Ethanone, 2,2-dichloro-1-(2,3-dihydro-3-methyl-4H-1,4-benzoxazin-4-yl)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 1987 to 3 April 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

A minimum of 5 strains were not tested. The test did not include S typhimurium TA102 or E.coli.

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2-dichloro-1-[(3R)-3-methyl-3,4-dihydro-2H-1,4-benzoxazin-4-yl]ethan-1-one; 2,2-dichloro-1-[(3S)-3-methyl-3,4-dihydro-2H-1,4-benzoxazin-4-yl]ethan-1-one

EC Number:

619-372-6

Cas Number:

98730-04-2

Molecular formula:

C11H11Cl2NO2

IUPAC Name:

2,2-dichloro-1-[(3R)-3-methyl-3,4-dihydro-2H-1,4-benzoxazin-4-yl]ethan-1-one; 2,2-dichloro-1-[(3S)-3-methyl-3,4-dihydro-2H-1,4-benzoxazin-4-yl]ethan-1-one

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

0.08, 0.31, 1.2, 4.9, 19.5, 78.1, 312.5, 1250, 5000 µg/0.1 mL (toxicity test)
20, 78, 313, 1250 and 5000 µg/0.1 mL (mutagenicity test). Analytical purity: 98.3%

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone, the vehicle was chosen due to its solvent properties and low toxicity to the bacteria.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: daunorubicin-HCl

Remarks:

TA98, 5 and 10 µg/0.1 mL, without S9

Controls 2

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

TA100, 0.125 and 0.25 µg/0.1 mL, without S9

Controls 3

Positive control substance:

sodium azide

Remarks:

TA1535, 2.5 and 5 µg/0.1 mL, without S9

Controls 4

Positive control substance:

other: 9(5)aminoacridine hydrochloride monohydrate

Remarks:

TA1537, 50 and 100 µg/0.1 mL, without S9

Controls 5

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA98, TA100 and TA1537, 5 µg/0.1 mL, with S9

Controls 6

Positive control substance:

cyclophosphamide

Remarks:

TA1535, 250 µg/0.1 mL, with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Each plate contained approximately 20 mL mimimum agar plus salts and glucose; 0.1 mL of a solution of the test substance or vehicle and 0.1 mL of bacterial culture in 2.0 mL of soft agar.
- The soft agar composed of 100 mL 0.6% agar with 0.6% NaCl and 10 mL of a solution of 0.5 mM 1-histidine and 0.5 mM +biotin.
- With metabolic activation, 0.5 mL S9 was also added.

DURATION
- Incubation: 48 hrs at 37±1.5°C in darkness

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

The test substance is considered positive if one or more of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA98, TA1535 and TA1537
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA100.
- a concentration-related effect should be demonstrable

Statistics:

not applicable

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Due to a growth-inhibiting effect of the test substance in the repeat experiments with and without S9, the number of back-mutant colonies was reduced with strain TA1537 at the upper concentrations.

At 1250 and 5000 µg/0.1 mL the test substance precipitated in soft agar.

Table 1: Test 1 without S9

	TA98	TA100	TA1535	TA1537
Control	29	95	12	6
20 µg/0.1 mL	34	98	12	6
78 µg/0.1 mL	20	88	10	7
313 µg/0.1 mL	16	110	10	11
1250 µg/0.1 mL	25	103	10	5
5000 µg/0.1 mL	17	97	11	5

Table 2: Test 1 with S9

	TA98	TA100	TA1535	TA1537
Control	41	131	18	17
20 µg/0.1 mL	44	131	14	26
78 µg/0.1 mL	43	128	15	14
313 µg/0.1 mL	41	129	17	15
1250 µg/0.1 mL	45	131	13	16
5000 µg/0.1 mL	41	126	17	13

Table 3: Test 2 without S9

	TA98	TA100	TA1535	TA1537
Control	27	129	10	7
20 µg/0.1 mL	27	122	15	5
78 µg/0.1 mL	24	114	16	6
313 µg/0.1 mL	27	94	16	7
1250 µg/0.1 mL	22	88	14	3
5000 µg/0.1 mL	17	80	12	2

Table 4: Test 2 with S9

	TA98	TA100	TA1535	TA1537
Control	34	129	14	13
20 µg/0.1 mL	32	131	12	10
78 µg/0.1 mL	36	158	23	10
313 µg/0.1 mL	40	147	14	8
1250 µg/0.1 mL	43	159	15	12
5000 µg/0.1 mL	45	126	9	6

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

There was no evidence of the induction of point mutations by the test substance , with or without S9 mix in the strains of S.typhimurium used in these experiments.

Executive summary:

In a bacterial mutagenicity study using S.typhimurium strains TA98, TA100, TA135 and TA1537 with 20, 78, 313, 1250 and 5000 µg test substance/0.1 mL, with and without S9 microsomal activation, there was no evidence of the induction of point mutations.