1,3-difluorobenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Structural specificity of aromatic compounds with special reference to mutagenic activity in Salmonella typhimurium - A series of chloro- or fluoro-nitrobenzene derivatives

Author:

Shimizu Makoto, Yasui Yoshiyuki and Matsumoto Nobuo

Year:

1983

Bibliographic source:

Mutation research, 116, 217-238

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3-difluorobenzene

EC Number:

206-746-5

EC Name:

1,3-difluorobenzene

Cas Number:

372-18-9

Molecular formula:

C6H4F2

IUPAC Name:

1,3-difluorobenzene

Test material form:

liquid

Details on test material:

- Name of the test material: m-difluorobenzene
- Purity: 97%
- Source: Aldrich Chemical Co. Inc.

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA1537, TA1538.

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation system S9 mix was prepared from a liver microsomal  fraction (S9 fraction) of male Sprague-Dawley rats induced with PCB at a dose of 500 mg/kg bw. 

Test concentrations with justification for top dose:

0, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56, 5.12, 10.24 µl/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

Plates were inverted and incubated at 37°C in the dark for 3 days. Colonies of his + revertants were counted after incubation, and chemicals inducing more than twice the number of revertant colonies on the control plate were considered as mutagenic.
All tests were performed in duplicate and repeated at least 3 times separately.
A contamination test was carried out through each experiment. The background bacterial lawn was routinely checked by microscopy, as high doses of the complexes proved toxic to all strains resulting in a thinning out of the bacterial lawn.
2 strains (TA98 and TA100) were checked routinely for the presence of the ampicillin resistance for the R factor.
First, the tests were carried out without metabolic activation and were terminated if the mutagenicity was positive. But if the results were negative, tests with metabolic activation were carried out additionally.

Results and discussion

Test results

Key result

Species / strain:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA1537, TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 10.24 µl/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Mutagenicity results

Compound	Dose per plate	His+ revertants/plate
		TA 98		TA1538		TA1537		TA100		TA1535
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
DMSO (control)	0.05 mL	28 ± 6 (16 – 23)	30 ± 5 (18 – 25)	22 ± 7 (13 – 17)	24 ± 5 (16 – 22)	8 ± 3 (5 – 8)	11 ± 4 (6 -10)	181 ± 23 (148 - 166	175 ± 36 (152 – 170)	32 ± 8 (18 – 26)	30 ± 9 (18 – 27)
ENNG	2 µg	-	-	-	-	-	-	1994 ± 377	-	-	-
	10 µg	-	-	-	-	-	-	-	-	2489 ± 287	-
2-NF	2 µg	1798 ± 258	-	-	-	-	-	-	-	-	-
	5 µg	-	-	1659 ± 228	-	-	-	-	-	-	-
9-AA	100 µg	-	-	-	-	1288 ± 198	-	-	-	-	-
2-AA	5 µg	-	1249 ± 202	-	753 ± 62	-	132 ± 36	-	1549 ± 269	-	174 ± 23
1 ,3-DFB	0.08 µL 0.16 µL 0.32 µL 0.64 µL 1.28 µL 2.56 µL 5.12 µL 10.24 µL	30 ± 4 34 ± 3 36 ± 4 31 ± 3 28 ± 2 26 ± 2 19 ± 3 0*	28 ± 4 31 ± 4 35 ± 6 35 ± 6 31 ± 4 22 ± 2 23 ± 3 0*	21 ± 2 22 ± 3 22 ± 2 23 ± 4 27 ± 7 30 ± 6 7 ± 3* 0*	26 ± 3 28 ± 4 24 ± 3 28 ± 4 33 ± 7 26 ± 3 18 ± 2 0*	12 ± 3 10 ± 2 8 ± 2 8 ± 1 9 ± 2 10 ± 3 4 ± 4* 0*	10 ± 2 9 ± 2 11 ± 3 7 ± 1 12 ± 4 9 ± 2 8 ± 1 0*	201 ± 24 188 ± 16 202 ± 18 201 ± 23 186 ± 20 177 ± 19 101 ± 38* 0*	224 ± 22 207 ± 32 196 ± 15 188 ± 24 199 ± 17 190 ± 14 167 ± 16 0*	28 ± 4 33 ± 6 38 ± 6 26 ± 3 39 ± 7 34 ± 4 30 ± 3 4 ± 8*	38 ± 5 28 ± 3 32 ± 4 31 ± 4 37 ± 8 35 ± 6 28 ± 3 9 ± 6*

Data represent the results of 3 separate experiments and give the mean values of 3 plates. The number of revertant colonies indicates mean ± standard deviation. All these revertant colonies were counted after 68-72 h incubation.

() indicates range in number of revertant colonies of control (DMSO) which was counted after 44-48 h incubation.

* indicates toxic effect.

0 indicates no revertants detectable.

ENNG: N-Ethyl-N'-nitro-N-nitrosoguanidine

2 -NF: 2 -nitrofluorene

9 -AA: 9 -aminoacridine

2 -AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:

Under the test conditions, 1,3-difluorobenzene did not induce gene mutations by base-pair changes or frameshifts in the genome of the strains used.