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Toxicological information

Genetic toxicity: in vitro

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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:
from 2011-11-10 to 2012-05-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study (OECD 471) well performed. No deviation from the protocol of the study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests For Pharmaceuticals, 1995. ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997
Deviations:
not specified
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines, OPPTS 870.5100 "Escherichia coli WP2 and WP2 uvrA Reverse Mutation Assays", EPA 712-C-96-247, June 1996 (Public Draft)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: compact

Method

Target gene:
Histidine gene (Salmonella typhimurium) and tryptophan gene (Escherichia coli WP2 uvrA)
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
other: See Table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
S9 from induced phenobarbital and beta-naphthoflavone rat liver
Test concentrations with justification for top dose:
- 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate (Informatory Toxicity Test, (+/- S9))
- 5000; 1581; 500; 158.1; 50 and 15.81 μg/plate (Initial Mutation Test, (+/-S9))
- 5000; 1581; 500; 158.1; 50 and 15.81; 5 and 1.581 μg/plate (Confirmatory Mutation Test, (+/- S9))
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO and WATER
- Justification for choice of solvent/vehicle: in this study, two vehicle control groups were used depending on the solubility of the test item and the solubility of strain specific positive chemicals. The test item is not soluble in Distilled water. Therefore, the solubility of the test item was examined in Dimethyl sulfoxide (DMSO), Acetone and N,N-Dimethylformamide (DMF). The test item was soluble in all of the three examined solvent. Due to the better biocompatibility to the test system, DMSO was selected for solvent of the study
Controls
Negative controls:
yes
Remarks:
(untreated)
Solvent controls:
yes
Remarks:
two vehicle control groups: DMSO and water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See Table 7.6.1/2
Remarks:
no remarks
Details on test system and conditions:
METHOD OF APPLICATION:
- In agar (plate incorporation): In the Range Finding Test as well as in the Initial Mutation Test
- preincubation: in the Confirmatory Mutation Test

DURATION
- Incubation period: at 37°C for 48 hours.
- Preincubation period: 20 min at 37ºC

NUMBER OF REPLICATIONS: 3 plates/dose/strain. Two independent experiments were performed

DETERMINATION OF CYTOTOXICITY
- Method: the cytotoxicity of the test material was determined using TA100 and TA98 in the presence and absence of metabolic activation system (+/-S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the test each samples (including the controls) were tested in triplicate. The concentrations examined were 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate. After 48 hours incubation at 37°C, the plates were scored for revertant colonies and examined for a thinning of the background lawn.

Evaluation criteria:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

- Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

Criteria for a Negative Response:
A test article was considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:
No data

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
inhibitory, slightly cytotoxic effect of the test item was observed in all examined bacterial strain +/- S9 during the study.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: in the range finding test the concentrations examined were 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate. The observed numbers of revertant colonies were mostly in the normal range, no insolubility or signs of cytotoxicity were observed. Based on these results, the test item concentrations in the initial mutation test were 5000, 1581, 500, 158.1, 50 and 15.81 µg/plate. Examined concentrations in the confirmatory test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: the number of revertants colonies was within the historical control range (the initial mutation test, confirmatory mutation test, vehicle and positive controls)

ADDITIONAL INFORMATION ON CYTOTOXICITY: inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in the initial mutation test in salmonella typhimurium TA100, TA1535 and TA1537 strains at 5000 µg/plate concentrations with metabolic activation; and in salmonella typhimurium TA100 bacterial strains at 5000 µg/plate concentration without metabolic activation. Similarly, inhibitory, cytotoxic effect of the test item (reduced/slightly reduced background lawn development) was observed in the confirmatory mutation test in all strains at 5000 and 1581 µg/plate concentrations with and without metabolic activation.

Any other information on results incl. tables

Table 7.6.1/3: Summary Table of the Initial Mutation Test

Concentrations

(µg/plate)

Mean
values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

22.3

25.3

96.0

103.7

6.7

8.7

9.7

8.0

19.3

37.0

MF

1.08

0.86

1.26

0.92

0.91

0.68

1.07

0.73

0.84

1.05

Distilled water control

Mean

-

-

87.3

-

8.0

-

-

-

25.0

-

MF

-

-

1.14

-

1.09

-

-

-

1.09

-

DMSO
control

Mean

20.7

29.3

76.3

112.3

7.3

12.7

9.0

11.0

23.0

35.3

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

17.0

23.7

62.7

77.7

6.7

7.7

6.7

6.7

21.7

27.3

MF

0.82

0.81

0.82

0.69

0.91

0.61

0.74

0.61

0.94

0.77

1581

Mean

20.3

23.0

83.3

97.7

7.7

13.7

9.7

10.3

23.7

30.0

MF

0.98

0.78

1.09

0.87

1.05

1.08

1.07

0.94

1.03

0.85

500

Mean

21.7

27.0

103.3

111.7

8.0

15.3

11.0

11.0

25.0

34.7

MF

1.05

0.92

1.35

0.99

1.09

1.21

1.22

1.00

1.09

0.98

158.1

Mean

17.7

26.7

118.3

92.7

7.0

10.0

11.0

11.0

24.3

32.0

MF

0.85

0.91

1.55

0.82

0.95

0.79

1.22

1.00

1.06

0.91

50

Mean

19.3

29.7

98.7

106.3

9.3

10.7

9.7

11.0

26.3

27.3

MF

0.94

1.01

1.29

0.95

1.27

0.84

1.07

1.00

1.14

0.77

15.81

Mean

17.3

24.7

95.7

112.0

6.0

10.0

10.0

13.0

24.0

26.3

MF

0.84

0.84

1.25

1.00

0.82

0.79

1.11

1.18

1.04

0.75

NPD (4µg)

Mean

369.7

-

-

-

-

-

-

-

-

-

MF

17.89

-

-

-

-

-

-

-

-

-

2AA (2µg)

Mean

-

3698.7

-

4240.0

-

258.7

-

233.7

-

-

MF

-

126.09

-

37.74

-

20.42

-

21.24

-

-

2AA (50µg)

Mean

-

-

-

-

-

-

-

-

-

299.7

MF

-

-

-

-

-

-

-

-

-

8.48

SAZ (2µg)

Mean

-

-

1153.3

-

1020.0

-

-

-

-

-

MF

-

-

13.21

-

127.50

-

-

-

-

-

9AA (50µg)

Mean

-

-

-

-

-

-

817.3

-

-

-

MF

-

-

-

-

-

-

90.81

-

-

-

MMS (2µL)

Mean

-

-

-

-

-

-

-

-

1073.3

-

MF

-

-

-

-

-

-

-

-

42.93

-

1.   Abbreviations: NPD: 4-nitro-1,2-phenylene-diamine; 2AA:2-aminoanthracene; SAZ: sodium azide;
9AA: 9-aminoacridine; MMS: Methyl-methanesulfonate

2.    Distilled water control was used because of SAZ and MMS

 

Applicant's summary and conclusion

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

Under the test conditions of this study, the test item 1,2-diethoxybenzene had no mutagenic activity in the bacterium tester strains.
Executive summary:

In a reverse gene mutation assay in bacteria (Hargitai, 2012) performed according to the OECD N° 471 guideline and in compliance with GLP,Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were exposed to the test item 1, 2-diethoxybenzene diluted in DMSO at concentrations ranging from 0 to 5000 µg/plate in the presence and absence of metabolic activation system from liver fraction of phenobarbital/beta-naphthoflavone-induced rats (S9-mix).

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

The positive controls induced the appropriate responses in the corresponding strains.

Under the test conditions, 1, 2-diethoxybenzene did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used with and without metabolic activation during the study as there was no evidence of induced mutant colonies over background. Inhibitory, slightly cytotoxic effect of the test item was observed in all examined bacterial strains with and without metabolic activation during the study.

In conclusion, 1,2-diethoxybenzene had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

This study is considered as acceptable as it satisfied the criteria of the OECD guideline N° 471.