Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19th Oct 2017 - 11 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial Reverse Mutation Test”, adopted 21st July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No. 440/2008, B.13/14. “Mutagenicity: Reverse Mutation Test Using Bacteria”, 30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
EPA Health Effects Test Guidelines, OPPTS 870.5100 “Bacterial Reverse Mutation Test” EPA 712-C-98-247, August 1998
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:
no
Principles of method if other than guideline:
BRUCE N. AMES, JOYCE MCCANN and EDITH YAMASAKI: Methods for Detecting Carcinogens and Mutagens with the Salmonella / Mammalian-Microsome Mutagenicity Test. Mutation Research, 31: 347-364, 1975
DOROTHY M. MARON and BRUCE N. AMES: Revised Method for the Salmonella Mutagenicity Test. Mutation Research, 113: 173-215, 1983
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
9,10-Anthracenedione, 1,4-diamino-, N,N'-mixed 2-ethylhexyl and 3-[(2-ethylhexyl)oxy]propyl and 3-methoxypropyl derivs.
EC Number:
290-505-4
EC Name:
9,10-Anthracenedione, 1,4-diamino-, N,N'-mixed 2-ethylhexyl and 3-[(2-ethylhexyl)oxy]propyl and 3-methoxypropyl derivs.
Cas Number:
90170-70-0
Molecular formula:
Not available
IUPAC Name:
9,10-Anthracenedione, 1,4-diamino-, N,N'-mixed 2-ethylhexyl and 3-[(2-ethylhexyl)oxy]propyl and 3-methoxypropyl derivs
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Solvent Blue 79B
- Physical state: Semi Solid/highly viscous liquid
- Lot/batch No.:TE2141
- Internal sample ref: S3493 and S3378
Specific details on test material used for the study:
No further details specified in the study report.

Method

Target gene:
histidine and tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
The examined test concentrations in the Initial Mutation Test in both E.Coli WPR uvr A and Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
The examined test concentrations in the Confirmatory Mutation Test in both E.Coli WPR uvr A Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test the different concentrations were used.
Vehicle / solvent:
The solubility of the test item was examined using distilled water, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF). At 100 mg/mL, insolubility was observed using distilled water and, partial dissolution was detected in DMSO. Solubility was observed at the same concentration for DMF, therefore DMF was selected as the vehicle solvent for the study.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water; Dimethyl sulfoxide (DMSO); N,N-Dimethylformamide (DMF).
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine (NPD); 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
Genotypes
In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damage. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair.

Storage
The strains are stored at -80 ± 10ºC in the Culture Collection of the Microbiological Laboratory of the CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.

Confirmation of Phenotypes of Tester Strains
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of CiToxLAB Hungary Ltd.

Spontaneous Reversion of Tester Strains
Each test strain reverts spontaneously at a frequency that is characteristic of the strain.
Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli WP2 uvrA strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011-2016 were (as guide) as follows: Salmonella typhimurium TA98: 9-50, TA100: 54-210, TA1535: 1-46, TA1537: 1-24, Escherichia coli WP2 uvrA: 11-82.

Procedure for Growing Cultures
The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory water bath shaker.

Viability of the Testing Cultures
The viability of each testing culture was determined by plating 0.1 mL of the 10E+5, 10E+6, 10E+7 and 10E+8 dilutions prepared by sterile physiological saline on Nutrient Agar plates.
The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

METABOLIC ACTIVATION SYSTEM
Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory in the CiToxLAB Hungary Ltd according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

DESCRIPTION OF THE TEST PROCEDURE
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, 100 mg/mL stock solution was prepared in DMF. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared of the test item with DMF, which was diluted by serial dilutions to obtain lower doses. The maximum test concentration was 5000 μg test item/plate.
The examined test concentrations in the Initial Mutation Test for all strains, with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
The examined test concentrations in the Confirmatory Mutation Test for all strains, with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

Control Groups Used in the Tests
Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, an untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

Test Method
Initial Mutation Test: This followed the standard plate incoporation procedure. Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45 °C).
The content of the tubes:
top agar 2000 μL
solvent or test item solution (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube.
The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hours.

Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test in the case of Salmonella typhimurium TA98 strain without metabolic activation and Salmonella typhimurium TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation. Since in the Initial Mutation Test a positive effect was observed in the case of Salmonella typhimurium TA98 with metabolic activation, the plate incorporation method was used.

Bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.

Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture, and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator.

After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.
Rationale for test conditions:
In accordance with the test guidelines.
Evaluation criteria:
The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting.

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are 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.
An increase was considered biologically relevant if:
- the number of reversions at least two times higher than the reversion rate of the solvent control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains
- the number of reversions at least three times higher than the reversion rate of the solvent control in Salmonella typhimurium TA1535 and TA1537 bacterial strains

Criteria for a Negative Response:
A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.
Statistics:
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
Postive results observed, but did not show a fully reproducible positive result.
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.

The fFollowing concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.

In the Preliminary Concentration Range Finding Test, a clear increase was observed in the number of revertant colonies in Salmonella typhimurium TA98 strain with and without metabolic activation. The calculated mutation factor values were over the biologically relevant threshold values 2 in most of the cases.

Slight precipitate was observed in the Preliminary Range Finding Test in all examined bacterial strains with and without metabolic activation on the plates at 5000 and 2500 μg/plate concentrations.

Inhibitory or toxic effects of the test item were not detected in the preliminary experiment.

Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 µg/plate.



INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method and the plate incorporation method were used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The Initial Mutation Test and Confirmatory Mutation Test were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.

Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and
1.581 μg/plate.

Slight precipitate was observed in all examined bacterial strains with and without metabolic activation in the Initial Mutation Test on the plates at 5000 and 1581 μg/plate concentrations.

Reduced / slightly reduced background lawn was detected in the Confirmatory Mutation Test in Salmonella typhimurium TA100, TA1535, TA1537 strains without metabolic activation on the plates at 5000 and 1581 μg/plate concentrations.

Reduced colony number was observed in the Initial Mutation Test in Salmonella typhimurium TA100 strain strains with and without metabolic activation on the plates at 5000 μg/plate concentration.

Taking into account the results at all concentrations under all conditions for each bacterial strain, the results for Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2 uvrA strains with and without metabolic activation and
Salmonella typhimurium TA1537 strain without metabolic activation were negative. The results for Salmonella typhimurium TA98 strain with and without metabolic activation using the plate incorporation method were reproducibly positive. Positive
results were observed in the Confirmatory Mutation Test using the pre-incubation method in Salmonella typhimurium TA1537 with metabolic activation. The results for Salmonella typhimurium TA1537 did not show a fully reproducible positive result, the
pattern of results indicates the result for this strain is equivocal (further investigation is not considered to be required because the results of one bacterial strain are clearly positive).


VALIDITY OF THE TESTS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid

Any other information on results incl. tables

Summary Table of the Range Finding Test

 

Concentrations

(mg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

TA98

TA100

-S9

+S9

-S9

+S9

Untreated control

Mean

21.7

23.3

106.0

114.7

MF

0.89

0.89

0.98

1.04

Distilled water control

Mean

--

--

113.7

--

MF

--

--

1.06

--

DMSO control

Mean

21.0

24.0

--

113.0

MF

0.86

0.91

--

1.02

DMF control

Mean

24.3

26.3

107.7

110.3

MF

1.00

1.00

1.00

1.00

5000

Mean

50.0

157.0

95.3

102.3

MF

2.05

5.96

0.89

0.93

2500

Mean

40.0

171.0

105.3

100.7

MF

1.64

6.49

0.98

0.91

1000

Mean

29.7

216.3

101.7

109.0

MF

1.22

8.22

0.94

0.99

316

Mean

24.0

165.0

103.3

104.3

MF

0.99

6.27

0.96

0.95

100

Mean

20.3

121.0

95.7

104.0

MF

0.84

4.59

0.89

0.94

31.6

Mean

26.7

87.3

96.7

97.7

MF

1.10

3.32

0.90

0.89

10

Mean

21.3

73.7

97.3

99.0

MF

0.88

2.80

0.90

0.90

NPD (4mg)

Mean

401.3

--

--

--

MF

19.11

--

--

--

2AA (2mg)

Mean

--

2389.3

--

2464.0

MF

--

99.56

--

21.81

SAZ (2mg)

Mean

--

--

1104.0

--

MF

--

--

9.71

--

 

Summary Table of the Initial Mutation Test

Concentrations

(mg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

22.0

19.3

107.0

95.7

13.3

13.0

12.7

14.7

42.0

43.3

MF

1.20

1.00

1.15

1.01

0.95

1.00

0.95

0.86

0.97

0.99

Distilled water control

Mean

--

--

98.0

--

14.0

--

--

--

44.7

--

MF

--

--

1.05

--

1.00

--

--

--

1.03

--

DMSO control

Mean

17.7

18.3

--

93.7

--

12.3

12.7

15.3

--

44.3

MF

0.96

0.95

--

0.99

--

0.95

0.95

0.90

--

1.02

DMF control

Mean

18.3

19.3

93.3

94.7

14.0

13.0

13.3

17.0

43.3

43.7

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

42.0

108.3

37.3

33.3

11.7

11.3

34.7

38.3

56.0

56.0

MF

2.29

5.60

0.40

0.35

0.83

0.87

2.60

2.25

1.29

1.28

1581

Mean

27.0

171.0

73.7

90.7

11.3

12.0

17.7

33.3

49.0

46.7

MF

1.47

8.84

0.79

0.96

0.81

0.92

1.33

1.96

1.13

1.07

500

Mean

19.7

200.7

78.3

95.0

12.0

12.7

17.3

32.0

49.3

48.7

MF

1.07

10.38

0.84

1.00

0.86

0.97

1.30

1.88

1.14

1.11

158.1

Mean

21.3

141.0

87.0

93.3

12.0

11.7

16.3

25.7

49.3

53.7

MF

1.16

7.29

0.93

0.99

0.86

0.90

1.23

1.51

1.14

1.23

50

Mean

18.0

99.0

91.0

94.7

9.7

13.3

15.0

23.3

47.0

49.7

MF

0.98

5.12

0.98

1.00

0.69

1.03

1.13

1.37

1.08

1.14

15.81

Mean

21.7

56.3

93.0

107.7

15.3

13.0

15.3

22.7

50.0

53.0

MF

1.18

2.91

1.00

1.14

1.10

1.00

1.15

1.33

1.15

1.21

5

Mean

18.0

40.7

92.7

95.3

15.0

13.0

14.7

18.7

48.7

48.3

MF

0.98

2.10

0.99

1.01

1.07

1.00

1.10

1.10

1.12

1.11

NPD (4mg)

Mean

380.0

--

--

--

--

--

--

--

--

--

MF

21.51

--

--

--

--

--

--

--

--

--

2AA (2mg)

Mean

--

2421.3

--

2392.0

--

224.0

--

206.7

--

--

MF

--

132.07

--

25.54

--

18.16

--

13.48

--

--

2AA (50mg)

Mean

--

--

--

--

--

--

--

--

--

249.3

MF

--

--

--

--

--

--

--

--

--

5.62

SAZ (2mg)

Mean

--

--

1130.7

--

1184.0

--

--

--

--

--

MF

--

--

11.54

--

84.57

--

--

--

--

--

9AA (50mg)

Mean

--

--

--

--

--

--

416.0

--

--

--

MF

--

--

--

--

--

--

32.84

--

--

--

MMS (2mL)

Mean

--

--

--

--

--

--

--

--

1066.7

--

MF

--

--

--

--

--

--

--

--

23.88

--

 

Summary Table of the Confirmatory Mutation Test

Concentrations

(mg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

17.3

21.3

103.0

97.0

12.7

9.3

8.3

9.3

69.3

69.3

MF

1.08

1.02

1.08

1.00

0.93

0.72

0.93

0.93

1.01

1.00

Distilled water control

Mean

--

--

94.0

--

8.7

--

--

--

67.0

--

MF

--

--

0.99

--

0.63

--

--

--

0.98

--

DMSO control

Mean

16.7

20.3

--

98.0

--

13.0

8.0

8.3

--

70.3

MF

1.04

0.97

--

1.01

--

1.00

0.89

0.83

--

1.02

DMF control

Mean

16.0

21.0

95.3

97.3

13.7

13.0

9.0

10.0

68.3

69.0

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

35.7

111.0

27.3

68.3

9.3

9.0

4.00

35.7

67.0

67.3

MF

2.23

5.29

0.29

0.70

0.68

0.69

0.44

3.57

0.98

0.98

1581

Mean

29.3

180.3

90.7

99.3

11.3

10.3

16.0

26.0

75.0

66.7

MF

1.83

8.59

0.95

1.02

0.83

0.79

1.78

2.60

1.10

0.97

500

Mean

17.7

179.0

138.3

121.0

10.7

10.0

13.0

22.7

67.3

68.0

MF

1.10

8.52

1.45

1.24

0.78

0.77

1.44

2.27

0.99

0.99

158.1

Mean

21.0

112.7

107.7

97.0

10.7

9.7

12.3

17.0

73.3

70.0

MF

1.31

5.37

1.13

1.00

0.78

0.74

1.37

1.70

1.07

1.01

50

Mean

19.0

89.0

97.0

105.3

12.0

10.0

9.3

16.0

71.3

70.3

MF

1.19

4.24

1.02

1.08

0.88

0.77

1.04

1.60

1.04

1.02

15.81

Mean

18.0

56.7

97.3

100.3

11.7

11.0

12.0

14.7

63.7

66.0

MF

1.13

2.70

1.02

1.03

0.85

0.85

1.33

1.47

0.93

0.96

5

Mean

16.7

32.3

94.3

93.3

10.3

9.7

10.3

10.7

61.0

69.0

MF

1.04

1.54

0.99

0.96

0.76

0.74

1.15

1.07

0.89

1.00

1.581

Mean

17.7

30.3

91.3

104.0

11.0

10.7

8.7

7.7

58.7

67.3

MF

1.10

1.44

0.96

1.07

0.80

0.82

0.96

0.77

0.86

0.98

NPD (4mg)

Mean

472.0

--

--

--

--

--

--

--

--

--

MF

28.32

--

--

--

--

--

--

--

--

--

2AA (2mg)

Mean

--

2473.3

--

2449.3

--

201.0

--

218.7

--

--

MF

--

121.64

--

24.99

--

15.46

--

26.24

--

--

2AA (50mg)

Mean

--

--

--

--

--

--

--

--

--

237.3

MF

--

--

--

--

--

--

--

--

--

3.37

SAZ (2mg)

Mean

--

--

1029.3

--

1076.0

--

--

--

--

--

MF

--

--

10.95

--

124.15

--

--

--

--

--

9AA (50mg)

Mean

--

--

--

--

--

--

422.0

--

--

--

MF

--

--

--

--

--

--

52.75

--

--

--

MMS (2mL)

Mean

--

--

--

--

--

--

--

--

1008.0

--

MF

--

--

--

--

--

--

--

--

15.04

--

Applicant's summary and conclusion

Conclusions:
The test item, Solvent Blue 79B was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method and the plate incorporation method were used.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by frameshifts in the genome of the strains used.

In conclusion, the test item Solvent Blue 79B was shown to be mutagenic in this study. The test item had mutagenic activity in Salmonella typhimurium TA98 bacterial strain with and without metabolic activation. Equivocal results were observed in Salmonella typhimurium TA1537 strain with metabolic activation. No mutagenic activity was observed in Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2 uvrA bacterial strains with and without metabolic activation and in Salmonella typhimurium TA1537 strain without metabolic activation under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced

rats.

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

Based on the results of the Compatibility Test, the test item was dissolved in N,N-dimethylformamide (DMF) at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding

Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50,

15.81 and 5 μg/plate, and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

In the Preliminary Concentration Range Finding Test, in the Initial Mutation Test and in the Confirmatory Mutation Test in the case of Salmonella typhimurium TA98 strain with and without metabolic activation, a clear, reproducible positive effect was

obtained using the plate incorporation method. In the Confirmatory Mutation Test a positive effect was obtained in Salmonella typhimurium TA1537 bacterial strain with metabolic activation using the pre-incubation method. Precipitate / slight precipitate was observed in the Preliminary Range Finding Test in all examined bacterial strains with and without metabolic activation on the plates at 5000

and 2500 μg/plate concentrations. The same effect was detected in all examined bacterial strains with and without metabolic activation in the main tests on the plates at 5000 and 1581 μg/plate concentrations.

Reduced / slightly reduced background lawn was detected in the Confirmatory Mutation Test in Salmonella typhimurium TA100, TA1535, TA1537 strains without metabolic activation on the plates at 5000 and 1581 μg/plate concentrations.

Reduced colony number was observed in the Initial Mutation Test in Salmonella typhimurium TA100 strain strains with and without metabolic activation on the plates at the 5000 μg/plate concentration.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by frameshifts in the genome of the strains used.

Categories Display