m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

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:

Experimental start date: 29 March 2017 Experimental completion date: 23 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

EC Number:

275-602-1

EC Name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Cas Number:

71550-21-5

Molecular formula:

C27H24N6O9S2.2C4H11NO2

IUPAC Name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Test material form:

liquid

Specific details on test material used for the study:

Physical state/Appearance: Amber coloured solid pieces
Expiry Date: 22 June 2018
Storage Conditions: Room temperature in the dark

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

hamster liver S9

Test concentrations with justification for top dose:

Experiment 1: The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item were tested: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Experiment 2: The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in house. Sterile distilled water was therefore selected as the vehicle. The homogeneity and stability was previously confirmed for the test item in sterile distilled water formulations (within Envigo study no. LR73TX).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

3 µg/plate for TA100, 5 µg/plate for TA1535

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Absence of S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

80 µg/plate for TA1537

Positive control substance:

9-aminoacridine

Remarks:

Absence of S9-mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/plate for TA98

Positive control substance:

other: 4-Nitroquinoline-1-oxide

Remarks:

Absence of S9-mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for TA102

Positive control substance:

mitomycin C

Remarks:

Absence of S9-mix

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Presence of S9-mix

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate for TA98

Positive control substance:

benzo(a)pyrene

Remarks:

Presence of S9-mix

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for TA102

Positive control substance:

other: 1, 8-Dihydroxyanthraquinone

Remarks:

Presence of S9-mix

Controls 8

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

50 µg/plate for TA100 and TA98

Positive control substance:

congo red

Remarks:

Presence of uninduced S9-mix

Details on test system and experimental conditions:

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in house. Sterile distilled water was therefore selected as the vehicle. The homogeneity and stability was previously confirmed for the test item in sterile distilled water formulations (within Envigo study no. LR73TX).
The test item was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 20 minutes at 40 °C on the day of each experiment. Formulated concentrations were adjusted to allow for the stated impurity content (10.4%) of the test item. All formulations were used within four hours of preparation. Analysis was carried out in Experiment 1 to determine the concentration of the maximum test item formulation (50 mg/mL). Details of the analytical determinations are presented in Appendix 2 of the report.

Test for Mutagenicity: Experiment 1 – Pre-Incubation Method
The Prival-Mitchell modification to the standard Ames Test is necessary for the testing of azo dyes which can contain mutagenic aromatic amines which are not readily detected using the standard method (Prival and Mitchell (1982)). The modification differs in five key areas from the standard plate incorporation Ames Test:
• Uninduced hamster liver S9 rather than induced rat liver S9.
• 0.15 mL of S9 rather than the maximum of 0.05 mL of S9 in the standard Ames Test.
• The use of flavin mononucleotide (FMN), nicotinamide adenine dinucleotide (NADH), four times the standard amount of glucose-6-phosphate, and the inclusion of exogenous glucose 6 phosphate dehydrogenase in the co-factor mix.
• A 30 minute pre-incubation prior to the addition of the molten top agar.
• Vogel-Bonner plates containing 0.5% glucose instead of the standard 2% glucose.

Only the test item concentrations, vehicle and the positive control, Congo Red, were dosed using the Prival Mitchell modification.

Without Metabolic Activation
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or vehicle. Each mixture was shaken gently at 37 ± 3 ºC for 30 ± 3 minutes. Then, 2 mL of molten, trace amino-acid supplemented, top agar was added to each tube. The mixture was vortexed and poured onto Vogel-Bonner minimal agar plates containing 0.5% glucose. Each concentration of the test item, appropriate positive control, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of hamster S9 mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer. In addition, 0.1 mL of TA98 or TA100, 0.5 mL of uninduced hamster liver S9 and 0.1 mL of Congo Red at 50 µg/plate was dispensed into dosing tubes, incubated and overlaid onto 0.5% glucose Vogel Bonner plates as previously described.

The standard Ames positive controls were dosed using the pre-incubation method (previously described) where 0.1 mL of bacterial culture was mixed with 0.5 mL of rat liver S9-mix (phenobarbitone/B-naphthoflavone) or phosphate buffer and 2 mL of amino-acid supplemented top agar before overlaying onto 2% glucose Vogel-Bonner agar plates.

The negative (untreated) controls were dosed using the plate incorporation method where 0.1 mL of bacterial culture was mixed with 2 mL of amino-acid supplemented top agar before overlaying onto 2% glucose Vogel-Bonner agar plates.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.


Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Without Metabolic Activation
Testing was performed as described above

With Metabolic Activation
Testing was performed as described above

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

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

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

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver and the uninduced hamster liver S9-mixes were validated.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A yellow test item induced colouration was noted from 15 µg/plate, becoming orange at 5000 g/plate, this observation did not prevent the scoring of revertant colonies.
There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

Any other information on results incl. tables

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		TA102		TA98		TA1537
93		24		305		11		11
90	(96)	34	(29)	247	(262)	12	(13)	15	(11)
104		30		234		17		8

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		TA102		TA98		TA1537
135		31		321		18		19
119	(126)	23	(26)	302	(321)	11	(18)	13	(15)
123		25		339		24		13

 

  Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 16 May 2017					To: 19 May 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		TA102			TA98		TA1537
	Solvent Control (Water)	85 83 76	(81) 4.7#	23 32 33	(29) 5.5	250 264 271		(262) 10.7	14 12 16	(14) 2.0	9 7 13	(10) 3.1
	1.5 µg	87 82 88	(86) 3.2	28 36 24	(29) 6.1	210 229 241		(227) 15.6	14 17 10	(14) 3.5	7 21 7	(12) 8.1
	5 µg	81 94 76	(84) 9.3	12 22 28	(21) 8.1	219 177 278		(225) 50.7	10 13 18	(14) 4.0	9 14 10	(11) 2.6
	15 µg	87 80 75	(81) 6.0	17 22 20	(20) 2.5	233 219 238		(230) 9.8	11 14 9	(11) 2.5	15 17 9	(14) 4.2
	50 µg	88 73 80	(80) 7.5	29 18 8	(18) 10.5	238 266 277		(260) 20.1	11 17 12	(13) 3.2	15 15 5	(12) 5.8
	150 µg	74 72 76	(74) 2.0	27 21 29	(26) 4.2	258 264 260		(261) 3.1	13 17 11	(14) 3.1	7 5 7	(6) 1.2
	500 µg	72 75 83	(77) 5.7	18 28 16	(21) 6.4	257 291 269		(272) 17.2	11 8 14	(11) 3.0	3 6 7	(5) 2.1
	1500 µg	90 77 80	(82) 6.8	24 19 18	(20) 3.2	271 236 274		(260) 21.1	14 9 9	(11) 2.9	7 5 11	(8) 3.1
	5000 µg	83 80 72	(78) 5.7	21 18 20	(20) 1.5	206 250 263		(240) 29.9	10 8 10	(9) 1.2	5 6 17	(9) 6.7
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		MMC			4NQO		9AA
		3 µg		5 µg		0.5 µg			0.2 µg		80 µg
		1557 1719 1536	(1604) 100.1	1037 963 1198	(1066) 120.2	1919 1813 2066		(1933) 127.1	349 386 369	(368) 18.5	311 398 355	(355) 43.5

ENNG       N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO       4-Nitroquinoline-1-oxide

9AA       9-Aminoacridine

MMC       Mitomycin C

#       Standard deviation

Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 16 May 2017					To: 19 May 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		TA102			TA98		TA1537
	Solvent Control (Water)	118 117 104	(113) 7.8#	29 30 21	(27) 4.9	359 257 320		(312) 51.5	40 28 32	(33) 6.1	21 6 5	(11) 9.0
	1.5 µg	112 95 125	(111) 15.0	25 19 21	(22) 3.1	247 277 319		(281) 36.2	21 32 22	(25) 6.1	10 5 7	(7) 2.5
	5 µg	111 100 94	(102) 8.6	21 21 20	(21) 0.6	291 263 262		(272) 16.5	24 24 13	(20) 6.4	5 3 7	(5) 2.0
	15 µg	99 93 115	(102) 11.4	17 27 12	(19) 7.6	349 218 249		(272) 68.5	12 31 43	(29) 15.6	11 12 9	(11) 1.5
	50 µg	102 103 111	(105) 4.9	16 22 14	(17) 4.2	320 305 328		(318) 11.7	27 40 9	(25) 15.6	6 11 13	(10) 3.6
	150 µg	84 103 92	(93) 9.5	26 22 23	(24) 2.1	299 299 357		(318) 33.5	27 43 26	(32) 9.5	14 12 6	(11) 4.2
	500 µg	91 92 111	(98) 11.3	24 30 26	(27) 3.1	283 318 309		(303) 18.2	20 33 26	(26) 6.5	6 4 16	(9) 6.4
	1500 µg	100 102 112	(105) 6.4	27 20 27	(25) 4.0	303 314 308		(308) 5.5	30 20 22	(24) 5.3	16 8 15	(13) 4.4
	5000 µg	80 99 85	(88) 9.8	24 23 18	(22) 3.2	254 283 302		(280) 24.2	14 27 28	(23) 7.8	12 11 11	(11) 0.6
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		DAN			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		301 285 302	(296) 9.5	374 451 478	(434) 54.0	850 782 759		(797) 47.3	122 161 153	(145) 20.6	329 275 320	(308) 28.9
Positive controls Hamster S9-Mix (+)	Name	CR							CR
	Dose Level	50 µg							50 µg
	No. of Revertants	484 533 358	(458) 90.3						341 317 300	(319) 20.6

BP       Benzo(a)pyrene

2AA       2-Aminoanthracene

DAN       1,8-Dihydroxyanthraquinone

CR              Congo Red

P       Test item precipitate

S       Sparse bacterial background lawn

T       Toxic, no bacterial background lawn

V       Very weak bacterial background lawn

#       Standard deviation

 

Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 20 June 2017					To: 23 June 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		TA102			TA98		TA1537
	Solvent Control (Water)	104 89 98	(97) 7.5#	24 23 21	(23) 1.5	289 303 285		(292) 9.5	11 16 11	(13) 2.9	19 10 9	(13) 5.5
	15 µg	115 91 110	(105) 12.7	18 20 30	(23) 6.4	292 258 310		(287) 26.4	11 10 13	(11) 1.5	10 15 7	(11) 4.0
	50 µg	117 93 90	(100) 14.8	24 19 25	(23) 3.2	284 302 309		(298) 12.9	12 9 16	(12) 3.5	5 12 8	(8) 3.5
	150 µg	109 84 101	(98) 12.8	24 22 27	(24) 2.5	305 272 268		(282) 20.3	10 14 11	(12) 2.1	9 5 14	(9) 4.5
	500 µg	98 107 95	(100) 6.2	22 20 19	(20) 1.5	264 226 268		(253) 23.2	11 12 12	(12) 0.6	11 6 4	(7) 3.6
	1500 µg	96 113 111	(107) 9.3	14 20 20	(18) 3.5	251 237 315		(268) 41.6	9 11 10	(10) 1.0	7 5 7	(6) 1.2
	5000 µg	93 87 105	(95) 9.2	26 23 23	(24) 1.7	248 236 300		(261) 34.0	13 10 13	(12) 1.7	2 7 5	(5) 2.5
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		MMC			4NQO		9AA
		3 µg		5 µg		0.5 µg			0.2 µg		80 µg
		761 663 680	(701) 52.4	799 755 744	(766) 29.1	2009 1633 1594		(1745) 229.2	263 253 299	(272) 24.2	381 267 303	(317) 58.3

ENNG       N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO       4-Nitroquinoline-1-oxide

9AA       9-Aminoacridine

MMC       Mitomycin C

#       Standard deviation

 

Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 16 May 2017					To: 19 May 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		TA102			TA98		TA1537
	Solvent Control (Water)	124 112 117	(118) 6.0#	29 21 23	(24) 4.2	349 308 314		(324) 22.1	18 21 22	(20) 2.1	20 18 20	(19) 1.2
	15 µg	113 127 109	(116) 9.5	26 23 17	(22) 4.6	332 316 340		(329) 12.2	23 26 17	(22) 4.6	27 18 17	(21) 5.5
	50 µg	110 114 102	(109) 6.1	27 25 22	(25) 2.5	294 343 348		(328) 29.8	24 14 16	(18) 5.3	16 32 23	(24) 8.0
	150 µg	129 117 123	(123) 6.0	27 23 25	(25) 2.0	340 334 353		(342) 9.7	17 14 22	(18) 4.0	24 18 18	(20) 3.5
	500 µg	107 120 112	(113) 6.6	20 28 21	(23) 4.4	328 304 305		(312) 13.6	18 21 16	(18) 2.5	26 10 15	(17) 8.2
	1500 µg	116 109 110	(112) 3.8	24 28 28	(27) 2.3	281 328 340		(316) 31.2	12 18 17	(16) 3.2	13 16 14	(14) 1.5
	5000 µg	115 110 116	(114) 3.2	25 29 18	(24) 5.6	344 236 270		(283) 55.2	16 18 18	(17) 1.2	19 13 9	(14) 5.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		DAN			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1035 908 927	(957) 68.5	207 213 241	(220) 18.1	718 627 693		(679) 47.0	133 124 109	(122) 12.1	119 173 322	(205) 105.1
Positive controls Hamster S9-Mix (+)	Name	CR							CR
	Dose Level	50 µg							50 µg
	No. of Revertants	816 778 792	(795) 19.2						220 223 232	(225) 6.2

BP       Benzo(a)pyrene

2AA       2-Aminoanthracene

DAN       1,8-Dihydroxyanthraquinone

CR              Congo Red

#       Standard deviation

Applicant's summary and conclusion

Conclusions:

Bayscript Gelb GGN was considered to be non-mutagenic under the conditions of this test.

Executive summary:

 Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA102, TA98 and TA100 were treated withBayscript Gelb GGNusing the Ames pre-incubation method (Prival Mitchell Modification for Azo Compounds)at up to eight dose levels, in triplicate, both with and without the addition of a hamster liver homogenate metabolizing system (30% liver S9 in modified co‑factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for Experiment 2 was amended, following the results of Experiment 1, and was 15 to 5000 µg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxic limit of the test item.

Formulation analysis was carried out in Experiment 1 to determine the concentration of the test item concentration (maximum dose). 

Results

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver and the uninduced hamster liver S9-mixes were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A yellow test item induced colouration was noted from 15 µg/plate, becoming orange at 5000mg/plate, this observation did not prevent the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. 

Conclusion

Bayscript Gelb GGNwas considered to be non-mutagenic under the conditions of this test.