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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Purity: > 98%

Method

Target gene:
his+
Species / strain
Species / strain:
other: Salmonella typhimurium TA1535, TA1538, TA98, TA1535, and TA100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix liver homogenates from Aroclor 1254 induced rats
Test concentrations with justification for top dose:
1, 3.3, 10, 33.3, 100, 333, 1000, 3330, and 5000 µg/plate; 5000 μg/plate is the maximum test substance concentration that should be used according to EEC guidelines
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylenediamine, daunomycine, 2-aminoanthracene
Details on test system and conditions:
plate-incorporation assay
Evaluation criteria:
Criteria for a positive response: A chemical was judged to have induced a positive response when a dose -related increase in revertants was observed in which the number of revertants exceeded the control values by at least two-fold in at least two successive concentrations of the test chemical

Results and discussion

Test results
Key result
Species / strain:
other: TA1535, TA1537, TA1538, TA98, AND TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
other: Due to the short incubation period (3 hours) the positive control chemicals did not produce mean plate counts of at least two times the concurrent vehicle control group mean in all tester strains.
Additional information on results:
Nine serial dilutions of the test substance, in approximately half-log steps, were plated with an appropriately diluted TA100 culture (equal numbers of bacterial cells/plate) onto non-selective agar (viability determination). The percentage survival of the TA100 culture is determined by comparing the number of colonies on the solvent control plate with those on the plates containing the test substance. However, even at the highest test substance concentration used, the survival of strain TA100 is not reduced (see Table 2 in the “Other information about results including tables" section).

Based on these data, the test substance was tested up to a concentration of 5000 μg/plate, which is the maximum test substance concentration that should be used according to the EEC guidelines. However, in the direct plate incorporation test (almost) no revertant colonies were present on the plates with test substance treated bacteria. The negative and strain specific positive control values fell within our laboratory historical ranges and the condition of the bacterial background lawn was normal. This result was reproducible in an independently repeated experiment. The results might indicate that the test substance is selectively cytotoxic for His+ revertant bacteria.

To investigate the possible selective cytotoxicity of the test substance, a second toxicity determination was performed in TA100 with 10(8) bacteria per selective agar plate (see Table 3 in the “Other information about results including tables" section).

It is clearly demonstrated that the test substance is more toxic for His+ revertants than for His- bacteria. A direct plate incorporation test with concentrations up to 100 μg/plate showed that the test substance was even more toxic to revertants from strains other than TA100 (see Table 4 in the “Other information about results including tables" section). It should be noted that no increase in the number of revertants was observed at any test substance concentration in any strain.

To avoid the toxic action of the test substance on His+ revertants, a liquid incubation test was performed in which bacteria were incubated in the presence of test substance for three hours, followed by extensive washing to remove the test substance. Subsequently, bacteria were plated in the absence of test substance. Using this assay high concentrations of test substance (up to 5000 μg/plate) could be used (see Table 1 the “Other information about results including tables" section).

All bacterial strains showed negative responses over the entire dose range of the test substance, i.e., no dose-related increase in the number of revertants. Due to the short incubation period the positive control chemicals did not produce mean plate counts of at least two times the concurrent
vehicle control group mean in all tester strains. The negative control values fell within our laboratory background historical ranges.

Based on these results, the test substance can be considered as not mutagenic under the experimental conditions described in this report.

Any other information on results incl. tables

Table 1. Mutagenic response of A7881 in the Ames Salmonella/microsome plate test

Treatment

µg/plate

Mean number of revertant (His+) colonies/3 replicate platesS.D.)

 

TA1535

TA1537

TA1538

TA98

TA100

                                                        Without S9-mix

DMSO

8 ± 1

8 ± 0

7 ± 3

34 ± 5

63 ± 5

A7881

100

12 ± 1

8 ± 2

5 ± 2

40 ± 9

53 ± 4

A7881

333

12 ± 1

3 ± 1

3 ± 2

27 ± 5

44 ± 12

A7881

1000

10 ± 1

7 ± 1

7 ± 3

38 ± 1

51 ± 4

A7881

3330

9 ± 4

12 ± 11

2 ± 1

35 ± 10

43 ± 6

A7881

5000

2 ± 2

4 ± 2

0 ± 0

41 ± 10

36 ± 2

Positive control (see list below)

10 ± 1

4 ± 1

77 ± 1

498 ± 50

45 ± 5

                                                            With S9-mix

DMSO

11 ± 3

6 ± 2

4 ± 3

30 ± 4

69 ± 14

A7881

100

15 ± 4

7 ± 3

5 ± 2

32 ± 5

73 ± 2

A7881

330

16 ± 3

4 ± 2

9 ± 4

25 ± 5

65 ± 5

A7881

1000

13 ± 4

4 ± 2

7 ± 3

32 ± 9

67 ± 6

A7881

3330

10 ± 5

3 ± 3

0 ± 1

24 ± 2

78 ± 7

A7881

5000

5 ± 3

1 ± 2

0 ± 0

31 ± 6

62 ± 4

DMN-B

13 ± 4

4 ± 1

4 ± 2

35 ± 4

74 ± 6

DMN-A

 

25 ± 3

5 ± 1

5 ± 2

25 ± 1

77 ± 4

 

Positive controls without S9-rnix

 

For TA1535: 5 μg Sodium azide (SA)/5 rnL exposition solution

For TA1537: 300 μg 9-Aminoacridine (9AC)/ 5 rnL exposition solution

For TA1538: 50 μg 4-Nitro-o-phenylenediamine (4NPD)/ 5 rnL exposition solution

For TA98: 20 μg Daunomycine (OM)/ 5 rnL exposition solution

For TA100: 3250 μg methylmethanesulfonate (MMS)/ 5 rnL exposition solution

 

Positive controls with S9-rnix for all strains: Dimethylnitrosamine (DMN);

DMN-A = 18500 μg/ 5 mL exposition solution and DMN-B = 9250 μg/ 5 mL exposition solution

Table 2 - Preliminary toxicity determination of the test substance in TA100

 

Concentration (μg/plate)                    Viable counts/plate (duplicate plates)

                                                           Without S9-mix         With S9-mix

 

Solvent control (DMSO)                        476;543                     550;580

1. 0                                                     536;500                      600:667

3.3                                                      610:616                      607;618

10.0                                                    582;642                      605;707

33.3                                                    553;600                      695;608

100                                                     570:453                      652:659

333                                                     514;545                      601;579

1000                                                   630;571                      686;648

3330                                                   581:571                      585;640

5000                                                   499;445                     410;568

Table 3 - Toxicity determination of the test substance in TA100

 

Concentration (μg/plate)                    Viable counts/plate (duplicate plates)

                                                           Without S9-mix         With S9-mix

 

Solvent control (DMSO)                    69; 95                        99; 88

1. 0                                                     121; 81                      84; 95

3.3                                                      75; 90                        76; 72

10.0                                                    89; 64                        85; 86

33.3                                                    44; 61                        70; 65

100                                                     26; 48                        41; 51

333                                                     29; 26                        39; 39

1000                                                   0; 0                            11; 10

3330                                                   0; 0                             0; 0

5000                                                   0; 0                             0; 0                 

Table 4- Mutagenic response of A7881 in the Ames Salmonella/microsome plate test

Treatment

µg/plate

Mean number of revertant (His+) colonies/3 replicate platesS.D.)

 

TA1535

TA1537

TA1538

TA98

TA100

                                                        Without S9-mix

DMSO

9 ± 6

7 ± 3

12 ± 4

23 ± 1

79 ± 19

A7881

1

11 ± 1

2 ± 1

4 ± 1

27 ± 6

105 ± 21

A7881

3.3

7 ± 5

2 ± 4

0 ± 0

21 ± 5

75 ± 3

A7881

10

3 ± 3

1 ± 2

0 ± 0

4 ± 5

52 ± 7

A7881

100

Microcolonies

0 ± 0

0 ± 0

1 ± 1

23 ± 6

Positive control

200 ± 16

7 ± 2

1912 ± 16

1979 ± 69

801 ± 20

                                                            With S9-mix

DMSO

16 ± 4

8 ± 5

23 ± 4

29 ± 8

96 ± 4

A7881

1

13 ± 2

6 ± 1

8 ± 4

29 ± 5

93 ± 1

A7881

3.3

6 ± 2

1 ± 2

2 ± 2

23 ± 4

71 ± 5

A7881

10

Plate infected with other bacteria

0 ± 1

0 ± 0

7 ± 5

23 ± 6

A7881

33.3

2 ± 2

0 ± 0

0 ± 0

6 ± 4

33 ± 12

A7881

100

1 ± 2

0 ± 0

1 ± 1

1 ± 1

25 ± 9

Positive control

114 ± 6

89 ± 4

694 ± 49

866 ± 75

1145 ± 48

Applicant's summary and conclusion

Conclusions:
The test substance was not mutagenic.
Executive summary:

The test substance was tested in the Ames Salmonella/microsome test up to 5000 μg/plate according to OECD Guideline 471. The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in any of the tester strains (TA1535, TA1537, TA1538, TA98 and TA100), neither in a direct plate incorporation test, nor in a liquid incubation test. The test substance can, therefore, be considered as not mutagenic under the experimental conditions described in this report.