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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:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
22 August 2005 to 15 September 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Reliability of 2 given since the data is based on read across, not the target substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

Method

Target gene:
Histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
The actual batch of the strain was tested for ampicillin resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.
Additional strain / cell type characteristics:
other: Histidine mutation G46; rfa; uvrB
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
The actual batch of the strain was tested for ampicillin resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.
Additional strain / cell type characteristics:
other: Histidine mutation D3052; rfa; uvrB; pkM101
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
The actual batch of the strain was tested for ampicillin resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.
Additional strain / cell type characteristics:
other: Histidine mutation G46; rfa; uvrB; pkM101
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
The actual batch of the strain was tested for ampicillin/tetracycline resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.
Additional strain / cell type characteristics:
other: Histidine mutation G428; rfa; pkM101
Species / strain / cell type:
S. typhimurium, other: TA 97a
Details on mammalian cell type (if applicable):
The actual batch of the strain was tested for ampicillin resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.
Additional strain / cell type characteristics:
other: Histidine mutation D6610; rfa; uvrB; pkM101
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (derived from rats induced with Aroclor 1254)
Test concentrations with justification for top dose:
Experiment 1
0, 62, 185, 556, 1667 and 5000 µg/plate in the presence and absence of metabolic activation for all strains tested.

Experiment 2
0, 62, 185, 556, 1667 and 5000 µg/plate in the presence and absence of metabolic activation in strains TA1535, TA98, TA100 and TA102.
0, 2.3, 7, 21, 62, 185 and 556 µg/plate in the presence and absence of metabolic activation in strain TA97a.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test material was not sufficiently soluble in water.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene; 1,8-Dihydroxy-anthraquinone; 4-Nitro-o-phenylenediamine; t-Butyl-hydroperoxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
NUMBER OF REPLICATIONS: The results of the first experiment were verified by a second, independent experiment. Triplicate repetitions were run for each dose group in each of the two separate experiments that were conducted and for the positive controls. For the vehicle control groups, six-fold repetitions were run.

PERFORMANCE OF THE TEST
- Conditions of cultivation: One day prior to the test, a small amount from each of the frozen bacterial cultures was transferred to nutrient broth. The liquid cultures were incubated in a shaker overnight at 37 °C and then used for the exposure. The mean number of viable cells was 2 to 3 x 10⁹ cells per mL.
- Exposure technique: For each sample the following solutions were combined: 0.1 mL of the overnight culture of the bacteria, 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation), 0.1 mL of the appropriate test or reference material solution and 2 mL of top agar
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).
- Colony counting: The plates with less than about 50 revertant colonies, with the exception of the positive controls, were counted visually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.
- Determination of the toxicity: The bacterial background of the plates was inspected visually. The following signs of toxicity, if present, were recorded: a reduced bacterial background lawn (mottled instead of homogeneous), microcolonies of bacteria instead of a homogeneous background lawn, no background lawn or clearly reduced numbers of revertant colonies.
Evaluation criteria:
The criteria for a positive result are a reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate (TA98 and TA1535) a 2½ fold increase of the amount of spontaneous revertants.
- For the strains with a high spontaneous revertant rate (TA97a, TA100 and TA102) a 1⅔ fold increase of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of the test laboratory’s historic data.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA1535, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test material was toxic at concentrations of 1667 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
MUTAGENICITY
There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.

TOXICITY
The test material was only toxic to strain TA97a, resulting in a completely or almost missing bacterial background lawn at 5000 and 1667 µg/plate. At 556 µg/plate and beneath, the bacterial background was normal. For the second experiment the concentrations were changed only for this strain. 556 µg/plate was the highest concentration used for the second experiment.

SOLUBILITY
A precipitate was visible when the test material was mixed with the agar at the 5000, 1667 and 556 µg/plate dose levels. The precipitate was still visible at 5000 µg/plate when the colonies were counted but did not impede the counting.

PROPERTIES OF THE BACTERIA
The strains showed the expected genetic properties and were sensitive against several mutagenic chemicals. The numbers of spontaneous revertants were comparable with the historic control data for the negative controls.

POSITIVE CONTROL SUBSTANCES
All positive control substances increased the mutation frequency to more than the threshold values. As 2-aminoanthracene, 1,8-dihydroxy-anthraquinone and 7,12-dimethyl-benz[a]anthracene require metabolic activation for mutagenicity, the results of these substances also demonstrated the efficiency of the metabolising system.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Experiment 1 Summary Data for all Strains Tested

+/- S9 Mix

Concentration

(µg/plate)

Mean number of revertants/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

TA102

TA98

TA97a

-

-

-

-

-

-

0

62

185

556

1667

5000

73.5

66.0

66.3

52.0

44.3

42.7

20.0

17.7

22.0

11.0

7.3

7.3

181.7

170.3

117.0

102.7

85.0

66.3

8.0

9.3

11.0

6.3

8.7

6.0

119.0

119.3

67.7

0.0

0.0

T

+

+

+

+

+

+

0

62

185

556

1667

5000

77.0

79.0

69.7

56.3

48.0

46.3

18.2

18.7

16.3

10.7

7.7

4.3

247.8

260.7

244.3

198.3

152.7

127.0

12.8

9.3

11.0

8.0

9.3

9.0

140.5

120.3

110.3

54.7

9.7

T

                                              Positive Controls

 

 

-

Name

SA

SA

tBHPO

2NF

4NOPD

Concentration (µg/plate)

2

1

50

2

10

Mean no. colonies/plate

422.3

207.7

487.7

109.7

374.0

 

 

+

Name

2AA

2AA

DHA

2AA

DMBA

Concentration (µg/plate)

2

2

50

1

10

Mean no. colonies/plate

1955.0

196.7

595.0

415.7

607.0

T = Toxic

 

Table 2 Experiment 2 Summary Data for Strains TA100, TA1535, TA102 and TA98

+/- S9 Mix

Concentration

(µg/plate)

Mean number of revertants/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

TA102

TA98

-

-

-

-

-

-

0

62

185

556

1667

5000

86.5

68.0

69.0

59.0

57.3

54.7

15.7

18.3

15.0

10.7

6.3

5.0

112.5

86.0

80.0

51.3

45.3

34.7

7.7

5.3

5.7

5.3

5.7

6.3

+

+

+

+

+

+

0

62

185

556

1667

5000

69.5

77.0

79.0

68.0

61.3

56.0

17.7

19.7

17.0

12.0

7.3

4.7

147.8

166.3

149.7

125.7

100.3

85.3

11.8

11.3

10.7

8.0

6.7

7.3

                                                      Positive Controls

 

 

-

Name

SA

SA

tBHPO

2NF

Concentration (µg/plate)

2

1

50

2

Mean no. colonies/plate

253.0

258.0

344.0

207.3

 

 

+

Name

2AA

2AA

DHA

2AA

Concentration (µg/plate)

2

2

50

1

Mean no. colonies/plate

1372.0

163.0

615.0

359.3

 

Table 3 Experiment 2 Summary Data for Strain TA97a

+/- S9 Mix

Concentration

(µg/plate)

Mean number of revertants/plate

Frameshift Type

TA97a

-

-

-

-

-

-

-

0

2.3

7

21

62

185

556

85.8

118.0

109.3

110.0

102.3

32.7

4.3

+

+

+

+

+

+

+

0

2.3

7

21

62

185

556

110.7

122.7

135.0

103.7

108.3

102.7

8.3

                                                Positive Controls

 

-

Name

4NOPD

Concentration (µg/plate)

10

Mean no. colonies/plate

229.7

 

+

Name

DMBA

Concentration (µg/plate)

10

Mean no. colonies/plate

351.7

 

Key to Positive Control Abbreviations

4NOPD: 4-Nitro-o-phenylene-diamine

tBHPO: t-Butyl-hydroperoxide

2AA: 2- Aminoanthracene

DHA: 1,8-Dihydroxy-anthraquinone

DMBA: 7,12-Dimethylbenz[a]anthracene

2NF: 2-Nitrofluorene

SA: Sodium azide

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material was non mutagenic to the Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102 both in the presence and absence of metabolic activation.
Executive summary:

The mutagenic activity of the test material was investigated in a reverse mutation test in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102 were exposed to the test material in DMSO using the direct plate incorporation method both in the presence and absence of exogenous metabolic activation (S9-mix derived from rat liver). The bacteria were also exposed to vehicle and appropriate positive controls.

The concentrations tested were 62, 185, 556, 1667 and 5000 µg/plate. The test material exhibited toxicity to the strain TA97a; as a consequence, the concentrations used for this strain only were altered for the independent, repeat experiment to 2.3, 7, 21, 62, 185 and 556 µg/plate.

There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.

Under the conditions of this study, the test material was non mutagenic to the Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102 both in the presence and absence of metabolic activation.