS PATHOLOGY: Yes
- All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetized using isoflurane and subsequently exsanguinated.
- After sacrifice, all animals were subjected to a full post mortem necropsy, 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. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at Charles River Den Bosch using Ammoniumsulfide-solution 20% and Milli-Ro water), and the number of corpora lutea were recorded in addition. Samples of the following tissues and organs 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), Colon (M/F), Coagulation gland (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area, inguinal region with skin (M/F), Femur with bone marrow including joint (M/F), Heart (M/F), Ileum (M/F), Jejunum (M/F), 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), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), Skin (M/F), Spinal cord -cervical, mid-thoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F).
- Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

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 following were examined:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
- For the testes of the selected 5 males of Groups 1 and 4 and all males that failed to sire or which died before mating detailed qualitative examination was made, taking into account the tubular stages of spermatogenic cycle.
- The preserved organs and tissues of the animals of all dose groups which were euthanized in extremis.
- The mammary gland of all females with total litter loss.
- All gross lesions of all animals (all dose groups).
- Liver, spleen and kidneys of all selected 5 animals of Groups 2 and 3 (males and females), adrenal glands, lung, ovaries, uterus, cervix, vagina and mammary gland of all selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- Nasal cavity level 3, 4 and 4 caudal and larynx of all selected 5 females of Groups 1 and 4 and Male no. 32 and Female no. 74 since the observation of rales may have been related to the moribundity of these animals.
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
- 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 were reported and organ to terminal body weights and organ to brain weights were calculated.
- 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, Testes, Thyroid (including parathyroid if detectable), Prostate, and Seminal vesicles including coagulating glands.
- Paired organs were weighed together.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
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, treatment-related

Description (incidence and severity):

At 330 mg/kg, the main clinical sign of toxicity was piloerection of the fur which started after about one week of treatment and was seen most frequently in females. Less frequent findings included rales (in several males and females on a few occasions), hunched posture (in 8 out 10 females, starting on treatment Day 6 for approximately 1-2 weeks), and slight lethargy, ptosis, pale appearance and red discharge/discoloration at the vulva (in a single female (no.78) towards the end of the treatment period). Slight lethargy was further noted once (Day 3) in two males (without other clinical signs). Additionally, yellow discoloration of the faeces was seen in all 330 mg/kg animals in Weeks 2-3. Piloerection of the fur and rales also occurred at 100 and 30 mg/kg, particularly in females. The incidence and time of onset of piloerection of the fur showed a dose-related trend. Such trends were not evident for rales (which occurred less frequently than piloerection of the fur). Salivation was noted after dosing at all dose levels, starting after seven (low dose) or three (higher dose levels) days of treatment. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its minor severity (generally slight) and the time of occurrence (i.e. immediately after dosing), and may be related to the irritancy of the test item. Any other clinical signs noted incidentally 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:

mortality observed, treatment-related

Description (incidence):

At 330 mg/kg there were three premature decedents, all sacrificed in moribund condition, which were considered to be related to treatment with the test item. Male no. 32 and female no. 74 were euthanized at study Day 8. These animals showed clinical signs of toxicity (hunched posture, piloerection of the fur, rales, ptosis) and body weight loss (10-12% between Days 1-8). Main necropsy findings consisted of gas distended parts of the intestines (no. 32) and emaciated appearance (no. 74). Moderate hepatocellular vacuolation of the liver was noted in both rats, similar as in the scheduled sacrificed rats. Examination of the nasal cavity revealed in both rats mucosal lesions indicative of reflux-related effects in the form of (sub)acute erosion/necrosis of the respiratory and/or olfactory epithelium, luminal exudate and granulocytic inflammatory cell infiltrate. These findings were considered to have attributed to the moribundity of these animals.
The third decedent (female no. 79) at 330 mg/kg was euthanized at Day 38 (Day 19 postcoitum) when she showed red discoloration in the inguinal region (blood was seen in her cage), increased respiratory rate, pale appearance (moderate), hunched posture, lethargy, piloerection of the fur and body weight loss (nearly 10% between Days 17-19 post-coitum). Main morphologic findings in female no. 79 consisted of two early resorptions and one dead fetus in the left uterus horn and slight inflammation of the endometrium of the uterus. This reproductive failure was considered to be related with the moribundity.
At 100 mg/kg, one female (no. 63) was sacrificed for humane reasons at Day 21 (Day 1 postcoitum). On the day of sacrifice, after dosing, she showed laboured respiration, rales, ptosis, felt cold and excessive salivation. Necropsy findings consisted of an emaciated appearance (body weight was normal) and gastric changes (distended with gas, isolated red foci in the glandular stomach mucosa). Microscopic examination revealed lesions indicative of a dosing error in the form of moderate to marked necrosis of bronchus and tracheal epithelium and the moribundity of this animal was therefore not considered test-item related.
Three females at 330 mg/kg were sacrificed due to total litter loss (nos. 62, 66 and 70 at PND 1, 3 and 4, respectively).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males and females at 330 mg/kg showed statistically significantly reduced body weight gain during the first week of treatment. Significant weight loss (between 6-12% of the initial weight) occurred in the two animals that were euthanized on Day 8 (male no. 32, female no. 74) and in two other animals (male no. 36, female no. 73). Mean body weights at Day 8 were
statistically significantly reduced in males (9% difference) while the difference in females (5%) did not attain statistical significance. Growth rate and mean body weights had recovered to normal values at Day 1 of the mating period and remained normal until the end of the treatment period in males and until Day 7 post-coitum in females. Thereafter, 330 mg/kg females gained less weight than controls (mean body weights were statistically significantly lower on post-coitum Days 11-20, 25% difference in body weight gain at Day 20 post coitum). This reduced gestational body weight gain was due to their abnormal pregnancies (i.e. 3 non-pregnant females and six females with implantations only).
No toxicologically relevant changes in body weight (gain) were observed in males and females treated up to 100 mg/kg. The statistically significantly higher body weight gain values noted in 30 mg/kg females at Days 4 and 7 of the lactation period (associated with higher food consumption values between lactation Days 1-4) were judged to be non-adverse as weight gain of these females was not significantly different at other time points. Moreover, mean body weights of 30 mg/kg females remained close to control values.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before and after allowance for body weight was reduced at 330 mg/kg in the first week of treatment in both sexes (by about 25%) and, to a lesser extent, in the second treatment week in females. Food consumption of 330 mg/kg females was also reduced during the post-coitum period, which was related to their abnormal pregnancy (i.e. 3 non-pregnant females and six females with implantations only). At 100 mg/kg, mean food consumption (before and after allowance for body weight) was statistically significantly reduced between Days 7-13 of the lactation period. The lower group means resulted from the lower food consumption of two females with very small litters (nos. 64 and 68). The remaining three lactating females at 100 mg/kg showed normal food consumption (and had normal litter sizes). This finding was therefore not regarded as toxicologically relevant, but a secondary effect due to the small litter sizes. No treatment-related changes in food consumption before or after allowance for body weight were observed in males treated up to 100 mg/kg and females treated at 30 mg/kg. The higher food consumption noted in 30 mg/kg females between Days 1-4 of the lactation period was considered unrelated to treatment as food consumption of these females was similar to that of controls at all other time points.

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):

Note: Females at 330 mg/kg had no offspring. Several haematology parameters are known to be influenced by physiological status (lactating versus implantations only). This was taken into account when assessing the possible relation with treatment of differences in haematology values between females of the 330 mg/kg group (all selected females had implantation sites, suggestive of former pregnancy, but no offspring) and controls (all lactating). The following changes in haematology parameters distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values relative to the concurrent control group are indicated between parentheses.
Male rats treated up to 330 mg/kg:
- Lower mean corpuscular haemoglobin (MCH) at 330 mg/kg (7%).
The statistically significantly lower number of platelets noted at 100 mg/kg was considered unrelated to treatment due to the absence of a dose-related trend and a somewhat high control value (resulting from an outlying value in one control male).
Female rats treated at 30 or 100 mg/kg:
- Lower mean corpuscular haemoglobin (MCH) at 100 mg/kg (8%).
- Higher red blood cell distribution width (RDW) at 100 mg/kg (11%). This change was not statistically significant.
Female rats treated at 330 mg/kg:
Note: Due to the difference in physiological status (lactating versus implantations only), no percentage differences from control values were calculated.
- Higher total white blood cell count (WBC; consisting of neutrophils, lymphocytes, monocytes, eosinophils and basophils)
- Higher number of reticulocytes.
- Lower haemoglobin concentration.
- Lower mean corpuscular haemoglobin volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC).
- A tendency towards higher red blood cell distribution width (RDW) and lower haematocrit.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Note on females at 330 mg/kg: see haematology section.
The following changes in clinical biochemistry parameters distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values relative to the concurrent control group are indicated between parentheses.
Male rats treated up to 330 mg/kg:
- Lower aspartate aminotransferase activity (ASAT) at 330 mg/kg (30%).
- Higher gamma glutamyl transferase activity (GGT) at 330 mg/kg. Statistical significance and magnitude of difference from control could not be determined as all control values were below the limit of detection (0.5 U/L). Values at 330 mg/kg were 2-3 fold higher than this detection limit and were outside the historical control range. Values at 30 and 100 mg/kg were within the historical control range.
- Lower total bilirubin at 330 mg/kg (20%).
Female rats treated at 30 or 100 mg/kg:
Clinical biochemistry parameters in lactating females were considered not to be affected by treatment up to 100 mg/kg.
Female rats treated at 330 mg/kg:
Note: Due to the difference in physiological status (lactating versus implantations only), no percentage differences from control values were calculated.
- Lower alanine aminotransferase activity (ALAT), alkaline phosphatase (ALP) activity (not statistically significant), bile acids (not statistically significant), urea, potassium and chloride.
- Higher gamma glutamyl transferase activity (GGT) (not statistically significant), glucose, total protein, albumin, creatinine, cholesterol (not statistically significant), sodium, calcium and inorganic phosphate (not statistically significant).
Thyroid hormone analyses:
Mean serum T4 levels of F0 males were statistically significantly decreased from 30 mg/kg onwards (relative differences from controls: 24, 40 and 48% at 30, 100 and 330 mg/kg, respectively). The mean value at 330 mg/kg, 2.75 ug/dL, was below the historical control range.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related findings were noted for functional observations up to 330 mg/kg. Mean hind limb grip strength values of treated males were statistically significantly lowercompared with the control value. The differences showed no dose-related response. Moreover, the concurrent control value was higher than normal. Therefore, the differences in hind limb grip strength in males were judged to be the result of a high control value rather than an effect of the test item. Fore limb grip strength in males and grip strength values in females were unremarkable. The non-statistical reduction in fore limb grip strength of females treated with 330 mg/kg was within normal range for nulliparous females. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. The higher mean values for total movements and ambulations noted in females at 100 mg/kg were considered unrelated to treatment since these values were within normal range and due to the lack of consistency in time (values were particularly high during the fifth interval). Moreover, the significant outcome at 100 mg/kg was likely the result of the lower control values in this study which were in the lower limit of the historical control data.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males rats treated up to 330 mg/kg:
Test item-related organ weight changes in males consisted of higher absolute and relative weights of the liver at 330 mg/kg.
Female rats treated at 30 or 100 mg/kg:
Females of the 100 mg/kg group showed the following statistically significant differences from controls: lower spleen weight (-18%, absolute) and higher ovary weight (+23%, absolute). The spleen weight and ovary weight were within the historical data range. No statistical significant changes in organ weights relative to body weights were observed. These differences and any other differences, were considered not to be test item-related due to the direction of the change and/or general overlap and variability in individual values.
Female rats treated at 330 mg/kg:
In females of the 330 mg/kg group, some organ weight differences were statistically significant when compared to the control group. Main organ weight differences consisted of lower liver weights (absolute and relative to body weight), higher adrenal gland weights (relative to body weight), higher thymus weights (relative to body weight), higher ovary weights (absolute and relative to body weight) and higher uterus weights (absolute and relative to body weight; uterus weights of two females were very high due to the presence of a dead fetus in their uterus). However, the physiologic status and treatment duration of the selected females of the control, 30 and 100 mg/kg groups (healthy offspring and dams necropsied PND 14 -16) differed from those of 330 mg/kg females (no living offspring and sacrificed about 1-2 weeks earlier). Therefore, a reliable interpretation of these organ weight differences is not possible.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic findings consisted of:
- Liver: enlargement in 7/10 males and accentuated lobular pattern in 1/10 males at 330 mg/kg (microscopic correlate: hepatocellular vacuolation and/or hepatocellular hypertrophy).
- Uterus: thickened horn and/or contents (early resorptions and/or presence of dead fetuses) in 4/10 females at 330 mg/kg (microscopic correlate: inflammation of the endometrium of the uterus).
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Results of males (treated for 29 days), females of the control, 30 and 100 mg/kg groups (selected females treated for 50 to 55 days and with normal offspring), and females of the 330 mg/kg group (selected females treated for 42 days and without healthy offspring) were evaluated separately.
Male rats of all groups:
Test item-related microscopic findings were noted in the liver of 100 and 330 mg/kg males and kidneys of 330 mg/kg males
Liver:
- Hepatocellular vacuolation was observed in males at 100 mg/kg (minimal) and 330 mg/kg (up to moderate). This vacuolation was variable in size (micro- and macro-vesicular) and in general present in zone 1 and 2.
- Hepatocellular hypertrophy was observed in males at 100 mg/kg (minimal, particularly centrilobular) and 330 mg/kg (up to slight, diffuse).
Kidneys:
Increased severity of hyaline droplets accumulation was noted in males at 330 mg/kg (all slight).
Selected female rats of the control, 30 and 100 mg/kg groups:
Test item-related microscopic findings were noted in the spleen of 30 and 100 mg/kg females. A dose-related increase of yellow-brown pigmentation of the spleen was observed in 30 and 100 mg/kg group females (up to moderate). This pigmentation was considered to represent hemosiderin, a degradation product of haemoglobin.
Female rats of the 330 mg/kg group (without living offspring):
Test item-related microscopic findings were noted in the liver, kidneys and lung of 330 mg/kg females as listed in the text below (shaded gray). Furthermore, there were possible test item related findings in the ovaries and adrenal glands of 330 mg/kg females.
Liver:
- Hepatocellular vacuolation was observed in 330 mg/kg females (up to slight). This vacuolation was variable in size (micro- and macro-vesicular) and in general present in zone 1 and 2.
- Hepatocellular hypertrophy was observed at increased incidence and severity in 330 mg/kg females (up to slight, diffuse). The presence of this finding in a single 100 mg/kg female at a minimal degree was considered to be a background finding.
Kidneys and lung:
Intravascular macrophage-like cellular emboli attached to the endothelium were observed at minimal degree in veins of the kidneys, and up to moderate degree in veins of the lung of 330 mg/kg females. The cells were characterized by oval to polygonal nuclei and a foamy or finely vacuolated cytoplasm, with frequent pigment and occasional eosinophilic granules. The cellular emboli were attached to the intima layer of the vessel wall, sometimes lining a larger area and sometimes more focally protruding into the lumen. Since they were only present in females, and in particular those that had experience embryo loss, a uterine or implantation site origin of the cellular emboli could not be ruled out. The remainder of the recorded microscopic findings 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:
- Accuracy of preparation: The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%). No test item was detected in the Group 1 formulation.

-Homogeneity
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

other: see organs listed below

Organ:

kidney

liver

spleen

uterus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

In conclusion, treatment with JNJ-123955-AAA (T000824) by oral gavage in male and female Wistar rats at dose levels of 30, 100, and 330 mg/kg revealed toxicological effects in several monitored endpoints. Based on these results, the NOAEL of the test item to rats in this study was found to be 30 mg/kg, based on clinical signs of toxicity (piloerection of the fur) and inflammation of the endometrium of the uterus (related to reproductive failure) at 100 mg/kg in females.
Therefore, the substance is classified as a repeated dose toxicant (STOT RE, Category 2) according to the CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

other: Autonomic nervous system (males and females) and female reproductive system.

Organ:

uterus

other: Clinical effects of piloerection in males and females may be linked to an autonomic nervous system effect.

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:

Critical effects observed:

not specified

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:

Critical effects observed:

not specified

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:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

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:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in Wistar Han rats, in which the rats were exposed to 0 (vehicle), 30, 100 and 330 mg/kg bw/day of the test substance daily by oral gavage (OECD 422; Meijer, 2018). The vehicle used was propylene glycol and the test solutions were prepared and administered daily. The parameters observed included mortality / viability, clinical signs, functional observations and locomotor activity, body weight, food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, macroscopy at termination, organ weights and histopathology.

Three animals treated at 330 mg/kg were prematurely euthanized for humane reasons. One male and one female (sacrificed at Day 8) had lesions in the mucosa of the nasal cavity, likely attributing to their moribundity, which were suggestive of gavage-related reflux and considered to represent unintended local adversity. Gavage-related reflux may also explain the respiratory symptoms (rales) noted in these animals. Another female was sacrificed at Day 19 post-coitum and showed signs of delivery problems. No test item related mortality was noted up to 100 mg/kg.

The main treatment-related clinical sign of toxicity was piloerection of the fur which occurred in a dose-related manner, starting at 30 mg/kg, most frequently in females. At 100 mg/kg, piloerection of the fur was considered adverse as this sign was noted in several females for approximately 1-2 weeks. The piloerection of the fur noted incidentally at 30 mg/kg was considered not to be toxicologically relevant. The majority of females in the 330 mg/kg group also showed hunched posture for several days.

Other treatment-related clinical findings were considered non-adverse, due to a physiological response or to gavage-related reflux.

Body weight gain was reduced at 330 mg/kg in both sexes in the first treatment week (with some weight loss in a few animals), resulting in slight reductions in mean body weights (less than 10%). This was accompanied by a transient decrease in food consumption. Growth rate had returned to the control level by the end of the second treatment week. Therefore, these effects were regarded as non-adverse.

Serum levels of thyroid hormone T4 in males were dose-dependently decreased from 30 mg/kg onward (by 24, 40 and 48% at 30, 100 and 330 mg/kg, respectively). These changes were observed in absence of accompanying changes in thyroid weight and morphology.

Microscopic examination revealed treatment-related changes in several organs as described below.

Hepatocellular vacuolation (up to moderate) and hepatocellular hypertrophy (up to slight) were observed in the liver at 100 mg/kg (males) and 330 mg/kg (both sexes). This correlated with enlargement of the liver in 330 mg/kg males (liver weight was increased by about 40%, absolute and relative to body weight). Possibly related clinical biochemistry changes noted at 330 mg/kg included non-statistically higher gamma glutamyl transferase activity in both sexes, statistically lower aspartate aminotransferase activity in males, and lower bile acids and cholesterol in females. These liver (related) findings were considered to be non-adverse as there were no degenerative or inflammatory hepatic changes.

Male rats at 330 mg/kg showed an increased severity (up to slight degree) of hyaline droplet accumulation in the kidneys (likely representing alpha2u-globulin, a male rat specific protein). In the absence of indicators of renal tubular damage, this renal change was regarded as non-adverse.

Increased severity (up to moderate) of yellow pigmentation in the spleen (hemosiderin) was observed in females at 30 and 100 mg/kg. Splenic hemosiderin pigmentation occurs as background finding in females subjected to this type of study. Based on the modest increase in severity and the absence of adverse haematological changes, this finding was regarded as non-adverse.

An unusual microscopic change was observed in the kidneys and lung of females treated at 330 mg/kg, consisting of macrophage-like cellular emboli attached to the intima layer of veins (up to moderate degree in the lung, slight in the kidneys). This change has not been described earlier and the underlying mechanism is unclear. However, this change indicates obstruction of the veins which could be considered an adverse finding.

Adverse microscopic changes were noted at high incidence in the adrenals (single cell necrosis of the zona fasciculate, minimal degree) and ovaries (cysts, up to slight degree) of females treated at 330 mg/kg. It remained unclear whether these findings were related with direct organ toxicity or secondary and related with the (stress-full) physiological state of these animals. Based on their high incidence and/or degenerative nature, these findings were regarded as adverse.

Females at 100 and 330 mg/kg had microscopic changes in reproductive organs, mostly the uterus, which were related to their reproductive failure and/or time of pregnancy/lactation. The main finding was inflammation of the endometrium of the uterus. Other findings consisted of endometrial hemorrhage, increased mucification of the vagina epithelium, and, in a single 330 mg/kg female, lobulo-alveolar development of the mammary gland. Macroscopic findings included a thickened horn, one or two early resorptions and/or a dead (large) fetus in the uterus of 330 mg/kg females.

Treatment-related changes in red blood cell parameters were considered non-adverse.

Clinical biochemistry showed changes in some liver-related parameters in both sexes at 330 mg/kg (see above). Additional findings in 330 mg/kg females consisted of higher plasma levels of albumin, total protein, creatinine, sodium, calcium and inorganic phosphate, and lower levels of alkaline phosphatase (ALP), potassium and chloride. Due to the lack of proper control data (due to different physiological state) for comparison, it could not be determined with certainty whether these findings were related to treatment with the test item. Also, their toxicological relevance was unclear.

Based on these results, the No Observed Adverse Effect Level (NOAEL) was concluded to be 30 mg/kg.

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 T000824, the NOAEL was established at 30 mg/kg bw/day, with a LOAEL of 100 mg/kg/day. Therefore, the test item is to be classified as STOT RE 2 according to the CLP Regulation.