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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

One Ames study in 1991 shows negative to S. typhimurium (TA1535, TA1537, TA98 and TA100) and one Ames study in 2009 shows negative to E. coli ( WP2 uvr A pKM 101) strains.

Link to relevant study records

Referenceopen allclose all

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
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
Specific details on test material used for the study:
Batch No.: 2008-9-16
Purity: >95.0%
Target gene:
trpE ochre
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.
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.
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.

 

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

 

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.

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 1991-07-16 to 1991-08-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study in accordance with recognised test guideline but: Ecoli not tested Test substance Lot/batch No.: not stated Test substance expiration date of the lot/batch: not stated NOTE: study deemed acceptable because spectral data for test item are available, covering approximately before and after the test period - see section 1.4 Analytical Information.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
First issued 1985, amended 1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Purity: Ca. 100%
Target gene:
histidine-dependent
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Test organisms
Cultures of the histidine-dependent strains of Salmonella typhimurium were derived from cultures provided by Prof. Bruce Ames, University of California. The characteristics of the individual strains are as follows:

TA 1535 - contains a histidine missense mutation but is also deficient in a DNA repair system (uvr B) and has a defective lipopolysaccharide coat on the cell wall. It is reverted by many agents causing base-pair substitutions, but is not sensitive to frameshift mutagens.

TA 100 - is the same as TA 1535 but contains a resistance transfer factor conferring ampicillin resistance and increasing sensitivity to some mutagens (plasmid pKM 101). In addition to base-pair substitutions, it is also able to detect certain frameshift mutagens.

TA 1537 - bears a histidine frameshift mutation. Like TA 1535, it is defective in a DNA repair system and lipopolysaccharide coat. It is sensitive to agents causing frameshift mutations involving insertion or deletion of a single base-pair.

TA 98 - contains another histidine frameshift mutation. Again it has a defective DNA repair system and lipopolysaccharide coat but also contains the pKM 101 plasmid. It is reverted by agents causing deletion of two adjacent base-pairs (double frameshift mutations), but not by simple alkylating agents causing base-pair substitutions.
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Details refer to appendix 2.
Test concentrations with justification for top dose:
50, 158, 500, 1580 and 5000 µg/plate
Vehicle / solvent:
The test item was found to be insufficiently soluble in solvents compatible with the test system (water, ethanol, dimethyl sulphoxide and acetone). Suspensions of test item in purified water (obtained by reverse osmosis) containing 0.15% agar, freshly prepared immediately before use with the aid of an Ultra-Turrax high-shear mixer, were therefore employed in this study.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
For TA1537, TA 100 and TA 98 with/without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For TA 98 without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
For TA 1537 without 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:
For TA 1535 with/without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For TA 1535 and TA 100 with/without S9-mix
Details on test system and experimental conditions:
Preliminary toxicity test
A suspension of test item was prepared at 25 mg/mL in purified water containing 0.15% agar, and an aliquot of this suspension (0.2 mL) was transferred to a sterile tube containing molten, histidine-deficient top-agar (2.0 mL) maintained at 45°C. An additional aliquot (0.1 mL) of the test material suspension was similarly transferred to another tube of molten top-agar (2.0 mL). Three serial ten-fold dilutions in molten top-agar were prepared from each of the above preparations, giving a series of eight different concentrations of test material from 2.5 µg to 5 mg per plate. All tubes were inoculated with an overnight culture of strain TA 98 (0.1 mL) and overlaid onto minimal medium plates. Control plates were prepared containing top-agar and culture alone, top-agar, 0.15% agar (0.2 mL) and culture, and top-agar and test material (0.1 mL) without bacterial culture.
The plates were incubated at 37°C for 2 days and were then examined for the presence of a background lawn of non-revertant colonies; toxicity of the test material is shown by absence or thinning of the background lawn. The level of test material chosen as the top level for pour-plate tests is normally the lowest level causing visible thinning of the lawn. (In the absence of such thinning, a top level of 5 mg/plate was selected).
The control plates were checked for the absence of growth on sterility checks or normal counts on negative controls in the presence and absence of the vehicle.

Pour-plate assay for mutagenesis
A suspension of test item was prepared at 50 mg/mL in purified water containing 0.15% agar, and four half-log dilutions were prepared from this suspension. An aliquot (0.1 mL) of each concentration of test item was placed in a sterile tube. Molten, histidine-deficient top-agar (2 mL) and bacterial suspension (0.1 mL), maintained at 45°C, were then added. The tubes were mixed by inversion and 0.5 mL rat liver microsomal preparation (5-9 mix) was added where appropriate. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (20 mL).
Further plates were prepared without the inclusion of the test organisms to verify the sterility of the 5-9 mix and the test material. A control series of plates was prepared to confirm the inability of purified water containing 0.15% agar (0.1 mL) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates.
Aliquots (0.1 mL) of a 10-6 dilution of culture were spread on the surface of plates of complete medium to measure the viability and cell-density of each culture.
All plates were prepared in triplicate, allowed to solidify and incubated at 37°C for 2 days. After incubation, numbers of revertant colonies were counted, either manually or with a Biotran II automatic colony counter. Total colonies on nutrient plates were counted in the same way.
Growth of the background lawn of non-revertant cells on minimal plates was verified.
Each test, in each strain, was conducted on two separate occasions.
All plates and tubes were identified by the use of numbers indelibly marked on the plates and test tube racks.

Positive controls
All positive control compounds were prepared as solutions in DMSO, except sodium azide, which was dissolved in purified water.
Evaluation criteria:
Increase in revertant colony numbers over control counts.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 100 and TA 98
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:
Main test:
The results obtained with test item and positive control compounds are presented in Tables 1 to 4 in attached background infomation. Note: the mean values quoted have been corrected to the nearest whole number.

Sterility checks, spontaneous reversion rate and viability checks:
The absence of colonies on test item and S-9 mix sterility check plates indicates that these preparations were free of microbial contamination. The total colony counts on plates number 18 confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects of the inclusion of purified water containing 0.15% agar on these rates.

Mutagenic activity of positive control chemicals:
Appropriate positive control chemicals (with S-9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S-9 mix.

Action of test item:
No increases in revertant co10ny numbers over control counts were obtained with any of the tester strains following exposure to test item at levels from 50 to 5000 µg per plate.

Preliminary toxicity test results:

Test material (µg per plate)

 

TA 98

 

 

Background lawn

 

 

Revertant colonies

Plate A

Plate B

Plate C

Plate A

Plate B

Plate C

2500
(sterility check)

A

A

A

0

0

0

5000

500

50

5

 

P

P

P

P

P

P

P

P

P

P

P

P

11

16

28

16

 

6

19

24

23

4

19

22

19

2500

250

25

2.5

P

P

P

P

P

P

P

P

P

P

P

P

13

23

16

19

16

16

12

10

18

13

10

11

0
 0 (0.2mL vehicle)

P

P

 

P

P

 

P

P

 

31

19

24

21

22

23

A  Absent

P   Present and normal

 

No visible thinning of the background lawn of non-revertant cells was obtained following exposure to test item. A top exposure level of 5 mg per plate was therefore selected for use in the main tests.

Conclusions:
The test item was devoid of mutagenic activity under the conditions of the test.
Executive summary:

The test item was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella Typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, using pour-plate assays in complied with OECD 471. Each test, in each strain, was conducted on two separate occasions.

 

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of test item (tested as a suspension) from 50 to 5000 µg per plate, selected following a preliminary toxicity test in strain TA 98. All tests included vehicle (0.15% agar in aqueous solution) controls with and without S-9 mix.

 

No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the test item levels tested, either in the presence or absence of S-9 mix.

 

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions. It was concluded that test item was devoid of mutagenic activity under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Negative results in two Ames study.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the substance should not be classified for this endpoint.