4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start: 13 July 2016
Experimental end (last day of pathology data recording): 14 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This short-term repeated dose toxicity study was generated to meet the data requirements of regulations not related to REACH in non-EEA countries.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Cas Number:

2061933-85-3

Molecular formula:

C23H19Cl2F4N3O4

IUPAC Name:

4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

other: RccHan:WIST

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHanTM;WIST) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Envigo RMS Limited
Age: 76 to 82 days
Weight: 268 to 324 g (males), 173 to 212 g (females)
Acclimatisation: 12 days
Housing: Animals were housed individually (main study) in polycarbonate cages with stainless steel mesh lid with wood bedding. Two pellets of diet were added directly to the shavings after dose administration each day as forage food. An Aspen chew block was provided to each cage throughout the study and replaced when necessary. Plastic shelter was provided to each cage throughout the study.
Diet: Teklad 2014C Diet, ad libitum
Water: potable water from public supply, ad libitum
Temperature: 20 to 24 °C
Humidity: 40 to 70%
Lighting: artificial lighting, 12 hours light to 12 hours darkness cycle

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

Approximately 24 hours before treatment commenced the dorsum between the limb girdles was clipped free of hair, as close as possible, using electric clippers. An area estimated to be at least 10% of the total body surface was clipped. The animals were clipped again as required, at a minimum frequency of weekly, avoiding damage to the site. Any re-clipping was undertaken at the end of the working day after removal of the Elizabethan collars.
The application site was moistened with approximately 0.2 mL of purified water and the dose was applied to the middle of the clipped site. Care was taken to ensure that the dose was distributed evenly across the test site. The sham control was treated similarly, but with the absence of test substance.
Unmedicated gauze dressing (2- or 4-ply) was applied immediately after dosing, held in position around the trunk with a cotton wool pad, a length of ‘Wide Open Weave (WOW)’ bandage and a Tubigrip bandage with sufficient tension to ensure dose remained in contact with the skin.
Not less than six hours (no more than six hours and 15 minutes). The semi-occlusive dressing was removed carefully and the exposed area cleansed with copious quantities of warm tap water (approximately 30-35°C) and dabbed-dry with disposable paper towels to reduce the risk of oral ingestion of the test material during grooming. After the application site had been washed and dried, an Elizabethan collar was fitted to each animal for a period of not less than 30 minutes (no more than 60 minutes).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Six hours

Frequency of treatment:

Five days per week for four weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Ten females and ten males

Control animals:

yes, sham-exposed

Details on study design:

Three days before the start of the treatment, variations in body weight of the animals were checked to ensure that they did not exceed ±20% of the mean for the appropriate sex.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

Blood samples were taken from the tail vein at defined time points after dosing for investigating the toxicokinetics. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Detailed observations were recorded daily during the first week of treatment and once weekly during the following weeks of treatment at defined time points before and following dosing. The dermal application site was examined for signs of dermal irritation before each administration. A detailed physical examination was performed on each animal before treatment commenced and during each week of treatment. Body weight was recorded before treatment commenced, on the day of first treatment, on the following day and weekly thereafter. The weight of food supplied to each cage, remaining food and an estimate of spilled food was recorded for the week before treatment commenced and for each week of treatment. Ophthalmic examinations were done before treatment started and in week 4. Haematology investigations on peripheral blood were carried out a necropsy, and bone marrow smears were prepared immediately following sacrifice on completion of the scheduled treatment period. Blood chemistry was analysed at necropsy. All animals were subject to a detailed necropsy at the end of the study. Organs were weighed and tissues were routinely preserved for histological investigations.

Sacrifice and pathology:

Animals were euthanised at the end of the scheduled treatment period, and tissues were routinely preserved for histological investigations.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. The following data types were analysed at each timepoint separately:
- Body weight, using gains over appropriate study periods
- Food consumption, over appropriate study periods
- Hematology
- Blood chemistry
- Organ weights, absolute and adjusted for terminal body weight
- Pathological findings, for the number of animals with and without each finding
The following sequence of statistical tests was used for body weight, food consumption, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. Treated groups were compared to Control using Dunnett's test (Dunnett 1955, 1964).
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to Control using Steel's test (Steel 1959). For macropathology and histopathology findings, the treatment groups were compared using pairwise comparisons of each dose group against the control using one-tailed Fisher’s exact tests (Fisher, 1973) for an increase. For histopathology findings, tissues with the majority of animals examined were analysed statistically. All groups were included in the statistical analysis of adrenals, duodenum and jejunum and control and high dose only for the other tissues. For organ weight data, analysis of covariance was also performed, in addition to analysing the absolute organ weight data, using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights. Significant differences between Control and treated groups were expressed at the

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The behaviour and appearance of the animals were unaffected by treatment.
There were no findings at the dermal application sites that were attributed to treatment with the substance. Intermittent eschar-formation occurred at the dermal application site of one male and two females given 100 mg/kg/day, two males and one female given 300 mg/kg/day and one male given 1000 mg/kg/day, and erythema was recorded in one female given 100 mg/kg/day, one male given 300 mg/kg/day and one male and one female given 1000 mg/kg/day. The low incidences of these findings indicated that they were incidental.
One female given 300 mg/kg/day (No. 121) was seen to be gasping upon return to the home cage following dosing on Day 2, and on Day 17 a female given 1000 mg/kg/day (No. 114) displayed signs of gasping upon return to the home cage, which continued until 1-2 hours after dosing but was then absent at the end of the working day. Given the lack of similar findings in any other animals, this transient finding was considered incidental to treatment.

Dermal irritation:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Control female No. 139 was killed for welfare reasons on Day 1 after a traumatic skin injury was observed adjacent to the application site. Macroscopic findings confirmed the presence of an open area on the skin; this animal was subsequently replaced with a spare animal (No. 141) which was treated from Day 2 in the same manner as the other control animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gains of females given 1000 mg/kg/day were lower than those of the controls, with the extent of the reduction being 35%, and this difference from controls was statistically significant. The effect was particularly marked during the final two weeks where there was a 48% reduction, compared to controls. There was also a reduction of weight gain in the final two weeks in females given 300 mg/kg/day.
There was no clear effect of treatment upon the body weights of males. At 1000 mg/kg/day there was a 42% reduction in the first two weeks but, in contrast, the weight gains of these animals were higher than controls in Week 3 and 4. The overall (Day 1 to 28) weight gains were similar to controls at all doses. There was considered to have been no effect of treatment upon body weight at 100 mg/kg/day in both sexes and at 300 mg/kg/day in males.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was persistently low, when compared to pre-treatment and controls, in females given 300 or 1000 mg/kg/day, with males given 1000 mg/kg/day showing a similar finding in the first two weeks of treatment only.
There was considered to have been no effect of treatment upon food intake at 100 mg/kg/day in both sexes and at 300 mg/kg/day in males.

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematological examination performed after four weeks of treatment revealed, when compared with controls, high neutrophil and lymphocyte counts, attaining statistical significance at 1000 mg/kg/day in the males and at 300 and 1000 mg/kg/day in the females, but there was no clear dose-relationship in either sex. There were also minor increases of eosinophil and basophil counts in males and females given 1000 mg/kg/day and monocyte counts in males given 1000 mg/kg/day but despite these differences attaining statistical significant in most cases, the degree of increase was unlikely to be of any toxicological significance. As a consequence of these findings the total leucocyte counts of females given 300 mg/kg/day and of males and females given 1000 mg/kg/day were higher than controls. Platelet counts were high, when compared to controls, at all doses in females and at 1000 mg/kg/day in males.
All other differences from control, including those attaining statistical significance, were considered due to natural biological variation and were therefore unrelated to treatment. Such differences included the statistically significantly high reticulocyte count and red cell distribution width in males given 1000 mg/kg/day since there were no alterations of any of the other erythrocyte indices and the majority of values were within or only marginally above the background control mean ranges (0.182x1012 to 0.226x1012/L for reticulocyte count and 11.6 to 13.0% for red cell distribution width). They also included minor variations of clotting times (low prothrombin times in males, where the majority of values were within or slightly below the background control mean range (22.7 to 25.6 seconds) and low activated partial thromboplastin times in the high dose females, where 8/10 values were within the concurrent control range, although the majority (9/10) were outside the background range and was considered to be of no toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of plasma after four weeks of treatment revealed, when compared with controls, slightly high alkaline phosphatase activities in females given 300 or 1000 mg/kg/day and high alanine amino transferase activities in females given 1000 mg/kg/day. There were no similar trends in the males. Females given 300 or 1000 mg/kg/day had higher plasma urea concentrations than the controls, though statistical significance was attained only at 1000 mg/kg/day. Males were unaffected.
There was a clear and statistically significant reduction of plasma cholesterol and triglyceride concentration in females given 1000 mg/kg/day. Although the majority of individual values were within or below the background control mean range (cholesterol 1.74 to 2.20 mmol/L and triglycerides 0.25 to 0.45 mmol/L) they were generally lower than concurrent controls and these changes were probably related to treatment. Similar findings were not observed in males.
There were a few disturbances of plasma electrolyte concentrations. There was a clear and dose-related reduction of chloride concentration in males given 300 or 1000 mg/kg/day which may have associated with to the small reduction of plasma sodium concentration in these animals. Plasma potassium concentrations were high in in males given 300 mg/kg/day and in both sexes given 1000 mg/kg/day. In addition, small reductions of plasma calcium concentration occurred at all doses and in both sexes. In the absence of any corroborative histopathological findings in the liver and kidney which could account for these changes, they are considered to be incidental and unrelated to treatment.
All other differences from control, including those attaining statistical significance, were minor, lacked any evidence of dose-relationship and, consequently, were attributed to normal biological variation. Such differences included the high aspartate aminotransferase activity in males given 1000 mg/kg/day, where the difference was due primarily to one animal (No. 13) with a particularly high value (212 U/L) that was considerably above the background range. They also included the increases of albumin/globulin ratios at all doses since there was no clear dose-response, the all values were within or only marginally above the background range and there were no significant changes of total protein and albumin concentration that would account for these differences from controls.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The analysis of organs weights after four weeks of treatment revealed, when compared with controls, a dose-related increase of absolute and body weight adjusted adrenal weights in males given 100 mg/kg/day and in males and females given 300 or 1000 mg/kg/day.
In addition, slightly high body weight adjusted liver weights were recorded in males given 300 mg/kg/day and in males and females given 1000 mg/kg/day, and high body weight-adjusted kidney weights in males given 1000 mg/kg/day.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Small intestine: Epithelial vacuolation within the duodenum was observed at 100, 300 or 1000 mg/kg/day in females, with the incidence and the extent of the finding being dose-related. Similar dose-related changes within the jejunum were also observed at 300 or 1000 mg/kg/day in females. The epithelial changes were characterized by cellular swelling and pale, vacuolated cytoplasm, typically distributed around the region of the villus tip. The changes within the small intestine positively correlated with statistically significant reductions in body weight gain, amongst females treated with 1000 mg/kg/day. These findings were not considered adverse where the incidence was low within the treatment group and where there was no correlating impact on any blood biochemistry parameters or on body weight gain. Epithelial vacuolation in duodenum in females: n = 0 in controls, n = 1 in 100 mg/kg bw dose group, n = 2 in 300 mg/kg bw dose group, n = 7 in 1000 mg/kg bw dose group; epithelial vacuolation in jejunum in females: n = 0 in controls, n = 0 in 100 mg/kg bw dose group, n = 2 in 300 mg/kg bw dose group, n = 7 in 1000 mg/kg bw dose group.
Adrenal glands: Hypertrophy of the zona fasciculata, generally associated with vacuolation, was observed at 300 or 1000 mg/kg/day in both sexes, with the incidence and extent of the hypertrophy being dose-related. The morphological changes within this organ correlated with a statistically significant increase in mean absolute organ weight at 300 and 1000 mg/kg/day in both sexes. Cortical hypertrophy in Zona fasciculata in males: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 4 in 300 mg/kg dose group, n = 7 in 1000 mg/kg dose group, and in females: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 3 in 300 mg/kg dose group, n = 9 in 1000 mg/kg dose group. Cortical vacuolation in Zona fasciculata in males: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 1 in 300 mg/kg dose group, n = 7 in 1000 mg/kg dose group, and in females: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 1 in 300 mg/kg dose group, n = 9 in 1000 mg/kg dose group.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

adrenal glands

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level following 4 weeks of semi-occluded dermal exposure for 6 hours per day, five days per week was 100 mg/kg bw/day.

Executive summary:

The potential systemic toxicity of the substance to female and male Han Wistar rats following sub-acute dermal exposure under semi-occlusion for a period of four weeks was studied under GLP to OECD TG 410. Animals were exposed to nominal doses of 100, 300 or 1000 mg/kg bw/day for six hours, on five days per week, during four weeks. The systemic exposure to the substance increased sub proportionally with respect to dose in male rats and proportionally in female rats. The systemic exposure to the substance was similar in males and females at 100 mg/kg/day, but higher in females at 300 and 1000 mg/kg/day. Body weight gain and food consumption were low in females given 1000 mg/kg/day, with the extent of the reduction of body weight gain being particularly marked during the final two weeks of treatment. There was also a persistent reduction of food consumption, resulting in reduced weight gain, in the final two weeks in females given 300 mg/kg/day. In males given 1000 mg/kg/day body weight gain and food consumption was low in the first two weeks of treatment. There were a number of effects with regards to haematology and blood chemistry, however there were no associated histological findings and these effects were therefore considered to be incidental and unrelated to treatment. After four weeks of treatment there was a dose-related increase of adrenal gland weight in males given 100 mg/kg/day and in males and females given 300 or 1000 mg/kg/day, slightly high liver weights in males given 300 mg/kg/day and in males and females given 1000 mg/kg/day, and high kidney weights in males given 1000 mg/kg/day. There were no treatment-related macroscopic findings.
Histopathological findings that were attributed to treatment were epithelial vacuolation in the duodenum in females given 100, 300 or 1000 mg/kg/day and in the jejunum of females given 300 or 1000 mg/kg/day, and hypertrophy and vacuolation of the adrenal gland zona fasciculata at 300 and 1000 mg/kg/day in both sexes.