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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:
key study
Study period:
13 August 2003 to 15 September 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

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
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
MHLW and MAFF
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: greasy solid
Details on test material:
- Physical state: Brown waxy solid
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
Histidine auxotrophs of S. typhimurium and tryptophan auxotrophs of E. coil
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: uvrB, rfa
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (10% liver S9 in co-factors)
Test concentrations with justification for top dose:
Preliminary Study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Main Study: 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controls
Untreated negative controls:
no
Remarks:
see table 1 in the field "any other information on material and methods inc tables"
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
see table 1 in the field "any other information on material and methods inc tables"
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation method

PRELIMINARY STUDY
- The dose range was determined through a preliminary toxicity assay
- Test strains, TA 100 and WP2uvrA
- 0.1 mL of the culture was added to 2 mL of molten top agar (either histidine or tryptophan supplemented), 0.1 mL of the test material and 0.5 mL of S9 mix or phosphate buffer. This was overlaid onto sterile agar plates of Vogel-Bonner Minimal agar (30 mL /plate).
- The solvent control and sterility controls were performed.
- The plates were incubated for 48 hours at 37 ºC.
- Revertant colonies were counted using a Domino colony counter.

MAIN TEST
- The main test was carried out twice on different days, following the same procedure, with fresh bacterial cultures.
- The procedure is the same as in the preliminary test; all plates were produced in triplicate.
- The colonies had to be counted by hand in the 2nd test as there was interference from the agar plates.
Evaluation criteria:
he test was considered positive if the following criteria was met:
The test induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

The mutation assay was considered to be valid if the following criteria were met:
All culture strains in the vehicle and untreated controls exhibit a characteristic number of spontaneous revertants per plate.
Strain characteristics have been confirmed i.e. rfa and pKM101 plasmid R-factor.
All strain cultures meet the approximate range of 1 to 9.9 x 10⁹ bacteria per mL.
Positive controls should be at least two times the respective vehicle control values.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.
Statistics:
Dunnett's method of linear regression.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.

MUTATION TEST:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments was shown to be sterile.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 pg/plate. A light, particulate precipitate was observed at 5000 pg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Table 2. Revertant Colony Counts from Experiment 1 and 2 Including Controls

Experiment No.

With or Without S9-mix

Concentration of Test Material

Mean Number of Revertants per Plate (standard deviation)

Strain

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

1

-

0

66 (13.1)

11 (1.0)

22 (6.1)

20 (3.5)

7 (1.2)

50

70 (6.0)

12 (3.1)

22 (6.1)

19 (3.5)

9 (2.1)

150

87 (13.2)

13 (3.5)

21 (4.6)

21 (7.5)

8 (0.6)

500

71 (13.0)

14 (10.1)

15 (1.2)

16 (7.1)

6 (2.6)

1500

71 (4.4)

13 (1.5)

21 (3.5)

17 (3.2)

8 (0.6)

5000

72 (13.9)

7 (2.3)

15 (3.5)

20 (5.0)

9 (2.5)

ENNG

514 (10.4)

275 (16.0)

797 (22.5)

 

4NQO

240 (13.1)

 

9AA

2316 (390.7)

+

0

71 (5.6)

13 (5.6)

24 (6.4)

27 (3.6)

15 (5.5)

50

71 (4.7)

10 (3.8)

18 (3.5)

22 (0.0)

16 (5.0)

150

68 (5.7)

8 (0.6)

22 (6.6)

23 (3.5)

15 (2.5)

500

69 (1.2)

11 (3.6)

18 (6.8)

25 (9.9

17 (3.5)

1500

67 (5.8)

9 (2.3)

20 (0.6)

21 (5.5)

17 (6.7)

5000

69 (4.7)

14 (4.0)

21 (2.1)

25 (7.8)

10 (3.5)

2AA

1440 (32.1)

156 (1.5)

791 (16.4)

502 (53.5)

BP

205 (15.1)

 

2

-

0

130 (9.5)

35 (3.5)

20 (2.0)

24 (3.8)

12 (3.5)

50

115 (2.1)

37 (3.5)

23 (5.2)

21 (2.3)

9 (3.2)

150

129 (14.4)

39 (0.0)

17 (2.3)

23 (5.0)

15 (2.5)

500

115 (15.7)

31 (6.1)

17 (3.5)

16 (2.6)

13 (1.5)

1500

99 (13.0)

27 (12.3)

19 (1.5)

22 (1.5)

11 (3.0)

5000

88 (4.6)

15 (9.5)

18 (4.6)

18 (1.7)

6 (2.1)

ENNG

381 (11.8)

137 (25.0)

817 (39.1)

 

4NQO

210 (9.9)

 

9AA

827 (65.5)

+

0

103 (17.2)

13 (4.4)

29 (2.3)

35 (5.5)

19 (2.1)

50

115 (9.1)

14 (1.7)

20 (0.0)

34 (4.0)

20 (4.0)

150

101 (9.8)

15 (2.6)

25 (4.5)

30 (2.1)

18 (2.6)

500

90 (3.1)

14 (1.5)

22 (2.1)

26 (3.0)

19 (6.7)

1500

77 (6.7)

13 (1.2)

17 (2.5)

20 (6.6)

15 (2.8)

5000

89 (11.0)

17 (1.7)

23 (1.5)

13 (0.6)

19 (1.2)

2AA

881 (50.6)

229 (59.7)

613 (64.6)

 

316 (40.8)

BP

 

 

 

260 (20.2)

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of the test, the test material was determined to be non mutagenic in several strains of S. typhimurium and E. coli confirmed in an Ames Test.
Executive summary:

In a GLP compliant study performed to standardised guidelines OECD 471 and EU Method B.13/14, the mutagenic potential of the test material was assayed in an Ames Test. S. typhimurium, TA 1535, TA 1537, TA 100, TA 98 and E. coli WP2 uvr A were exposed to the test material in varying concentrations with and without metabolic activation. Under the conditions of the test it was determined that the test material is non-mutagenic as there was no significant increase in revertant colony counts.