5-chloro-1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one

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 150 mg/kg body weight/day (OECD 422, Van Otterdijk, 2016). The NOAEL is established as 50 mg/kg body weight/day and the LOAEL as 150 mg/kg body weight/day. The substance is therefore classified as STOT RE 2, according to 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:

2015-08-24 to 2015-12-23

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

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

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 : A14JB3414 (pilot phase), A15CB1464 (main phase)
- Expiration date of the lot/batch: 2015-10-01 (pilot phase), 2016-05-18 (main phase)
- Purity test date: 2014-12-22 (pilot phase), 2016-02-23 (main phase)

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 of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was confirmed as part of the analytical method development and validation study (Test Facility Study No. 509776).

FORM AS APPLIED IN THE TEST (if different from that of starting material): White suspension (pilot phase; group 1); white turbid fluid (pilot phase; group 2); Suspension (main phase)

OTHER SPECIFICS: correction factor: 1.00

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: Crl:WI(Han) (outbred, SPF-Quality) from Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
pilot phase: females approx. 8 weeks (group 1) or 10 weeks (group 2)
main phase: females approx. 11 weeks (at start pretest) and 13 weeks (at start F0-treatment); males approx. 9 weeks (at start F0-treatment)
- Weight at study initiation: 295-331 g (males, main phase), 207-239 g (females, main phase)
- 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: 2015-10-07 (start pretest; females aged approx. 11 weeks); 2015-10-21 (start treatment; males aged approx. 9 weeks); 2015-11-27/28/29/30 and 2015-12-1/10 (delivery of litters)
To: 2015-12-11/14/15/23 (necropsy females); 2015-11-19 (necropsy males); 2015-12-9/10/11/14/22 (necropsy pups)

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 (specific gravity 1.00)

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 1.00 as used. 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 and on information provided by the Sponsor.
- Concentration in vehicle: 0 mg/mL (group 1), 3 mg/mL (group 2), 10 mg/mL (group 3), 30 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 (27 October 2015; Day 21 of treatment), according to a validated method (Test Facility Study no. 509776). Sextuplicate samples (i.e. 3 sets of duplicate samples) were collected. Two sets of duplicate samples were stored 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% compared to those obtained during the method validation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration.
Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Stability of the test item under test conditions was demonstrated in the method validation study (Test Facility Study no. 509776). Density was determined from all formulations on a single day during the treatment phase to express analytical concentrations in mg/mL.

Duration of treatment / exposure:

29 days (males), 51-63 days (females), 38-55 days (females that failed to deliver healthy offspring)
pups: were not doses 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.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

group 1

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

group 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

group 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

group 4

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were based on the results of a 10-day range finding study with T000990 (Test Facility Study no. 509027; pilot phase) in which doses of 100 and 300 mg/kg/day were administered. No mortality occurred. At 300 mg/kg/day, hunched posture, piloerection, lethargy and/or salivation were noted for all animals from Day 6 onwards. At 100 mg/kg/day, clinical signs were confined to the occurrence of piloerection for two out of three animals on Day 7. At 300 mg/kg/day, weight loss was recorded for two out of three animals between Days 1-5 (-2 and -5%) and for one out of three animals also between Days 5-10 (-10%), and food intake was reduced. At 100 mg/kg/day, body weight and food intake were considered to be normal for all animals. At both 100 and 300 mg/kg/day, liver weights were increased, and kidney weights were considered to be normal. No macroscopic abnormalities were noted at 100 and 300 mg/kg/day. Based on these data, the highest dose level to be administered in the main study was selected to be 150 mg/kg/day upon consultation with the Sponsor.
- 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 (early in the morning and close to the end of the working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least immediately 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.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure 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 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 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 partial 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, 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)
- Maternal animals: All surviving animals, on PND 14-16 (following delivery), Post-coitum day 26-27 (females with evidence of mating, but which failed to deliver) or within 24h after total litter loss (females with total litter loss)

GROSS PATHOLOGY: Yes
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated and 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: Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F) (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin: Cervix (F), Clitoral gland (F), Coagulation gland (M), 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 (for Group 4 females only reproductive organs were examined histopathologically
from selected females which had implantation sites only (nos. 75 and 79) or were not pregnant (no. 80). No further females were available with live pups; Additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males that failed to sire (see table below) to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups; Histopathological examination of the mammary gland was also conducted for female nos. 71, 74, 77 and 78 (Group 4) that had a total litter loss; Thyroid glands, liver, thymus and spleen of all selected 5 animals of Groups 2 and 3 (males and females), based on (possible) treatment-related changes in these organs in Group 4.
- 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg, hunched posture and/or piloerection were observed in 6 out of 10 males on some occasions during Weeks 3 and 4 of treatment and among all females from Week 2 of treatment onwards. Pale faeces was additionally noted for females at 150 mg/kg from Week 5 of treatment onwards. Salivation seen after dosing of both sexes at 50 and 150 mg/kg and for males also at 15 mg/kg 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). This sign may be related to taste of the test item.
Any other clinical signs noted during the treatment period 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 did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No unscheduled mortality occurred during the study. At 150 mg/kg, 4 females (nos. 71, 74, 77 and 78) were sent for necropsy due to total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg, females had statistically significantly lower body weights and body weight gain or slight weight loss during the first week (up to 4%) over the 14-day premating period. Weight gain was lower on Days 17 and 20 of the post-coitum (statistically significant), and absolute body weights were statistically significantly lower on several occasions during post-coitum. Body weights of the 150 mg/kg group females were also statistically significantly lower on Day 1 of lactation, but weight gain during lactation (based on Day 1 lactation body weights) was similar between the groups. At 15 and 50 mg/kg (both sexes) and at 150 mg/kg (males), body weights and body weight gain remained essentially in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg, females had a lower food intake (before and after correction for body weights) over the premating period, and also during the last week of the post-coitum and during the lactation period (being statistically significant over Days 0-4 and 14-20 of the post coitum period). At 15 and 50 mg/kg (both sexes) and at 150 mg/kg (males), food intake (before and after correction for body weights) remained in the same range as controls over the treatment period.

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):

No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significantly lower activated partial thromboplastin time (APTT) for males at 150 mg/kg was considered not toxicologically relevant since the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
− Higher alanine aminotransferase activity (ALAT) in males at 150 mg/kg (approximately 28% higher than control mean). Mean ALAT levels of female dose groups also appeared to show a trend towards an increase, but no level of statistical significance was achieved.
− Higher mean aspartate aminotransferase activity (ASAT) of females at 15, 50 and 150 mg/kg (approximately 45, 28 and 16% higher than control mean, respectively). A relatively high individual variation was noted across the dose groups, no clear dose-related response was noted and no level of statistical significance was achieved.
− Higher total bilirubin in males at 150 mg/kg (approximately 17% higher than control mean), and in females at 50 and 150 mg/kg (approximately 42% higher than control mean at both dose levels), not statistically significant at 150 mg/kg.
− Higher bile acid in males at 150 mg/kg (approximately 459% higher than control mean), and in females at 15, 50 and 150 mg/kg (approximately 329, 733 and 11,336% higher than control mean, respectively); a relatively high individual variation was noted across the female dose groups, and statistical significance was only achieved at 150 mg/kg.
− Lower sodium in males at 150 mg/kg (approximately 2% lower than control mean).
− Higher potassium in males at 150 mg/kg (approximately 8% higher than control mean).
− Lower chloride in males at 150 mg/kg (approximately 2% lower than control mean.
Thyroid hormone analyses: Mean serum T4 levels in F0 males appeared lower than controls (not statistically significant; mean approximately 24% lower than control mean).
The statistically significantly lower inorganic phosphate level of males at 15 mg/kg occurred in the absence of a dose-related trend and was therefore not considered to be related to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Females at 150 mg/kg showed a statistically significantly lower grip strength of the forelimbs (approximately 29% lower than controls). This was primarily due to a low value for one female (no. 72). Without the value for this female, the mean was 846±67 gram, and not statistically different from the control mean (approximately 21% lower than controls). Hindlimb grip strength of these females also appeared slightly lower (approximately 22% lower than controls), but did not achieve a level of statistical significance.
In addition, motor activity (total movements and ambulations) of males and females at 150 mg/kg appeared lower than controls (approximately -30% and -37% for males and females, respectively). For females, motor activity showed an apparent trend towards a decrease over the dose groups. None of these variations achieved a level of statistical significance, and all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Grip strength of males was considered to have been unaffected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following changes in organ weights (before and after correction for body weight) were noted at 150 mg/kg:
− Higher liver weight (absolute and relative) for males and females (mean relative weights were approximately 37 and 22% higher than control means, respectively).
− Higher kidney weights (relative) for males (mean relative weight was approximately 16% higher than control mean).
Other statistically significant differences in organ weights and organ to body weight ratios among the dose groups occurred in the absence of a dose-related trend and were therefore not considered to be related
to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following test item-related microscopic findings were noted:
− Increased incidence and severity of hepatocellular vacuolation and hepatocellular hypertrophy in the liver in males starting at 50 mg/kg and in females in the 150 mg/kg group only.
− Thymus cortical atrophy in 40-50% of the animals of the males and females in the 150 mg/kg group and in 20% of the females of the 15 and 50 mg/kg group.
− Increased severity of follicular cell hypertrophy in the thyroid gland in males and females of the 50 and 150 mg/kg group.
− Increased severity of extra-medullary hematopoiesis in the spleen in males of the 150 mg/kg group.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

50 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

histopathology: non-neoplastic

other: lower fore- and hindlimb grip strength and lower motor activity

Critical effects observed:

not specified

Conclusions:

Based on these results, the following No Observed Adverse Effect Level (NOAEL) was derived:
Parental NOAEL: 50 mg/kg.
The substance is therefore classified as STOT RE 2, according to CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

other: Neurological system, Reproductive system, Hepatobiliary system

Organ:

liver

thyroid gland

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

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:

other justification

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 toxicity: oral

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats were exposed to 0 (vehicle), 15, 50, 150 mg/kg bw/day via gavage (OECD 422, Van Otterdijk, 2016). The vehicle used was CMC (carboxymethyl cellulose) 1% aqueous carboxymethyl cellulose and the test solutions were prepared daily and administered within 6 hours after preparation.

The test item did not cause mortality (treatment-related) during the study; however, male and female rats exposed to 150 mg/kg/day presented clinical signs (hunched posture and/or piloerection and/or pale faeces) from week 2 of treatment and onwards. Also, lower body weight, lower food intake, hepatocellular vacuolation, cortical atrophy of the thymus, thyroid gland follicular hypertrophy, lower fore- and hindlimb grip strength and lower motor activity were observed in male and female rats exposed to 150 mg/kg/day. Non-adverse treatment-related changes in clinical biochemistry parameters occurring essentially at 150 mg/kg/day consisted of lower sodium and chloride, and higher total bilirubin, potassium, bile acid and aspartate aminotransferase activity.

Based on the abovementioned considerations, the NOAEL was considered to be 50 mg/kg bw/day (nominal dose received).

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

Based on the available data and according to the criteria of the CLP Regulation, T000990 should be classified as STOT RE 2 via the oral route.