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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:
key study
Study period:
9th August 1985 to 27th August 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study is conducted by recognised test house, to experimental procedure in compliance with OECD Guideline 471. Full data set and rationale provided. GLP compliance is specified, although no certificate is available. The report is subject to Quality Assurance Procedures.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Remarks:
Claimed.
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Bontron E-84
- Substance type: Solid
- Physical state: White powder
- Analytical purity: Not specified
- Impurities (identity and concentrations): Not specified
- Lot/batch No.: Not specified
- Stability under test conditions: Not specified
- Storage condition of test material: Ambient temperature

Method

Target gene:
N/A
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Araclor 1254 induced rat liver homegenate S9 mix
Test concentrations with justification for top dose:
Range Finding Test: 5000, 500, 50, 5 µg/plate
Mutation Test: 1500, 500, 150, 50, 15 µg/plate
The test substance was found to be toxic to the tester strains at the highest dose in the range finding test, hence 1500 µg/plate was chosen as the top level dosage in the mutation tests.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Not specified. Anticipated that experience and historical usage are the justification.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2-aminoanthracene
Positive control substance:
other: 2-aminoanthracene
Remarks:
Used at 2 ug/plate for strains TA1535 and TA1537 with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2-aminoanthracene
Positive control substance:
other:
Remarks:
Used at 0.5 ug/plate for strains TA98 and TA100 with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2-nitrofluorene
Positive control substance:
2-nitrofluorene
Remarks:
Used at 1 ug/plate for strains TA98 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
9-aminoacridine
Positive control substance:
9-aminoacridine
Remarks:
Used at 80 ug/plate for strains TA1537 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
N-ethyl-N-nitro-N-nitrosoguanidine
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Used at 5 ug/plate for strains TA1535 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
N-ethyl-N-nitro-N-nitrosoguanidine
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Used at 3 ug/plate for strains TA100 without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

PROCEDURE:

Without metabolic activation:
Carried out on each tester strain.
0.1ml aliquots of bacterial suspension and 0.5ml of sterile 0.1M sodium phosphate buffer (pH7.4) are added to each of one set of sterile bijou bottles. 0.1 ml of the test compound is added to cultures at five concentrations separated by half-log 10 intervals. The negative control is the chosen solvent. The appropriate positive control is also included. 3 bottles are used at each dose level.
2.0ml of histidine deficient agar is added to each of the bottles, thoroughly mixed and then overlaid onto previously prepared plates containing 15ml of minimal agar. Plates are incubated for 72 hours at 37 ®C.
Colonies are counted using a Biotran Automatic Colony Counter, and the mean number of revertant colonies per treatment group assessed.

With metabolic activation:
Methodology is described as above, except that 0.5ml of liver homogenate S-9 mix is added to bijou bottles in place of sterile buffer.

Second mutation test:
The procedure outlined above is repeated at a later date. Results are documented as specified below.


POSITIVE CONTROLS
With S-9 Mix:
2-Aminoanthracene at 2 µg/plate for strains TA1535 and TA1537
2-Aminoanthracene at 0.5 µg/plate for strains TA98 and TA100
Without S-9 Mix:
2-Nitrofluorene at 1 µg/plate for strains TA98
9-Aminoacridine at 80 µg/plate for strains TA1537
N-ethyl-N’-nitro-N-nitrosoguanidine at 5 µg/plate for strain TA1535
N-ethyl-N’-nitro-N-nitrosoguanidine at 3 µg/plate for strain TA100

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
Evaluation Criteria:
The mean number of revertant colonies for all treatment groups was compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction of each treatment group.
A compound is deemed to provide evidence of mutagenic potential if:
1) A statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments
and
2) The increase in the number of revertant colonies is at least twice the concurrent solvent control value
Statistics:
None specified.

Results and discussion

Test results
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:
cytotoxicity
Remarks:
observed at 5000 µg/plate in range finding study
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The revertant colony counts for the substance obtained in the dose range finding test are referenced in Table 1 (attached, below). The substance was toxic towards the tester strains at the highest dose level. Therefore, 1500 µg/plate was selected as the top dose level in the mutation study.
The mean number of revertant colonies, together with the individual plate counts for the substance obtained in the first mutation test with tester strains TA1535, TA1537, TA98 and TA100 are referenced in Table 2 (attached, below). Compound sterility and positive control mutability checks are shown in Table 3 (attached, below).
The mean number of revertant colonies, together with the individual plate counts for the substance obtained in the second mutation test with strains TA1535, TA1537, TA98 and TA100 are referenced in Table 4 (attached, below). Compound sterility and positive control mutability checks are shown in Table 5 (attached, below).
No substantial increases of revertant colony numbers of any of the four tester strains were observed following treatment with the substance at any dose level, either in the presence of or absence of metabolic activation (S-9 mix).
It is concluded that no evidence of mutagenic potential of the substance was obtained in the bacterial system at the dose levels assessed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

 

Dose range finding test on Bontron E-84 – revertant colony counts obtained withS typhimuriumstrains TA 1535, TA 1537, TA 1538, TA 98, TA 100

Dose level (µg/plate)

Metabolic Activation

Strain ofS typhimurium

TA 1535

TA 1537

TA 1538

TA 98

TA 100

5000

-

IL

1

IL

IL

IL

500

-

5

9

10

6

36

50

-

20

6

13

15

64

5

-

11

11

8

12

63

Solvent

-

12

7

5

16

66

 

 

 

 

 

 

 

5000

+

IL

8

IL

NL

12

500

+

13

7

18

7

51

50

+

13

7

14

8

56

5

+

13

21

12

15

73

Solvent

+

8

10

10

14

73

 -      Absence

+     Presence

NL  No bacterial lawn

IL    Incomplete bacterial lawn

 

 

Test 1

Bontron E-84 – revertant colony counts obtained withS typhimuriumstrains TA 1535, TA 1537, TA 1538, TA 98, TA 100

 

Strain

Dose level (µg/plate)

Metabolic Activation

Mean revertant colony counts

S.D

Individual revertant colony counts

TA 1535

1500

-

5

1.0

5,4,6

500

-

10

3.0

10.7.13

150

-

13

5.1

17,14,7

50

-

11

2.5

11,9,14

15

-

10

3.2

6,11,12

0

-

8

2.0

8,6,10

Solvent

-

12

3.2

8,14,13

 

 

 

 

 

1500

+

7

2.5

7,9,4

500

+

12

3.8

9,10,16

150

+

8

1.7

6,9,9

50

+

8

0.6

7,8,8

15

+

13

3.0

16,10,13

0

+

11

3.1

14,10,8

Solvent

+

8

4.0

6,13,6

TA 1537

1500

-

9

2.5

11,9,6

500

-

7

0.6

7,8,7

150

-

9

2.9

7,12,7

50

-

8

2.9

5,10,10

15

-

9

1.5

8,11,9

0

-

9

1.5

9,7,10

Solvent

-

11

4.0

7,15,12

 

 

 

 

 

1500

+

5

3.5

2,5,9

500

+

11

1.2

12,10,12

150

+

11

5.3

13,15,5

50

+

12

5.0

11,7,17

15

+

14

1.7

13,13,16

0

+

12

3.5

16,9,12

Solvent

+

14

3.6

18,11,13

  TA 98

1500

-

6

2.0

6,4,8

500

-

11

1.0

11,12,10

150

-

13

5.6

18,7,14

50

-

12

3.5

16,10,10

15

-

14

5.1

20,13,10

0

-

20

5.9

18,27,16

Solvent

-

17

3.6

13,18,20

 

 

 

 

 

1500

+

3

2.5

3,1,6

500

+

15

2.1

13,14,17

150

+

17

2.3

20,16,16

50

+

15

4.0

19,14,11

15

+

19

3.0

16,19,22

0

+

14

4.6

13,19,10

Solvent

+

17

4.7

21,12,19

TA 100

1500

-

48

5.0

49,43,53

500

-

59

8.4

69,54,55

150

-

70

5.7

68,76,65

50

-

73

10.4

65,70,85

15

-

83

16.6

68,81,101

0

-

84

15.6

68,86,99

Solvent

-

74

5.0

69,79,75

 

 

 

 

 

1500

+

40

8.0

41,48,32

500

+

68

7.1

60,69,74

150

+

80

7.8

71,84,85

50

+

82

8.1

91,75,81

15

+

97

4.7

102,93,95

0

+

97

8.1

106,93,91

Solvent

+

85

4.6

89,80,86

 

-      Absence

+     Presence

SD  Standard Deviation

 

 

Test 1

Mutability and sterility tests withS typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100

Strain

Compound

Concentration of compound (µg/plate)

Metabolic Activation

Mean revertant colony counts

SD

Individual revertant colony counts

TA 1535

N-ethyl-N’-nitro-N-nitroso-guanidine

5

-

196

17.0

213,179,195

TA 1537

9-aminoacridine

80

-

1754

96.2

1649, 1775,1838

TA 98

2-nitrofluorene

1

-

66

2.6

68,67,63

TA 100

N-ethyl-N’-nitro-N-nitroso-guanidine

3

-

288

74.3

231,372,261

TA 1535

2-amino-anthracene

2

+

80

14.4

72,72,97

TA 1537

2

+

66

5.8

59,69,69

TA 98

0.5

+

223

11.2

236,215,219

TA 100

0.5

+

354

32.3

317,373,373

-

S-9 Mix

500 µl

 

0

 

0

-

Bontron E-84

1500

 

0

 

0

 

-      Absence

+     Presence

SD  Standard Deviation

 

Applicant's summary and conclusion

Conclusions:
It was concluded that there was no evidence of mutagenic potential of the test substance was obtained at the dose levels examined within the test.
Executive summary:

The reverse mutation of 5 strains of salmonella was tested according to the Ames test. No evidence of mutagenic potential was observed during the study. No classification and labelling is applicable to this substance for this endpoint.