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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Feb - 03 Mar 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only 2-aminoanthracene was used to test the efficacy of the S9 mix. Additional mutagens requiring metabolic activation such as benzo(a)pyrene or dimethylbenzanthracene were not used to characterise the S9 mix. Historical control data was not provided.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Current version adopted in 2020

Deviations:

yes

Remarks:

Only 2-aminoanthracene was used to test the efficacy of the S9 mix. Additional mutagens requiring metabolic activation such as benzo(a)pyrene or dimethylbenzanthracene were not used to characterise the S9 mix. Historical control data was not provided.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Guideline in place during study conduct: adopted in 1983

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

Guideline in place during study conduct: adopted in 1983

Deviations:

not applicable

Test guideline 4

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

Adopted in 1985

Deviations:

no

Test guideline 6

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Adopted in 1985

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

Method

Target gene:

his operon for S. typhimurium strains, trp operon for E. coli strain

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver homogenate of 5 - 6 male Sprague Dawley rats (200 - 300 g) pretreated with Aroclor 1254 (single i.p. injection of 500 mg/kg bw) 5 days prior to sacrifice
- method of preparation of S9 mix: The S9 homogenate was diluted 1:10 with a co-factor solution. The resulting S9 mix contained the following components and concentrations: 33 mM KCl, 8 mM MgCl2, 5 mM, glucose-6-phosphate, 4 mM NADP, and 100 mM phosphate buffer (pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 concentration in the S9-mix was 10%. The volume of S9 mix in the final culture medium was 0.5 mL.
- quality controls of S9: Sterility of the S9 mix was indicated by the absence of contamination on the S9 mix sterility check plates.

Test concentrations with justification for top dose:

First experiment (= range-finding experiment), second experiment, and cytotoxicity experiment: 4, 20, 100, 500, 2500, and 5000 µg/plate, with and without metabolic activation, all strains (except for the cytotoxicity experiment, here only TA 100 was tested)
5000 µg/plate was selected as the highest test concentration based on the results of the range-finding experiment, in which no cytotoxicity was observed up to and including the highest concentration of 5000 µg/plate tested.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: N-methyl-N-nitro-N-nitrosoguanidine (MNNG, 2.5 µg/plate, -S9, WP2uvrA), 2-aminoanthracene (2-AA, 0.5 to 10 μg/plate, +S9, all strains)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2 independent experiments were performed.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Following exposure, his+ and trp+ revertant colonies were counted.

Evaluation criteria:

A test substance is considered mutagenic if either of the following conditions under a) and b) is achieved:
a) a test substance produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test substance induces a dose-dependent increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test substance at complete bacterial background lawn.
The test results must be reproducible.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(precipitation was observed at concentrations of 2500 µg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(precipitation was observed at concentrations of 2500 µg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(precipitation was observed at concentrations of 2500 µg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(precipitation was observed at concentrations of 2500 µg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(precipitation was observed at concentrations of 2500 µg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: visible precipitation of the test substance on the plates has been observed at 2500 µg/plate and above
- Definition of acceptable cells for analysis: Identification of the different bacterial strains was performed periodically and all criteria for a valid assay were achieved as described in B.N. Ames, J. McCann and E. Yamasaki: Methods for detecting carcinogens and mutagens with the Salmonella / mammalian-microsome mutagenicity test (Mutation Res. 31 (1975) 347 - 364) and M.H.L. Green and W.J. Muriel: Mutagen testing using trp reversion in Escherichia coli (Mutation Res. 38 (1976) 3 - 32).

RANGE-FINDING/SCREENING STUDY:
The test substance was tested at concentrations of 4 to 5000 µg/plate in TA 100 and proved to be not toxic to the bacterial strain, therefore 5000 µg/plate was selected as the highest concentration tested.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see Table 1 and 2 under "Any other information on results incl. tables".

Ames test:
- Signs of toxicity : No signs of toxicity were noted in any strain up to and included the highest concentration tested, i.e 5000 µg/plate.
- Mean number of revertant colonies per plate and standard deviation : The mean number of revertant colonies was not significantly increased for any strain at any concentration tested. See Table 1 and 2 under "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1. Test results of first experiment (plate incorporation)           

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± standard deviation)
		Base-pair substitution type		Cross-linking type	Frameshift type
		TA 100	TA 1535	WP2 uvr A	TA 98	TA 1537
–	0	173.0 ± 16.1	10.0 ± 2.0	40.7 ± 6.8	36.0 ± 5.2	17.7 ± 2.5
–	0 (DMSO)	162.3 ± 17.2	14.0 ± 6.1	37.0 ± 6.2	32.0 ± 5.3	18.0 ± 4.6
–	4	176.0 ± 7.5	14.0 ± 4.4	37.3 ± 9.6	27.3 ± 4.0	15.0 ± 2.6
–	20	175.7 ± 1.5	13.0 ± 3.6	35.3 ± 3.5	34.0 ± 5.3	20.7 ± 2.5
–	100	173.3 ± 7.5	15.3 ± 2.3	33.0 ± 0.0	30.0 ± 7.2	12.3 ± 1.5
–	500	187.3 ± 22.1	18.3 ± 6.0	34.3 ± 2.1	30.3 ± 8.5	17.0 ± 1.7
–	2500	190.7 ± 11.0 P	14.0 ± 3.5 P	36.7 ± 1.5 P	29.7 ± 2.1 P	12.7 ± 3.5 P
–	5000	206.7 ± 13.1 P	12.0 ± 4.0 P	38.7 ± 3.5 P	30.7 ± 3.8 P	14.0 ± 3.0 P
Positive controls, -S9	Name	SA	SA	MNNG	2-NF	9-AA
	Concentrations (μg/plate)	1	1	2.5	2.5	50
	Mean No. of colonies/plate (average of 3 ± SD)	762.7 ± 29.9	537.0 ± 32.1	225.3 ± 12.6	643.0 ± 83.0	114.3 ± 4.6
+	0	170.7 ± 26.6	13.7 ± 5.9	43.3 ± 6.4	36.3 ± 9.1	10.3 ± 0.6
+	0 (DMSO)	159.0 ± 12.1	11.7 ± 0.6	42.3 ± 4.7	41.3 ± 6.1	11.7 ± 3.2
+	4	158.7 ± 15.6	12.0 ± 1.0	37.3 ± 7.4	41.0 ± 2.6	9.7 ± 4.0
+	20	166.0 ± 17.1	14.3 ± 1.2	48.0 ± 7.5	34.7 ± 2.3	9.3 ± 4.5
+	100	167.3 ± 18.6	12.0 ± 4.6	40.0 ± 7.2	43.0 ± 7.8	10.0 ± 1.0
+	500	180.3 ± 11.5	17.3 ± 4.5	42.0 ± 5.2	40.3 ± 11.6	12.0 ± 1.0
	2500	168.7 ± 6.7 P	16.3 ± 6.4 P	39.7 ± 1.5 P	37.3 ± 10.3 P	10.7 ± 1.5 P
+	5000	192.0 ± 12.8 P	12.0 ± 5.3 P	36.0 ± 2.6 P	33.3 ± 4.5 P	13.0 ± 5.0 P
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	0.5	1	10	0.5	1
	Mean No. of colonies/plate (average of 3 ± SD)	1194.7 ± 49.4	137.7 ± 10.1	273.7 ± 6.7	996.0 ± 82.3	244.0 ± 12.5

2-NF = 2-nitrofluorene

SA = sodium azide

MNNG= N-methyl-N-nitro-N-nitrosoguanidine

9-AA = 9-aminoacridine

2-AA = 2-aminoanthracene

P = precipitate

 

Table 2. Test results of second experiment (plate incorporation)                                  

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± standard deviation)
		Base-pair substitution type		Cross-linking type	Frameshift type
		TA 100	TA 1535	WP2 uvr A	TA 98	TA 1537
–	0	222.3 ± 23.4	15.3 ± 2.1	33.0 ± 7.0	36.0 ± 2.6	20.3 ± 2.1
–	0 (DMSO)	190.7 ± 16.8	12.3 ± 4.0	32.3 ± 5.5	37.0 ± 2.6	24.0 ± 6.6
–	4	188.3 ± 11.6	19.3 ± 5.1	30.0 ± 4.4	35.3 ± 4.0	16.0 ± 9.2
–	20	186.3 ± 14.5	10.7 ± 5.5	32.7 ± 9.0	32.7 ± 10.1	17.7 ± 4.6
–	100	175.0 ± 18.5	12.0 ± 4.6	36.3 ± 3.5	37.7 ± 2.3	17.3 ± 4.5
–	500	187.3 ± 1.2	15.7 ± 7.5	37.0 ± 8.5	32.3 ± 7.0	16.3 ± 4.2
–	2500	199.7 ± 30.0 P	18.3 ± 2.5 P	34.3 ± 8.5 P	27.7 ± 2.9 P	20.3 ± 3.5 P
–	5000	233.7 ± 15.8 P	15.7 ± 1.2 P	34.7 ± 3.2 P	26.7 ± 3.8 P	19.7 ± 3.5 P
Positive controls, -S9	Name	SA	SA	MNNG	2-NF	9-AA
	Concentrations (μg/plate)	1	1	2.5	2.5	50
	Mean No. of colonies/plate (average of 3 ± SD)	949.7 ± 64.5	731.7 ± 16.8	242.7 ± 62.5	1334.7 ± 82.7	155.7 ± 27.0
+	0	186.0 ± 10.1	16.7 ± 6.0	40.7 ± 6.8	39.0 ± 8.2	21.0 ± 3.0
+	0 (DMSO)	163.0 ± 6.0	16.7 ± 1.5	38.0 ± 4.6	40.0 ± 4.4	19.0 ± 2.6
+	4	179.3 ± 17.2	13.0 ± 1.0	36.3 ± 11.2	44.7 ± 3.8	15.7 ± 4.9
+	20	199.3 ± 4.2	14.3 ± 1.5	36.0 ± 5.3	41.0 ± 9.2	14.3 ± 1.2
+	100	199.0 ± 8.2	18.3 ± 2.5	38.7 ± 1.5	42.7 ± 4.9	14.7 ± 0.6
+	500	198.0 ± 7.8	13.3 ± 1.2	39.3 ± 2.3	46.0 ± 7.2	19.0 ± 2.6
	2500	199.0 ± 24.3 P	14.7 ± 3.5 P	39.7 ± 9.0 P	40.0 ± 7.8 P	18.3 ± 3.5 P
+	5000	207.7 ± 4.5 P	16.3 ± 2.9 P	34.0 ± 6.1 P	28.0 ± 7.5 P	10.7 ± 3.1 P
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	0.5	1	10	0.5	1
	Mean No. of colonies/plate (average of 3 ± SD)	2041.7 ± 80.7	167.7 ± 7.6	275.3 ± 57.5	1712.3 ± 42.2	289.7 ± 0.6

2-NF = 2-nitrofluorene

SA = sodium azide

MNNG= N-methyl-N-nitro-N-nitrosoguanidine

9-AA = 9-aminoacridine

2-AA = 2-aminoanthracene

P = precipitate

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study, the test substance was negative for genotoxicity in bacteria with and without metabolic activation.