ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Jul - 18 Aug 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study was performed in accordance with an old version of the OECD TG 474; some parameters do not meet current requirements.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Current version adopted in 2016

Deviations:

yes

Remarks:

1000 polychromatic erythrocytes were counted for each animal. Body weight, justification for single dose treatment, and historical control data not provided. Dose spacing exceeds currently recommended factor.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Guideline in place during study conduct: adopted in 1983

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Not specified

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

Adopted in 1985

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-870-0

EC Name:

-

Cas Number:

163520-33-0

Molecular formula:

C18H17NO3

IUPAC Name:

ethyl 5,5-diphenyl-4,5-dihydro-1,2-oxazole-3-carboxylate

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse has been chosen for this study since it provides a convenient in vivo mammalian model.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Tierzucht Schönwalde GmbH i.G., Schönwalde, Germany
- Age at study initiation: 8 weeks
- Body weight at study initiation: males: 33 - 44 g (mean: 38.1 g), females: 28 - 43 g (mean: 32.6 g)
- Assigned to test groups randomly: yes
- Housing: group housed, 5 animals per cage, Makrolon cages Type 3, on softwood granulate
- Diet: rat/mice diet ssniff R/M-H (V 1534) (ssniff GmbH, Soest, Germany), ad libitum
- Water: tap water in drinking water quality in plastic bottles, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): housing in air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 0.2, 1 and 2% (w/v)
- Amount of vehicle (gavage): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test substance was suspended in sesame oil at appropriate concentrations. A magnetic stirrer was used to keep the preparations homogeneous until the completion of dosing.

Duration of treatment / exposure:

single treatment by gavage

Frequency of treatment:

single treatment by gavage

Post exposure period:

animals were sacrificed 12, 24 or 48 h post dosing and bone marrow smears were obtained

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw (total dose)

No. of animals per sex per dose:

5 animals per sex per dose and per sampling time point

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (Endoxan)
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw, single oral dose

Examinations

Tissues and cell types examined:

Tissue: bone marrow
Cell type: erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a preliminary dose range finding study, oral administration of 400 mg/kg bw test substance resulted in mortality in male and female mice. Clinical signs of toxicity (spontaneous activity decreased, increased activity when
touched) were still observed at 200 mg/kg bw. Therefore, the highest sublethal dose of 200 mg/kg bw was selected for the main study.

TREATMENT AND SAMPLING TIMES: In conformity with the test procedure, the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 h after administration of the single, oral dose.

DETAILS OF SLIDE PREPARATION: Femoral bone marrow was flushed into the centrifuge tubes containing 3 mL of fetal bovine serum. A suspension was formed. The mixture was then centrifuged for 5 min at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 h. Slides were fixed with methanol and stained according to May-Grünwald-Giemsa staining.

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes, and the number of normocytes with micronuclei occurring in the 1000 normocytes, were evaluated statistically.

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes.
A test substance was considered positive if there was a significant dose-related increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points compared with the concurrent negative control group. Individual and/or group mean values should exceed the laboratory historical control range. A test substance producing no significant dose related increase in the number of micronucleated polychromatic erythrocytes, and the values within the historical control range, is considered not mutagenic in this system.
For the assay to be valid, individual and/or group mean values for the positive control should exceed the laboratory's historical control range.

Statistics:

One-sided Wilcoxon-Test was performed for each treatment interval and for polychromatic and normochromatic erythrocytes. These tests are performed sequentially with a multiple level of significance of 5%. Tests on lower dose groups are only performed if the higher dose group is significantly different from the control. If there is a difference between the positive and negative control (24 h) (two-sided Wilcoxon-Test with a 5%-level of significance) two-sided Wilcoxon-Tests are also performed sequentially for the ratio of polychromatic erythrocytes for each treatment interval (12 h, 24 h, 48 h) at a multiple level of significance of 5%. Actual data were also compared with historical controls. Lower dose groups are only tested if the higher dose group is significantly different from the control.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

200 mg/kg bw: 24 h sampling time point: 1 male with spotted/patterned appearing hepatic tissue

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 200 - 1200 mg/kg bw in 3 animals per sex and dose
- Clinical signs of toxicity in test animals:
1200 mg/kg bw: spontaneous activity decreased, squatting posture, ataxic gait, prone position, forward crawling, staggering gait, palpebral fissure narrow, irregular respiration, panting, flanks pinched in, clonic convulsions, no macroscopic findings were observed; mortality: 2/3 males, 3/3 females
1000 mg/kg bw: spontaneous activity decreased, squatting posture, unsteady gait, staggering gait, prone position, flanks pinched in, coat bristling, panting, clonic convulsions, palpebral fissure narrow, palpebral fissure very narrow, no macroscopic findings were observed; mortality: 1/3 males, 3/3 females
800 mg/kg bw: spontaneous activity decreased, squatting posture, prone position, staggering gait, stilted gait, ataxic gait, panting, clonic convulsions, no macroscopic findings were observed; mortality: 3/3 males, 2/3 females
600 mg/kg bw: spontaneous activity decreased, increased activity when touched, squatting posture, unsteady gait, forward crawling, stilted gait, ataxic gait, prone position, flanks pinched in, marked flank respiration, tonic convulsions when touched, palpebral fissure narrow, lacrimation clear, colourless; macroscopic findings: hepatic tissue lightly coloured, distinctly patterned appearence of hepatic tissue; mortality: 2/3 males, 2/3 females
400 mg/kg bw: spontaneous activity decreased, increased activity when touched, stilted gait, forward crawling, unsteady gait, squatting posture, prone position, coat bristling, marked flank respiration, panting, palpebral fissure narrow, palpebral fissure very narrow, twitching; macroscopic findings: hepatic tissue lightly coloured, distinctly patterned appearence of hepatic tissue; mortality: 2/3 males, 1/3 females
200 mg/kg bw: spontaneous activity decreased, increased activity when touched; no macroscopic findings were observed; mortality: 0/6 animals

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups was within the normal range of the negative control groups.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test substance.
- Appropriateness of dose levels and route: In a preliminary dose range finding study, oral administration of 400 mg/kg bw test substance resulted in mortality in male and female mice and 200 mg/kg bw clinical signs of toxicity. Therefore, the highest sublethal dose of 200 mg/kg bw was selected for the main study.
- Statistical evaluation: One-sided Wilcoxon-Test was performed for each treatment interval.

For details see results tables under "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay in male animals.

			Mean PCEs / 1000 NCEs at sampling time ± SD			Total micronuclei per 1000 PCEs at sampling time			Total micronuclei per 1000 NCEs at sampling time
Exp group	Number of animals	Dose [mg/kg bw]	12 h	24 h	48 h	12 h	24 h	48 h	12 h	24 h	48 h
Vehicle control (corn oil)	5	0	1.1±0.08	0.8±0.07	0.8±0.10	1±0.0	1.2±0.11	1.4±0.09	1.8±0.08	0.8±0.08	1.6±0.09
Positive control (Cyclophosphamide)	5	50	n.d	0.8±0.19	n.d.	n.d.	40±0.73	n.d.	n.d.	1.60±0.05	n.d.
Test substance	5	20	1±0.22	1.0±0.09	0.8±0.10	1±0.1	2±0.12	0.8±0.04	0.6±0.05	0.80±0.04	0.8±0.11
Test substance	5	100	0.8±0.09	0.9±0.07	0.8±0.07	1.2±0.11	1±0.07	0.8±0.08	1.4±0.09	0.8±0.08	1±0.10
Test substance	5	200	1.1±0.09	1.1±0.09	0.9±0.19	2.4±0.11	1.8±0.11	0.8±0.04	0.8±0.13	0.6±0.09	0.8±0.13

n.d. = not determined

 

Table 2: Results of the in vivo micronucleus assay in female animals.

			Mean PCEs / 1000 NCEs at sampling time ± SD			Total micronuclei per 1000 PCEs at sampling time			Total micronuclei per 1000 NCEs at sampling time
Exp group	Number of animals	Dose [mg/kg bw]	12 h	24 h	48 h	12 h	24 h	48 h	12 h	24 h	48 h
Vehicle control (corn oil)	5	0	1.1±0.13	1±0.10	1±0.16	2±0.17	1.2±0.11	0.8±0.08	0.4±0.05	1.2±0.13	0.4±0.09
Positive control (Cyclophosphamide)	5	50	n.d.	0.7±0.18	n.d.	n.d.	29.8±0.73	n.d.	n.d.	0.8±0.08	n.d.
Test substance	5	20	1.0±0.21	1.1±0.14	0.9±0.13	1.2±0.08	2.4±0.11	1±0.1	1±0.07	0.2±0.04	1±0.07
Test substance	5	100	0.8±0.10	0.8±0.20	0.9±0.17	1.8±0.08	1.2±0.11	1±0.17	0.6±0.09	0.6±0.05	0.6±0.09
Test substance	5	200	1.0±0.09	1.0±0.13	0.8±0.14	1.2±0.13	2.2±0.18	1.2±0.11	1±0.07	0.8±0.08	0.6±0.05

n.d. = not determined

 

Table 3: Results of the in vivo micronucleus assay.

Treatment group	Dose [mg/kg]	Sampling time [h]	PCE with MN ± SD	Mean frequency of PCE with MN	PCE/NCE ratio
Vehicle control	0	12	1.5±0.13	0.2	1.1±0.11
	0	24	1.2±0.10	0.1	0.9±0.15
	0	48	1.1±0.09	0.1	0.9±0.15
Test substance	200	12	1.8±0.13	0.2	1.1±0.13
	200	24	2.0±0.14	0.2	1±0.12
	200	48	1.0±0.08	0.1	0.9±0.16
Positive control (Cyclophosphamide)	40	24	34.9±0.87*	3.5	0.8±0.18

* statistically significant (p <0.05)

Applicant's summary and conclusion

Conclusions:

The test substance was not mutagenic (clastogenic) in the present mouse bone marrow micronucleus test.