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
Study period:
From May 14 to August 3, 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 471 and GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 4-Aminoquinaldine
- Physical state: Slightly yellow powder
- Analytical purity: 98.1%
- Purity test date: 13th April 2015
- Lot/batch No.: 9213/R.83
- Expiration date of the lot/batch: April 2018
- Storage condition of test material: Controlled room temperature (15-25ºC, below 70 RH%)

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The strains differentiate between base-pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations.
The E.coli WP2 uvrA tryptophan (trp) reversion system measures trp- to trp+ reversions. It detects mutagens that cause base-pair substitutions (AT to GC).
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver Homogenate S9 Fraction
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver Homogenate S9 Fraction
Test concentrations with justification for top dose:
Preliminary Concentration Range Finding test: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
Main tests: 5000, 2500, 1250, 625, 312.5, 156.25, 78.125 and 29.0625 µg/plate.
Complementary Confirmatory Mutation Test: 625; 312.5; 156.25; 78.125; 39.0625; 19.5313, 9.7656 and 4.8828 µg/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO).
- Justification for choice of solvent/vehicle: Based on the available information and the results of a solubility test.
Controlsopen allclose all
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 4-nitro-1,2-phenylenediamine (NPD) SIGMA-ALDRICH
Remarks:
TA98. 4 µg in DMSO.
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
TA100 and TA1535. 2 µg in Distilled water
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
TA1537. 50 µg in DMSO.
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli WP2 uvrA. 2 µL in Distilled water.
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
Salmonella strains. 2 µg in DMSO.
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
E.coli WP2 uvrA. 50 µg in DMSO.
Solvent controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
yes
Remarks:
Distilled water
Details on test system and conditions:
METHOD OF APPLICATION:
Initial mutation test: Plate incorporation method.
Confirmatory mutation test: Plate incorporation/Preincubation method.
Complementary confirmatory mutation test: Preincubation method.

DURATION
- Preincubation period: Overnight.
- Exposure duration: 20 minutes
- Expression time (cells in growth medium): 48±1 hours.

NUMBER OF REPLICATIONS: Triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Reduced/slightly reduced background lawn.
Evaluation criteria:
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
(at all concentrations)
Cytotoxicity:
yes
Remarks:
(5000 μg/plate, without metabolic activation)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA1537
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
(at all concentrations)
Cytotoxicity:
yes
Remarks:
(5000, 2500 and 1250 μg/plate, without metabolic activation)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA98
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(1250 μg/plate, without metabolic activation)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA1535
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(1250 μg/plate, without metabolic activation)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA100
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(Complementary confirmatory test: 625 μg/plate without metabolic activation).
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: E.coli WP2 uvr A
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was detected in the Preliminary Range Finding Test.
Slight precipitate was detected on the plates in Initial Mutation Test in S.typhimurium TA1537 at 5000 μg/plate concentration with metabolic activation.

RANGE-FINDING/SCREENING STUDIES: It was observed a dose-related increase in the number of revertant colonies in S.typhimurium TA98 with metabolic activation. In S.typhimurium TA100 mutation factor values above the respective biological threshold value were also observed but the revertant counts were within the historical negative control range.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Inhibitory, cytotoxic effect was seen in the preliminary experiment in the both tester strains at 5000 μg/plate without metabolic activation.
In the Confirmatory Mutation Test using the pre-incubation method, excessive cytotoxicity was observed in the E.coli WP2 uvrA strain without metabolic activation at several concentrations. Therefore an additional experiment was performed in this strain.
Inhibitory, cytotoxicity was observed in the Confirmatory Mutation Test in S.typhimurium TA98 at 5000, 2500 and 1250 μg/plate, without metabolic activation, and in S.typhimurium TA100 and TA1535 at 1250 μg/plate without metabolic activation, and in S.typhimurium TA1537 at 5000 μg/plate without metabolic activation, and in E.coli WP2 uvrA at 625 μg/plate without metabolic activation.

Any other information on results incl. tables

Table 1. Summary table of the Confirmatory Mutation Test and Complementary Confirmatory Mutation test.

Concentrations

(µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimurium

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

16.0

19.0

117.0

113.7

8.0

9.7

5.0

3.3

47.7

54.3

MF

0.83

0.85

1.06

0.98

0.96

1.00

0.83

0.42

0.99

1.10

DMSO control

Mean

19.3

22.3

110.3

116.0

8.3

9.7

6.0

8.0

48.3

49.3

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

122.0

--

12.3

--

--

--

51.3

--

MF

--

--

1.11

--

1.48

--

--

--

1.06

--

5000

Mean

1.0

99.3

0.0

3.3

0.0

0.0

1941.3

806.7

--

8.0

MF

0.05

4.45

0.00

0.03

0.00

0.00

323.56

100.83

--

0.16

2500

Mean

2.3

141.0

3.0

129.0

0.7

4.3

261.0

131.0

--

14.7

MF

0.12

6.31

0.03

1.11

0.08

0.45

43.5

16.38

--

0.30

1250

Mean

7.7

123.0

32.3

219.3

4.3

9.3

21.7

23.7

--

56.3

MF

0.40

5.51

0.29

1.89

0.52

0.97

3.61

2.96

--

1.14

625

Mean

19.0

100.7

116.3

183.7

6.7

11.0

14.7

20.3

17.0

63.0

MF

0.98

4.51

1.05

1.58

0.80

1.14

2.44

2.54

0.35

1.28

312.5

Mean

13.0

86.3

108.3

162.3

8.3

12.7

6.7

8.7

52.3

55.7

MF

0.67

3.87

0.98

1.40

1.00

1.31

1.11

1.08

1.08

1.13

156.25

Mean

17.0

52.3

110.7

137.7

9.0

10.0

7.0

8.7

45.7

52.0

MF

0.88

2.34

1.00

1.19

1.08

1.03

1.17

1.08

0.94

1.05

78.125

Mean

18.0

40.0

117.7

116.7

8.3

12.0

7.0

9.0

48.0

50.3

MF

0.93

1.79

1.07

1.01

1.00

1.24

1.17

1.13

0.99

1.02

29.0625

Mean

16.7

35.3

128.7

111.7

9.0

10.3

8.0

8.0

51.0

48.0

MF

0.86

1.58

1.17

0.96

1.08

1.07

1.33

1.00

1.06

0.97

19.5313

Mean

--

--

--

--

--

--

--

--

51.0

--

MF

--

--

--

--

--

--

--

--

1.06

--

9.7656

Mean

--

--

--

--

--

--

--

--

49.7

--

MF

--

--

--

--

--

--

--

--

1.03

--

4.8828

Mean

--

--

--

--

--

--

--

--

54.0

--

MF

--

--

--

--

--

--

--

--

1.12

--

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
ambiguous

The substance had mutagenic activity in the Salmonella typhimurium TA98 strain in the presence of metabolic activation and in Salmonella typhimuriumTA1537 strain in the presence and absence of metabolic activation system. However , no clear mutagenic activity was observed in the other three experimental strains.
Executive summary:

This study intended to evaluate the mutagenic potential of the test item. A Bacterial Reverse Mutation Assay was performed according to OECD Guideline 471 and following GLP.

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

In the Initial Mutation Test and Confirmatory Mutation Test, clear, reproducible positive effect was obtained in Salmonella typhimurium TA98 bacterial strain with metabolic activation and Salmonella typhimurium TA1537 bacterial strain with and without metabolic activation as the observed revertant colony numbers were above the respective biological threshold value and dose-related trends were also observed.

Inhibitory, cytotoxicity of the test item was observed in the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 bacterial strain at 5000, 2500 and 1250 μg/plate concentration without metabolic activation; and in Salmonella typhimurium TA100 and TA1535 strains at 5000, 2500 μg/plate concentration with and without metabolic activation; and in Salmonella typhimurium TA100 and TA1535 strains at 1250 μg/plate concentration without metabolic activation; and in Salmonella typhimurium TA1535 strains at 1250 μg/plate concentration with metabolic activation; and in Salmonella typhimurium TA1537 strains at 5000 μg/plate concentration without metabolic activation; and in Escherichia coli WP2 uvrA strain at 5000, 2500 μg/plate concentration with metabolic activation and in Escherichia coli WP2 uvrA strain at 625 μg/plate concentration without metabolic activation.

The substance had mutagenic activity in Salmonella typhimurium strains TA98 in the presence of metabolic activation system and in TA1537 strain in the presence and absence of metabolic activation system. However, no clear mutagenic activity was observed in the other three experimental strains.