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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-09-07 to 1995-10-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
before 2002
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
92/69/EEC
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium dicyanoamide
EC Number:
217-703-5
EC Name:
Sodium dicyanoamide
Cas Number:
1934-75-4
Molecular formula:
C2HN3.Na
IUPAC Name:
sodium dicyanamide
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Aggregate State at room temperature: powder
Colour: white to off-white
Storage Conditions: at room temperature

Method

Target gene:
histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: sensitive to agents inducing frame-shift mutations
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: sensitive to agents inducing base-pair substitution
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: sensitive to agents inducing base-pair substitution
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: sensitive to agents inducing frame-shift mutations
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: sensitive to agents inducing frame-shift mutations
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from livers of male Sprague-Dawley rats induced with Arochlor 1254
Test concentrations with justification for top dose:
0, 50, 150, 500, 1500 and 5000 µg/plate. Potential toxicity of the test item was determined with tester strain TA100 in a pre-experiment.
Vehicle / solvent:
sterile distilled water
Controls
Untreated negative controls:
no
Remarks:
Concurrent solvent control only
Negative solvent / vehicle controls:
yes
True negative controls:
no
Remarks:
Concurrent solvent control only
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other:
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY: visible reduction in the growth of the bacterial lawn
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained, then a third experiment may be used to confirm the correct response.
To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.
Statistics:
All data are statistically analysed using the methods recommended by the UKEMS and normally Dunnett's method of linear regression is used to evaluate the result.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium 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 examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 1538
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolarity: not reported
- Evaporation from medium: not reported
- Water solubility: yes, test item was dissolved in sterile distilled water
- Precipitation: not reported
RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined with tester strain TA 100 in a pre-experiment. Six concentrations were tested: 0, 50, 150, 500, 1500 and 5000 µg/plate. The test material caused no visible reduction in the growth of the bacterial lawn at any of the dose levels in any of the strains of Salmonella tested. The test material was, therefore, tested up to the maximum recommended dose of 5000 µg/plate.
HISTORICAL CONTROL DATA: not reported
ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused no visible reduction in the growth of the bacterial lawn at any of the dose levels in any of the strains of Salmonella tested.

Any other information on results incl. tables

Preliminary Toxicity Study

The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 µg/plate. The test material was non-toxic in the strain of Salmonella used (TA 100).

The mean numbers of revertant colonies for the toxicity assay were:

Strain

Dose (µg/plate)

0

50

150

500

1500

5000

TA 100

111

110

108

118

114

115

 Mutation study

The results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.

No toxicity was exhibited to any of the strains of Salmonella used.

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

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Results for the negative spontaneous mutation rates are presented in Table 1.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls both with and without metabolic activation are presented in Tables 2 and 3 for experiment 1 and Tables 4 and 5 for experiment 2.

Table 1: spontaneous mutation rates

EXPERIMENT 1

Number of revertants (average number of colonies per plate)

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

TA1538

TA98

TA1537

115

19

16

13

13

EXPERIMENT 2

103

16

15

16

11

 Table 2: experiment 1 without metabolic activation

S9-mix

Test substance (µg/plate)

Number of revertants (average number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA1538

TA98

TA1537

-

0

109

15

14

17

8

-

50

106

14

6

17

7

-

150

99

22

9

13

5

-

500

108

16

12

7

7

-

1500

105

14

7

14

3

-

5000

97

16

5

13

6

Positive controls

without S9-mix

ENNG

ENNG

4NOPD

4NQO

9AA

-

Concentration (µg/plate)

3

5

5

0.2

80

-

No. colonies per plate

444

220

461

147

289

 Table 3: experiment 1 with metabolic activation

S9-mix

Test substance (µg/plate)

Number of revertants (average number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA1538

TA98

TA1537

+

0

103

10

20

55

7

+

50

110

8

26

38

7

+

150

97

6

20

39

11

+

500

104

10

21

31

9

+

1500

102

7

19

31

9

+

5000

109

8

22

42

9

Positive controls

without S9-mix

2AA

2AA

2AA

2AA

2AA

+

Concentration (µg/plate)

1

2

0.5

0.5

2

+

No. colonies per plate

389

143

297

308

173

 Table 4: experiment 2 without metabolic activation

S9-mix

Test substance (µg/plate)

Number of revertants (average number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA1538

TA98

TA1537

-

0

98

15

11

17

13

-

50

103

13

10

20

9

-

150

100

19

11

24

18

-

500

103

17

12

19

13

-

1500

92

15

15

24

10

-

5000

104

15

11

17

8

Positive controls

without S9-mix

ENNG

ENNG

4NOPD

4NQO

9AA

-

Concentration (µg/plate)

3

5

5

0.2

80

-

No. colonies per plate

473

172

412

174

374

 Table 5: experiment 2 with metabolic activation

S9-mix

Test substance (µg/plate)

Number of revertants (average number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA1538

TA98

TA1537

+

0

109

15

19

27

16

+

50

105

12

15

22

16

+

150

122

11

19

27

15

+

500

108

19

21

26

18

+

1500

103

18

18

27

13

+

5000

90

16

20

21

15

Positive controls

without S9-mix

2AA

2AA

2AA

2AA

2AA

+

Concentration (µg/plate)

1

2

0.5

0.5

2

+

No. colonies per plate

494

108

226

194

185

 

Applicant's summary and conclusion

Conclusions:
No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was found to be non-mutagenic under the conditions of this test.
Executive summary:

In a Bacterial Reverse Mutation Assay according to OECD Guideline 471, 5 strains of Salmonella typhimurium were exposed to sodium dicyanoamide at concentrations of 0, 50, 150, 500, 1500 and 5000 µg per plate in the presence and absence of mammalian metabolic activation (S9-mix). The maximum dose was determined in a pre-experiment.

This study was conducted in 1995 but satisfies the requirements for Test Guideline OECD 471. The results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.

No toxicity was observed in any of the strains of Salmonella used.

No significant increase in the frequency of revertant (His+) colonies of bacteria was recorded for any of the strains of Salmonella used at any dose level with or without metabolic activation when tested up to the predetermined maximum concentration of 5000 µg per plate.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Since there was no evidence of induced mutant colonies over background, sodium dicyanoamide is considered non-mutagenic in this Bacterial Reverse Mutation Assay.

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