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

Carcinogenicity

Description of key information

There were no substance-related neoplastic findings noted in rats receiving dietary exposure over a period of 104 weeks or in mice receiving dietary exposure over a period of 80 weeks.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 12 April 2016
Experimental termination date: 15 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

other: Han Wistar, Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River UK Limited, Margate, Kent, UK
Age: 6-7 weeks old
Weight: males between 130 to 197 g, females between 103 to 158 g
Acclimatisation period: 14 days
Housing: animals were housed up to five per cage per sex prior to stratification. Following stratification, animals were housed 4 per cage per sex (unless reduced by mortality) in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings which were provided with a certificate of analysis for significant contaminants. Animals were socially housed for psychological/environmental enrichment and were provided with items such as a device for hiding in and an object for chewing, except when interrupted by study procedures/activities.
Temperature: 18 to 31 °C
Humidity: 27 to 82%
Photoperiod: Lighting was controlled to provide a 12 hour light/darkness cycle
Air changes: at least 10 per hour
Food: SDS Rat and Mouse (modified) No. 1 Diet SQC Expanded (Ground) was provided ad libitum
Water: ad libitum from the public supply

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

Applied in diet: Rat and Mouse No. 1 Maintenance Diet SQC Expanded (Ground)

Details on exposure:

Diet formulations were prepared as serial dilutions from a more concentrated stock premix of at least 200 g. The premix was prepared by firstly mixing the test substance with the required amount of untreated control diet manually at first and then in an automated mortar and pestle and mixing until visibly homogeneous. Diet for the high dose group at the requested concentration was prepared by adding the premix to a suitably sized diet bin, containing the required amount of untreated diet, and this was then mixed for 20 minutes in a diet mixer (Winkworth Change drum mixer). The diets at lower concentrations were prepared as a dilution from the high dose group using the same procedure as above. Diet formulations were prepared at appropriate intervals, stored at ambient temperature and used within the conditions established in a separate study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were sampled at regular intervals during the study. All samples were transferred at ambient temperature to the analytical laboratory at the test facility. The concentrations of the test substance were determined by a suitable UP liquid chromatography method with UV detection. Analysis of samples taken at various intervals throughout the study showed that the analytically determined dietary concentrations ranged from -6.8% to +4.3% of the target concentrations; all samples were within the acceptance criteria of ± 10% of target. For homogeneity, the RSD of concentrations for all samples in each group ranged from 0.7 to 9.5%; all samples were within the acceptance criteria of <10%.

Duration of treatment / exposure:

104 weeks (carcinogenicity study), 52 weeks (chronic toxicity study)

Frequency of treatment:

Daily (dietary)

Dose / conc.:

20 ppm

Dose / conc.:

50 ppm

Dose / conc.:

150 ppm

No. of animals per sex per dose:

Carcinogenicity study: 52 females, 52 males
Chronic toxicity study: 12 females, 12 males

Control animals:

yes, plain diet

Details on study design:

Animals were assigned to groups by a stratified randomisation scheme designed to achieve similar group mean body weights. Males and females were randomised separately. Cages were racked in a random order by placing on racks in horizontal rows starting from the top left corner of the rack.
During the week before the commencement of dosing, individual body weights were checked to ensure all animals were within ± 20% of the mean weight of each sex.

Positive control:

Not applicable

Observations and examinations performed and frequency:

The animals were monitored regularly for general health/mortality and moribundity as well as clinical observations. Body weights and food consumption were measured and recorded at pre-determined intervals. Achieved dosage was determined for each period of food consumption throughout the course of the study. Food utilisation was determined for Weeks 1 to 13. Ophthalmoscopy examinations were carried out during pretreatment for all animals and in Week 52 for Groups 1 and 4 Toxicity Study animals and in Week 104 for Groups 1 and 4 Carcinogenicity Study animals. Detailed functional observations and functional tests (including motor activity) were carried out during pretreatment and Week 51/52. Blood and urine samples were collected from all animals at pre-determined intervals throughout the course of the study to evaluate haematology, coagulation, clinical chemistry and urinalysis parameters.

Sacrifice and pathology:

All animals were euthanised after at least 52 weeks (Toxicity Study) or 104 weeks (Carcinogenicity Study) of treatment and subjected to a detailed necropsy examination, with selected organs being weighed. Unscheduled decedents were subjected to a necropsy examination. Tissues from all animals were subjected to a comprehensive histopathological evaluation.

Statistics:

See any other information

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Chronic study: one female in the 150 ppm dose group was terminated prematurely in week 39 due to adverse clinical signs (caused by adenoma in the pars distalis of the pituitary gland, not treatment related.
Carcinogenicity study: the number of unscheduled deaths in the control, 20 ppm, 50 ppm and 150 ppm were 18/52, 16/52, 14/52 and 19/52 for females and 14/52, 13/52, 19/52 and 12/52 for males. There were no statistically significant differences in mortality between the control and any
other groups. The trend test was not statistically significant for either males or females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain on the end of the study was lower (11%) in high group females (150 ppm), when compared to control animals, however statistical significance was not achieved.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The total number of white blood cells were slightly higher in females receiving 150 ppm at Week 14 which was mainly attributable to a slightly higher number of lymphocytes when compared to concurrent controls. The total number of red blood cells, haemoglobin concentration and haematocrit were slightly lower in females receiving 150 ppm at Week 14 when compared to concurrent controls. Fibrinogen was slightly higher in males receiving 150 ppm at Week 53 when compared to concurrent controls.
The total number of white blood cells were slightly higher in males receiving 50 or 150 ppm at Week 105/106 which was mainly attributable to a slightly higher number of lymphocytes, neutrophils and monocytes when compared to concurrent controls. In addition, the haemoglobin concentration and haematocrit were slightly lower in males receiving 150 ppm at Week 105/106.
As all of these changes were of low magnitude with no correlating histopathology and there were no similar changes at subsequent timepoints, they were considered not to be related to the administration of the substance.
Any other changes in haematology and coagulation parameters were considered to be within normal biological variation and therefore were considered not to be related to the administration of the substance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At Week 27, calcium was slightly lower in males receiving ≥ 20 ppm at Week 27, however no dose relationship was noted and there were no similar changes at other timepoints. In addition, at Week 27 phosphate was slightly higher in males receiving 150 ppm at Week 27. Bile acids and creatinine were slightly higher in females receiving 150 ppm at Week 14 when compared to concurrent controls. Globulin was slightly lower and albumin/globulin ratio was slightly higher in females receiving 150 ppm when compared to concurrent controls at Week 27 only.
As all of these changes were of low magnitude with no correlating histopathology and there were no similar changes at subsequent timepoints, they were considered not to be related to the administration of the substance.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were incidences of statistical significance noted such as: lower basic and fine movement and x-ambulation at Week 51, segment 1 (0-5 min) in females at 20 and 50 ppm, and basic and fine movement at Week 51 segment 2 in females at 20 ppm. However, due to the lack of any dose relationship, no consistency through the observation period or across timepoints these were considered not to be related to the test substance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and covariant kidney weight was slightly higher in males receiving 150 ppm. As this change was of small magnitude and in the absence of any correlating clinical chemistry and histopathological changes, they were considered not to be related to the administration of the substance.
No other test substance-related organ weight changes were noted. There were isolated organ weight values that were different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and unrelated to administration of the substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Chronic toxicity study (52/53 weeks): No test substance related gross findings were noted.
Carcinogenicity study (104 weeks): Small testis was observed in a small number of males administered the substance at equal to or greater than 20 ppm: n = 0 in controls, n = 2 in 20 ppm group, n = 1 in 50 ppm group, n = 3 in 150 ppm group.
This change correlated microscopically with tubular degeneration but of those animals noted with small testis at 20 ppm, the reported small testis was considered secondary to a tumour (Animal 2020) – left testis was small due to a Leydig cell adenoma in the right testis: Animal 2040) – testis was small due to a hemangiosarcoma in the epididymis. In addition, one of the animals at 150 ppm was reported with a small left testis due to a Leydig cell adenoma, in the right testis (Animal 4048).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Chronic toxicity study (52/53 weeks): an increased incidence and severity of minimal to mild centrilobular vacuolation was observed in the liver of males in the 150 ppm group: n = 1 in controls, n = 1 in 20 ppm group, n = 2 in 50 ppm group, n = 10 in 150 ppm group
In the testis, minimal tubular degeneration was observed in 2/12 males at 150 ppm and one male from the control group. In the epididymis, minimal cellular debris was also noted in 1/12 males at 150 ppm. Although the incidence of these testis and epididymis findings at 150 ppm was low and did not achieve statistically significant differences, these findings were considered to be test substance-related since tubular degeneration of the testis and cellular debris of the epididymis was noted in the previous 13 Week Oral (Dietary) Toxicity Study in males at ≥150 ppm. Minimal tubular degeneration in testis: n = 1 in controls, n = 0 in 20 ppm group, n = 0 in 50 ppm group, n = 2 in 150 ppm group. Minimal cellular debris in epididymis: n = 0 in controls, n = 0 in 20 ppm group, n = 0 in 50 ppm group, n = 1 in 150 ppm group
Carcinogenicity study (104 weeks): In the liver, a dose-related, statistically significant increase in the incidence and severity of vacuolation (minimal to marked) was observed in males at ≥50 ppm and females at 150 ppm. Vacuolation in the liver, males: n = 14 in controls, n = 14 in 20 ppm group, n = 27 in 50 ppm group, n = 37 in 150 ppm group; vacuolation in liver, females: n = 6 in controls, n = 8 in 20 ppm group, n = 10 in 50 ppm group, n = 16 in 150 ppm group
Hepatic vacuolation per se, in the absence of any alteration in liver enzymes, organ weight or accompanying microscopic changes, is not considered to be an adverse finding.
In the male reproductive tissues, a statistically significant increase in the incidence of tubular degeneration (minimal to marked) in the testis and reduced sperm (minimal to moderate) in the epididymis were observed at 150 ppm. Tubular degeneration/atrophy (minimal to severe) occurs spontaneously in aged rats (the historical control data range for tubular degeneration/atrophy is 3.8-20.0%). The incidence and severity of tubular degeneration at 20 and 50 ppm was consistent with the concurrent control and the historical control data range, and was therefore not considered to be related to treatment with the substance. Tubular degeneration in testes: n = 6 in controls, n = 7 in 20 ppm group, n = 10 in 50 ppm group, n = 22 in 150 ppm group
In the epididymis, in addition to a reduction in sperm, an increased incidence of minimal cellular debris was also noted in males at 150 ppm, without reaching statistical significance. Both reduced sperm and cellular debris findings are considered secondary to tubular degeneration in the testis. The concurrent control incidence of reduced sperm is considered low compared to the historical control data range and the incidence of findings at 20 and 50 ppm are considered consistent with the background incidence of this finding in this strain of rat at this laboratory. Reduced sperm in epididymis: n = 0 in controls, n = 4 in 20 ppm group, n = 4 in 50 ppm group, n = 6 in 150 ppm group; minimal cellular debris in epididymis: n = 1 in controls, n = 2 in 20 ppm group, n = 1 in 50 ppm group, n = 6 in 150 ppm group

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Equivalent to 2.3 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Equivalent to 3.0 mg/kg bw/day

Key result

Critical effects observed:

no

Non-neoplastic findings in the toxicity study

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (ppm)	0	20	50	150	0	20	50	150
# animals examined	12	12	12	12	12	12	12	11
Liver (# examined)	12	12	12	12	12	12	12	11
Centrilobular vacuolation	1	1	2	10*	12	12	12	11
Minimal	1	0	1	6	0	0	0	0
Mild	0	1	1	4	0	0	0	0
Testis (# examined)	12	12	12	12	0	0	0	0
Minimal tubular degeneration	1	0	0	2	-	-	-	-
Epididymis (# examined)	12	12	12	12	0	0	0	0
Minimal cellular debris	0	0	0	1	-	-	-	-

* Significantly different from controls: p≤0.01 (Fisher’s exact test)

Non-neoplastic findings in the carcinogenicity study

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (ppm)	0	20	50	150	0	20	50	150
# animals examined	52	52	52	52	52	52	52	52
Liver (# examined)	52	52	52	52	52	52	51	51
Vacuolation	14	14	27*	37**	6	8	10	16*
Minimal	11	9	19	13	5	5	7	9
Mild	0	3	7	10	1	2	3	5
Moderate	3	1	1	13	0	1	0	1
Marked	0	1	0	1	0	0	0	1
Testis (# examined)	52	52	52	52	0	0	0	0
Tubular degeneration	6	7	10	22**	-	-	-	-
Minimal	5	3	5	14	-	-	-	-
Mild	1	2	2	3	-	-	-	-
Moderate	0	2	2	4	-	-	-	-
Marked	0	0	1	1	-	-	-	-
Epididymis (# examined)	52	52	52	52	0	0	0	0
Reduced sperm	0	4	4	6*	-	-	-	-
Minimal	0	1	1	0	-	-	-	-
Mild	0	2	2	5	-	-	-	-
Moderate	0	1	1	1	-	-	-	-
Minimal cellular debris	1	2	1	6	-	-	-	-

* Significantly different from controls: p≤0.05 (Fisher’s exact test)

** Significantly different from controls: p≤0.01 (Fisher’s exact test)

Conclusions:

Dietary administration of the substance to Han Wistar Rats at 0, 20, 50 or 150 ppm for a period of up to 104 weeks was well tolerated, with no effects on survival and did not result in any test-substance related neoplastic findings.

Executive summary:

The chronic toxicity and carcinogenicity potential of the substance to Han Wistar rats when administered via diet for 52 and 104 weeks, respectively, was studied under GLP to OECD TG 453. Female and male rats were assigned randomly to the control group (receiving plain diet not containing test substance) or to one of the treatment groups receiving diet containing 20, 50 or 150 ppm test substance. Each group consisted of 12 females and 12 males (toxicity study) or 52 females and 52 males (carcinogenicity study). The analysis of dietary formulations confirmed the correct concentrations of test substance corresponding to nominal concentrations, and homogeneous distribution of test substance in the dietary formulations. The overall mean achieved dosages at week 52 were 1.4, 3.4 and 10.3 mg/kg bw/day in females and 1.1, 2.7 and 8.1 mg/kg bw/day in males; mean achieved dosages at week 104 were 1.2, 3.0 and 9.2 mg/kg bw/day in females and 0.9, 2.3 and 7.0 mg/kg bw/day in males.

One female in the 150 ppm group of the chronic toxicity study was euthanised prematurely due to clinical signs. The cause of morbidity was pars distalis adenoma of the pituitary gland. However, this death was considered to be unrelated to test substance administration. The number of unscheduled deaths in the carcinogenicity study was comparable in the controls and the treatment groups. There were no statistically significant differences in mortality between the control and any other groups.
There were no clinical observations or differences in the number of animals with palpable masses that were considered to be related to the substance.
Body weight gain at the end of the study was lower (11%) in high group females (150 ppm), when compared to control animals, however statistical significance was not achieved. There were no changes in food consumption, food utilisation, water consumption, ophthalmoscopy findings, functional observation, motor activity, haematology, coagulation, clinical chemistry, urinalysis or organ weights that were attributable to administration of the substance.
In the toxicity study, on microscopic evaluation, substance-related changes were observed in the liver (vacuolation), testis (tubular degeneration) and epididymis (cellular debris) in males at 150 ppm. In the carcinogenicity study, on microscopic evaluation, substance-related, non-neoplastic findings were observed in the liver (vacuolation) in males at ≥50 ppm and females at 150 ppm, testis (tubular degeneration) and epididymis (reduced sperm, cellular debris) in males at 150 ppm. Vacuolation in the liver, in the absence of any alteration in liver enzymes, organ weight or other accompanying histopathological changes was considered not to be adverse.
Overall, dietary administration of the substance to Han Wistar rats at 0, 20, 50 or 150 ppm for a period of up to 104 weeks was well tolerated, with no effects on survival, and did not result in any substance-related neoplastic findings.
The dietary administration for a period of 104 weeks resulted in a lower body weight gain in females receiving 150 ppm, testis (tubular degeneration) and epididymis (reduced sperm, cellular debris) in males at 150 ppm. Based on these effects in females and males at 150 ppm, the no observed adverse effect level (NOAEL) was established at 50 ppm, which is equivalent to 2.3 in males and 3.0 mg/kg bw/day in females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

Quality of whole database:

Valid and reliable chronic carcinogenicity study

Justification for classification or non-classification

In the absence of neoplastic effects in the available reliable and valid carcinogenicity studies in rats and mice, a classification of the substance for carcinogenicity is not warranted according to Regulation (EC) No 1278/2008.

Additional information

The carcinogenicity potential of the substance was investigated under GLP in a combined chronic toxicity/carcinogenicity study to OECD TG 453 in Han Wistar rats (Strepka 2019a) and in a carcinogenicity study to OECD TG 451 in Crl-CD1 mice (Strepka 2019b. Test animals were treated with the test substance via the diet for a period of 104 weeks (rats) or 80 weeks (mice). No substance-related neoplastic findings were noted in any of the studies, and the incidences and types of tumours in the treatment groups were not different from the controls. Therefore, the substance was found to not exhibit a carcinogenicity potential in these two rodent species under the conditions of these valid GLP tests.