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Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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.

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