Sodium dicyanoamide

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

before 2002

Test guideline 2

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

Test material information

Constituent 1

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 / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: sensitive to agents inducing frame-shift mutations

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: sensitive to agents inducing base-pair substitution

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: sensitive to agents inducing base-pair substitution

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: sensitive to agents inducing frame-shift mutations

Species / strain 5

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.