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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed GLP and OECD guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
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): 2,5- Dimethoxynitrobenzol

Method

Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
pre-experiment/experiment I: 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation test

DURATION
- Exposure: After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY:
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 1535, TA 1537, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
other: postive at toxic concentrations without activation
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Experiment I
Strain: without S9 mix / with S9 mix
TA 1535: 2500 - 5000 / 1000 - 5000
TA 1537: 1000 - 5000 / 2500 - 5000
TA 98: 2500 - 5000 / 2500 - 5000
TA 100: 2500 - 5000 / 2500 - 5000
WP2 uvrA: 5000 / 2500 - 5000

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1535, TA 1537 at 5000 µg/plate and in strain WP2 uvrA from 2500 µg/plate up to 5000 µg/plate in the presence of metabolic activation.

Any other information on results incl. tables

  Summary of Results Pre-Experiment and Experiment I

Study Name: 1261801

Study Code: Harlan-CCR 12614801

Experiment: 1261801 VV plate

Date Plated: 20/04/2009

Assay Conditions:

Date Counted: 23/04/2009

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

DMSO

11 ± 2

9 ± 1

26 ± 6

145 ± 16

51 ± 0

Untreated

14 ± 1

7 ± 1

26 ± 5

167 ± 15

54 ± 9

2,5-

3 µg

13 ± 1

9 ± 4

26 ± 7

142 ± 23

50 ± 12

Dimethoxynitrobenzol

10 µg

16 ± 4

6 ± 4

25 ± 4

139 ± 2

53 ± 11

33 µg

11 ± 5

8 ± 1

24 ± 7

162 ± 4

55 ± 4

100 µg

20 ± 7

10 ± 5

28 ± 7

180 ± 11

53 ± 5

333 µg

17 ± 2

9 ± 1

30 ± 4

228 ± 6

48 ± 3

1000 µg

23 ± 3

17 ± 7 R

36 ± 6

370 ± 7

42 ± 3

2500 µg

28 ± 3 R

9 ± 3 R

59 ± 11 R

847 ± 68 R

50 ± 7

5000 µg

14 ± 8 R

17 ± 2 R

69 ± 13 R

657 ± 35 R

36 ± 14 R

NaN3

10 µg

1978 ± 80

2297 ± 65

4-NOPD

10 µg

354 ± 22

4-NOPD

50 µg

79 ± 3

MMS

3.0 µL

1307 ± 64

With Activation

DMSO

16 ± 3

11 ± 1

34 ± 3

146 ± 15

63 ± 6

Untreated

18 ± 2

13 ± 6

34 ± 9

167 ± 24

59 ± 12

2,5-

3 µg

19 ± 3

17 ± 7

28 ± 7

134 ± 15

58 ± 1

Dimethoxynitrobenzol

10 µg

16 ± 5

12 ± 2

32 ± 4

149 ± 7

53 ± 5

33 µg

18 ± 2

8 ± 4

28 ± 6

165 ± 12

65 ± 6

100 µg

16 ± 4

8 ± 1

32 ± 3

214 ± 13

63 ± 4

333 µg

18 ± 5

13 ± 1

37 ± 5

307 ± 31

60 ± 11

1000 µg

34 ± 6 R

17 ± 5

51 ± 3

657 ± 35

51 ± 13

2500 µg

36 ± 6 R

12 ± 2 R

57 ± 1 R

951 ± 179 R

24 ± 6 R

5000 µg

0 ± 0 R

4 ± 2 R

67 ± 24 R

140 ± 26 R

13 ± 5 R

2-AA

2.5 µg

311 ± 4

275 ± 2

2066 ± 205

2487 ± 70

2-AA

10.0 µg

319 ± 30

Key to Positive Controls

Key to Plate Postfix Codes

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

R

Reduced background growth

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations in the genome of the strains TA 98 and TA 100.
Therefore, 2,5-Dimethoxynitrobenzol is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The test item 2,5-Dimethoxynitrobenzol was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:            3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Strain

Experiment I

without S9 mix

with S9 mix

TA 1535

2500 - 5000

1000 - 5000

TA 1537

1000 - 5000

2500 - 5000

TA 98

2500 - 5000

2500 - 5000

TA 100

2500 - 5000

2500 - 5000

WP2 uvrA

5000

2500 - 5000

No precipitation of the test item occurred up to the highest investigated dose.

Substantial and dose dependent increases in revertant colony numbers were observed following treatment with 2,5-Dimethoxynitrobenzol in strains TA 98 and TA 100. The number of colonies exceeded the threshold of twice in strain TA 98 at 2500 µg/plate and above in the absence of metabolic activation at concentrations showing reduced background growth. The number of colonies exceeded the threshold of twice in strain TA 100 at 1000 µg/plate and above in the absence of metabolic activation and at 333 µg/plate and above in the presence of metabolic activation.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.