Registration Dossier

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP study but the determination of mutagenic activity was carried out according to Ames' procedure.

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 M.,YASUI Y. and MATSUMOTO N.
Year:
1983
Bibliographic source:
MUTAT RES 116:217-238.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
A strain as S. typhimurium TA102 or E. coli WP2 or WP2(pKM101) allowing the detection of cross-linking mutagens has not been tested.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): o-Fluoronitrobenzene
- Analytical purity: 98 %

Method

Species / strain
Species / strain / cell type:
other: S. typhimurium TA98, TA1538, TA1537, TA100 and TA1535
Metabolic activation:
with and without
Metabolic activation system:
Male Sprague-Dawley rats, weighing 100-200 g (5 weeks old), were injected with PCB at a dose of 500 mg/kg bw, 5 days before they were killed.
Test concentrations with justification for top dose:
0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28 and 2.56 µl
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
DMSO served as control
Negative solvent / vehicle controls:
yes
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:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 15 min
- Exposure duration: 3 days

MUTATION TEST PROTOCOL:
A poure-plate method for quantitative determination of mutagenic activity was carried out according to Ames' procedure (Ames et al., 1973). In brief, a volume of 0.1 ml of various concentrations of test compounds were added to a sterile test-tube containing 3-6 x 10exp7 bacterial cells, 0.5 ml of S9 mix or sodium phosphate buffer (pH 7.4). This mixutre was preincubated in a shaker water-bath at 37 °C for 15 min, then added to 2 ml molten top agar (45 °C). The contents of each tube were mixed well and immediataly poured onto the surface f a minimal-agar plate. A control plate contained 01.05 ml of DMSO. 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. Test 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
Evaluation criteria:
Chemical inducing more than twice the number of colonies on the control plate were considered as mutagenic.
Statistics:
No statistic was performed.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA98, TA1538, TA1537, TA100 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1.28 µl
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mutagenicity of o-fluoronitrobenzene compound in S. typhimurium TA98, TA 1538, TA1537, TA100 and TA1535

Compound Dose per plate His+ revertants/plate
TA98 TA1538 TA1537 TA100 TA1535
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 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 5 µg 1798 +/-258 - - - - - - - - -
2 µg - - 1649 +/-228 - - - - - - -
9-AA 100 µg - - - 1288 +/-198 - - - - -
2-AA 5 µg - 1249 +/-202 - 753 +/-62 - 132 +/-36 - 1549 +/-269 - 174 +/-23
2-fluoronitrobenzene 0.02 µl 32 +/-3 34 +/-5 23 +/-3 27 +/-6 8 +/-2 12 +/-3 189 +/-17 198 +/-21 27 +/-4 32 +/-4
0.04 µl 30 +/-3 33 +/-3 26 +/- 2 30 +/-4 9 +/-2 12 +/-4 186 +/-18 201 +/-24 31 +/-5 35 +/-7
0.08 µl 31 +/-4 34 +/-3 23 +/- 3 26 +/-4 12 +/-4 8 +/-2  184 +/- 15 190 +/-18 32 +/-6 31 +/-4
0.16 µl 26 +/-3 31 +/- 4 24 +/-2 26 +/-3 10 +/-3 10 +/-2 189 +/-18 194 +/-14 36 +/-4 40 +/-6
0.32 µl 33 +/- 2 22 +/- 3 26 +/-4 33 +/-4 9 +/- 2 9 +/-2 196 +/-17 178 +/- 19 30 +/- 5 32 +/-4
0.64 µl 22 +/- 3 34 +/-6 23 +/-2 24 +/-3 11 +/-2 10 +/- 3 158 +/-12 194 +/-22 29 +/-4 36 +/-6
1.28 µl 2 +/-8* 15 +/- 3 12 +/-5* 19 +/- 3 2 +/-3* 6 +/- 2 133 +/- 18* 156 +/-9* 22 +/- 3 28 +/-6
2.56 µl 0* 0* 0* 0* 0* 0* 0* 46 +/-28* 3 +/-5* 10 +/-8*

Data represents 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 -48h incubation.

* indicates toxic effect.

0 indicates no revertants detectable.

Control: average value of all control groups in each assay.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this test, 2-fluoronitrobenzene did not show any mutagenic effect.
Executive summary:

The mutagenicity of 2-fluoronitrobenzene compound in Salmonella typhimurium (strains TA98, TA1538, TA1537, TA100 and TA1535) was examined. A pour-plate method for quantitative determination of mutagenic activity was carried out according to Ames' procedure (Ames et al., 1973). All tests were performed in duplicate and repeated at least 3 times separately. The tests were carried out under the conditions of absence and presence of liver microsomal activation. Compound tested was dissolved in sterilized dimethyl sulphoxide (DMSO). In all tests the current vehicle and positive control were within acceptable ranges. It was concluded that 2-fluoronitrobenzene did not demonstrate mutagenic potential in this in vitro bacterial reverse mutation assay, under the experimental conditions described.