Reaction products of 7-amino-4-hydroxynaphthalene-2-sulfonic acid reacted with 2-((1,3,5-triazin-2-yl)amino)ethan-1-ol, coupled with diazotised 3-amino-4-hydroxybenzenesulfonic acid, subsequently coupled with diazotised 2-amino-5-methoxybenzenesulfonic acid, chelated with copper, sodium salt

Genetic toxicity: in vitro

Some information in this page has been claimed confidential.

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:

1991-03-27 to 1991-04-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Direct Red 83:1

Method

Target gene:

Histidine (Salmonella)
Tryptophan (E. coli WP2 uvr A-)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix was prepared as follows:S9 5.0 mL, 1.65 M KCL 1.0 mL, 0.4 MMgCl2 1.0mL, 0.1 M Glucose-6-phosphate 2.5 mL, 0.1 M NADP 2.0 mL, 0.2 M Sodium Phosphate buffer (pH 7.4) 25.0 mL, sterile distilled water 13.5 mL

Test concentrations with justification for top dose:

With and without the addition of a rat liver homogenate metabolising system at 10% in standard co-factors.

Dose range determined in preliminary toxicity assay: 8 – 5000 µg/plate in first experiment
Dose range used in preliminary: 0, 312.5, 625, 1250, 2500, 5000µg/plate

Experiment repeated on separate day using different cultures of back-strains and fresh chemical solutions. 312.5 – 5000 µg/plate

.

Vehicle / solvent:

The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range.sterile distilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

(-S9) -0.2 ug/plate For tester strain TA 98 Migrated to IUCLID6: 0.1 mL of 4NQO was added to a test tube containing 2.0 mL of molten, trace histidine or tryptophan supplemented top agar and 0.5 ml of pH 7.4 buffer.

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

(-S9) 80 ug/plate for tester strain TA 1537 Migrated to IUCLID6: 0.1 mL of 9AA was added to a test tube containing 2.0 mL of molten, trace histidine or tryptophan supplemented top agar and 0.5 ml of pH 7.4 buffer.

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

(-S9). 2ug/plate for tester strain WP2 uvr A-, 3ug/plate for tester strain TA 100 and 5 ug/plate for tester strain TA 1535 Migrated to IUCLID6: 0.1 mL of ENNG was added to a test tube containing 2.0 mL of molten, trace histidine or tryptophan supplemente

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

0.1 mL of 2AA was added to a test tube containing 2.0 mL of molten, trace histidine or tryptophan supplemented top agar.

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

(+S9) at 2ug/plate for tester strain TA 1535 and 20 ug/plate for tester strain WP2uvrA-

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

0.1 mL of BP was added to a test tube containing 2.0 mL of molten, trace histidine or tryptophan supplemented top agar.

Positive control substance:

benzo(a)pyrene

Remarks:

(+S9) at 5 ug/plate for tester strains: TA100, Ta 1537 and TA 98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
In This assay, overnight Sub-cultures of the master slopes were prepared in nutrient broth( oxoid limited) and incubated at 37 °C for 10 hours. These overnight cultures yielded approximately 10 Exp 8-10 Exp9 bacteria per mL.

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS:
3
NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method:relative total growth

Evaluation criteria:

Criteria for evaluating Results:
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 (of at least twice the spontaneous reversion 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 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 or solubility or up to the maximum recommended dose of 5000 ug/plate. In this case the limiting factor was the maximum recommended dose.

Statistics:

A statistical analysis of the data was not required for the evaluation of the result.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Kayarus Supra Rubine BLN caused no reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation. Kayarus Supra Rubine BLN was therefore tested up to the maximum recommended dose level of 5000 µg/plate.

Any other information on results incl. tables

Judgement	Negative
Reason for judgement and referential Matters	No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used in two separate experiments either with or without metabolic activation. An appropriate allowance of the purity of the test substance (93.7%) was made when the test material solution were prepared. A statistical analysis of the data was not required for the evaluation of the result.
Test condition for the Plate method
Composition		Bacterial suspension 0.1 mL
		Test substance solution 0.1 mL
		Na-phosphate Buffer   0.5 mL
		S9 Mix (incase of metabolic activation method)      0.5 mL
		Top Agar solution:    2.0 mL
Incubation		Temperature/Time  37 °C/ 48 hours

 

KEY TO TABLE OF RESULTS:

 

 

1.     When bacterial growth is found, the applicable value is marked with an asterisk.

2.     The average number of colonies for each concentration is recorded in parenthesis

3.     “Number of revertants”: The observed values and average value are shown in order, beginning with the low concentration of the test substance.

4.     The following postfixes are used when required:

 

 

C: Contaminated

 P: precipitate

 X: plate unscorable

ENNG= N-ethyl-N-nitro-N-nitrosoguanidine

 4NQO= 4-nitroquinoline-N-oxide

 9AA= 9-aminoacridine

 2AA= 2-Aminoanthracene

BP= Benzo(a) pyrene

 

 

 

 

TABLE OF TEST RESULTS: EXPERIMENT I NAME OF THE TEST SUBSTANCE: KAYARUS SUPRA RUBINE BLN
Number of revertants (number of colonies/plate)
With (+) S9 or Without (-) S9	Test substance concentration (ug/plate)	Base-pair substitution type					Fragmentg-type
	0	TA 100		TA 1535		W2uvrA-	TA 98		TA 1537
-		72		15		13	16		15
		100	(84.3)	9	(11.7)	19 (15.7)	17	(16.7)	16	(12.7)
		81		11		15	17		9
-	8.0	61		13		14	16		14
		87	(83.0)	16	(14.7)	13(13.7)	14	(15.7)	9	(10.0)
		101		15		14	17		7
-	40	84		5		15	17		14
		79	(76.7)	13	(10.3)	16(15.0)	15	(16.0)	10	(11.3)
		67		13		14	16	15	10
-	200	89		13		11	15		14
		88	(85.3)	15	(14.3)	13(11.3)	16	16(15.7)	9	(12.7)
		79		15		10	16		15
-	1000	96		11		7	18		11
		85	(86.9)	10	(10.3)	14(11.3)	17	(17.3)	6	(8.7)
		79		10		13	17		9
-	5000	60		11		11	15		12
		76	(74.3)	11	(10.0)	13(11.0)	18	(16.0)	16	(13.7)
		78		8		9	15		13
	Name	ENNG		ENNG		ENNG		4NQO		9AA
Positive control not requiring S9 mix	Concentration (ug/plate)	3.0		5.0		2.0		0.2		80.0
-	Number of colonies/plate	510		178		544		166		148
		546	(534.7)	189	(193.3)	520	(534.0)	156(158.3)		152	(151.7)
		548		215		538		153		155
With (+) S9 or Without (-) S9	Test substance concentration (ug/plate)	Base-pair substitution type					Fragmentg-type
		TA 100		TA 1535		W2uvrA-	TA 98		TA 1537
+	0	131		14		19		17		12
		123	(116.0)	19	(15.7)	19	(20.3)	18 (17.7)		15	(13.7)
		94		14		23		18		14
+	8.0	98		16		15		19		14
		94	(100.7)	11	(14.0)	14	(14.0)	22 (19.7)		12	(14.0)
		110		15		13		18		16
+	40	106		11		12		19		16
		99	(95.3)	12	(10.3)	9	(10.3)	16 (16.3)		14	(15.0)
		81		8		10		14		15
+	200	71		13		18		16		13
		110	(85.3)	7	(10.3)	15	(17.3)	15(17.3)		10	(13.0)
		75		11		19		21		16
+	1000	73		16		7		12		10
		108	(97.7)	17		14		17 (14.7)		11	(11)
		112		13	(15.3)	15	(12.0)	15		12
+	5000	85		8		13		14		9
		71	(75.3)	8	(7.3)	21	(15.7)	14(14.3)		9	(8.7)
		70		7		13		15		8
	Name	BP		2AA		2AA		BP		BP
Positive control not requiring S9 mix	Concentration (ug/plate)	5.0		2.0		20.0		5.0		5.0
	Number of colonies/plate	339		206		505		326		123
		343	(353.3)	197	(192.7)	570	(473.3)	362	(369.0)	128	(125.0)
		378		175		439		419		124

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used with any dose of Kayarus Supra Rubin BLN in two separate experiments either with or without metabolic activation.


No significant Increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of Kayarus Supra Rubine BLN, either with or without metabolic activation. Kayarus Supra Rubine BLN was found to be non-mutagenic under the conditions of this test

Executive summary:

A genetic toxicity test was performed ( in the period of 1991 -03 -27 to 1991 -04 -12) according to Safepharm Definitive Protocol number TX 3094 and was designed to assess the mutagenic potential of KAYARUS SUPRA RUBINE BLN using a bacterial/microsome test system. This in vitro study was based on a technique in which a mutagenic activity is assessed by exposing histidine auxotroph of Salmonella thyphimorium to various concentrations of the test material.

This method is conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EEC an USA, APA (TISCA) guidelines.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium ( Salmonella typhimurium TA98, TA 100, TA100, TA 1535 and TA 1537) and the trytophan-requiring auxotroph strains of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from rat liver.

Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems, not available in the bacterial cell, the test material and the bacteria were also incubated in the presence of a liver microsomal fraction (S9) obtained from rats previously treated with a compound known to induce and elevated level of these enzyme.

The test study included a:

1)Preliminary toxicity study

The dose range of KAYARUS SUPRA RUBINE BLN used in the preliminary toxicity study was 0, 312.5, 625, 1250, 2500 and 5000 µg/plate. The test material was non-toxic in the strains of bacteria used (TA100 and WP2uvrA).

2) Mutation study

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

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

 

No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used with any dose of Kayarus Supra Rubine BLN in two separate experiments either with or without metabolic activation. An appropriate allowance of the purity of the test substance (93.7%) was made when the test material solution were prepared. A statistical analysis of the data was not required for the evaluation of the result.

The positive control substance all produced marked increased in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Therefore Kayarus Supra Rubine BLN was found to be non-mutagenic under the conditions of this test