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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:
weight of evidence
Study period:
From 2008-12-22 to 2009-01-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch No.: 2008-9-16
Purity: >95.0%

Method

Target gene:
trpE ochre
Species / strain
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : purchased from a commercial source and stored at approximately -80℃.
- method of preparation of S9 mix: The S9 mix contained S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water. All the cofactors were filter-sterilised before use.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500 and 5000 µg/plate
No evidence of toxicity was obtained following exposure to test item up to 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Suspensions of test item in water (purified in-house by reverse osmosis) containing 0.15% bacteriological agar were used in this study.

- Justification for choice of solvent/vehicle: It has been shown in a previous study conducted in this laboratory that the test item is insufficiently soluble in solvents compatible with the test system.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide
Remarks:
In the absence of S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
In the presence of S9 mix
Details on test system and experimental conditions:
Mutation test procedure
- First test:
Aliquots of 0.1 mL of the test substance suspensions (seven concentrations up to 5000 μg/plate), positive control or negative control were placed in glass vessels. The negative control was the chosen vehicle, aqueous 0.15% agar solution. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).
Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be obtained, unless otherwise justified by the Study Director.

- Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.

- Stability, homogeneity and formulation analysis
The stability of test item and the stability and homogeneity of test item in the vehicle were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice the concurrent vehicle controls, with some evidence of a positive dose response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fai1 to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained. Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgment.

Results and discussion

Test results
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
First test
No evidence of toxicity was obtained following exposure to test item. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control counts were obtained following exposure to test item at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Second test
No evidence of toxicity was obtained following exposure to test item.
No substantial increases in revertant colony numbers over control counts were obtained following exposure to test item at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Any other information on results incl. tables

 

Results of Ames test for test item using Escherichia coli

 

 Table 1– results obtained in test 1 without metabolic activation 

 

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard deviation

Fold increase relative to vehicle

Individual revertant colony counts

 

WP2 uvrA (pKM101)

Water + 0.15% agar

 

196.3

4.7

 

200, 191, 198

Test item

5 µg

182.3

8.1

0.9

175, 191, 181

15 µg

149.0

5.6

0.8

150, 154, 143

50 µg

175.3

12.3

0.9

165, 189, 172

150 µg

207.0

9.6

1.1

218, 200, 203

500 µg

197.0

26.5

1.0

177, 227, 187

1500 µg

197.3

22.2

1.0

221, 177, 194

5000 µg

176.3

15.6

0.9

160, 191, 178

WP2 uvrA (pKM101)

NQO

(positive control)

2 µg

1950.7

65.6

9.9

2022, 1893, 1937

 

NQO = 4-Nitroquinoline-1-oxide

 

 

Table 2– results obtained in test 1 with metabolic activation 

 

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard deviation

Fold increase relative to vehicle

Individual revertant colony counts

 

WP2 uvrA (pKM101)

Water + 0.15% agar

 

186.0

9.2

 

188, 194, 176

Test item

5 µg

176.3

21.5

0.9

198, 155, 176

15 µg

200.3

26.9

1.1

231, 181, 189

50 µg

191.3

27.2

1.0

205, 160, 209

150 µg

185.3

23.5

1.0

204, 159, 193

500 µg

204.7

30.9

1.1

240, 183, 191

1500 µg

200.3

11.0

1.1

209, 188, 204

5000 µg

168.7

18.9

0.9

177, 147, 182

WP2 uvrA (pKM101)

AAN

(positive control)

10 µg

667.7

45.2

3.6

663, 625, 715

 

AAN = 2-Aminoanthracene

 

 

 Table 3– results obtained in test 2 without metabolic activation 

 

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard deviation

Fold increase relative to vehicle

Individual revertant colony counts

WP2 uvrA (pKM101)

Water + 0.15% agar

 

141.0

3.5

 

137, 143, 143

Test item

50 µg

156.0

10.1

1.1

167, 154, 147

150 µg

195.0

22.3

1.4

213, 170, 202

500 µg

140.0

31.0

1.0

172, 110, 138

1500 µg

168.3

12.4

1.2

176, 175, 154

5000 µg

150.0

27.5

1.1

174, 120, 156

WP2 uvrA (pKM101)

NQO

(positive control)

2 µg

2053.3

811.8

14.6

2890, 2001, 1269

 

NQO = 4-Nitroquinoline-1-oxide

  

 

Table 4– results obtained in test 2 with metabolic activation 

 

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard deviation

Fold increase relative to vehicle

Individual revertant colony counts

WP2 uvrA (pKM101)

Water + 0.15% agar

 

183.7

4.7

 

189, 182, 180

Test item

50 µg

170.0

36.2

0.9

136, 166, 208

150 µg

191.7

33.5

1.0

203, 154, 218

500 µg

181.7

27.8

1.0

213, 160, 172

1500 µg

167.3

17.0

0.9

180, 174, 148

5000 µg

166.7

32.3

0.9

172, 196, 132

WP2 uvrA (pKM101)

AAN

(positive control)

10 µg

559.7

54.9

3.0

608, 500, 571

 

AAN = 2-Aminoanthracene

 

Applicant's summary and conclusion

Conclusions:
It is concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

In this in vitro assessment of the mutagenic potential of test item, a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), was exposed to test item suspended in aqueous 0.15% agar solution, which was also used as a negative control The test procedure was in compliance with OECD 471.

 

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.

 

Concentrations of test item up to 5000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strain in either mutation test following exposure to test item.

 

No evidence of mutagenic activity was seen at any concentration of test item in either mutation test.

 

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.

 

It is concluded that test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.