Reaction mass of [5-methyl-2-[[4-(4-methylanilino)-9,10-dioxo-1-anthryl]amino]phenyl]sulfonyloxypotassium and [5-methyl-2-[[4-(4-methyl-2-potassiooxysulfonyl-anilino)-9,10-dioxo-1-anthryl]amino]phenyl]sulfonyloxypotassium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 June to 1 July, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Salmonella typhimurium TA 98 and TA 1535 were obtained from Prof. B. Ames, Berkeley, USA. Strain TA 100 was obtained from Dr. M. Schüpbach, Hoffmann-La Roche Limited, Basel, Switzerland. Strain TA 1537 was obtained from Dr. S. Albertini, Hoffmann-La Roche Limited, Basel, Switzerland. Escherichia coli (WP2 uvrA) was obtained from the National Collection of lndustrial Bacteria, Aberdeen, Scotland.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + DMSO (10ml + 1ml) in a deep freezer at about -80 °C.
- Precultures: aliquots from frozen stocks were grown in liquid nutrient broth medium for 8 hours and then used for the experiment. The bacterial cultures were incubated in a time-temperature controlled incubater at about 37 °C.

MAINTAINANCE
- Periodically characteristic check: the strains were checked every two to six months. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene was demonstrated by the sensitivity for UV-light.
- Periodically 'cleansed' against high spontaneous background: yes
- Periodically ampicillin resistance check: the Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance.

Metabolic activation:

with and without

Metabolic activation system:

rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

Main experiments: 187.5, 375.0, 750.0, 1500.0 and 3000.0 µg/plate

Vehicle / solvent:

- Vehicle: bidistilled water.
- Test solution: on day of experiment test item was dissolved in bidistilled water after warming up to 50 °C in a water bath and strongly shaken. The test item was soluble up to the concentration of 30 mg/ml.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

other: 2-aminoanthracene

Details on test system and experimental conditions:

PLATES TEST
The following materials were mixed in a test tube and poured onto the minimal agar plates: 100 µl of test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control); 500 µl 59 mix (for test with metabolic activation) or 59 mix substitution buffer (for test without metabolic activation); 100 µl bacteria suspension (cf. test system, pre-culture of the strains), 2000 µl overlay agar.

PREINCUBATION TEST
In the pre-incubation assay 100 µl test solution,500 µl S9 mix and 100 µl bacterial suspension were mixed in a test tube and shaken at about 37 °C for 30 minutes. After pre-incubation 2.0 ml overlay agar (about 45 °C) was added to each tube and well mixed. The mixture was poured on minimal agar plates. In addition, the respective controls (solvent) and positive controls were run together with each strain.

INCUBATION :after solidification the plates were incubated upside down for at least 48 hours at 37 °C ± 2 °C in the dark.

NUMBER OF REPLICATIONS: three replicates x concentration x strain.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male rats (HanBrl:WIST SPF). The animals were treated with Aroclor 1254, 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content of the S9 fraction was 42.5 mg/ml.

On day of experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % vlv in the mixture. Cofactors were added to the 59 mix to reach the following concentrations in the 59 mix: 8 mM MgCl2; 33 mM KCI; 5 mM Glucose-6-Phosphate; 4 mM NADP, in 100 mM sodium phosphate-buffer, pH7.4.
Before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator.

PRE-EXPERIMENT FOR TOTOXICITY - range finding test
- Strains: S. typhimurium TA 100 and E. coli WP2 uvrA
- Metabolic activation: with and without
- Concentrations: 12.3, 37.0,111.1, 333.3, 1000.0 and 3000.0 µg/plate
- Incubation: plates were inverted and incubated for about 48 hours at 37 ± 2 °C in darkness.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment.
However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis was not required.

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA 98 and 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

Species / strain:

E. coli WP2 uvr A

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:

The plates incubated with the test item showed normal background growth up to 3000 µg/plate with and without S9 mix in both experiments. No reduction in the growth of the bacterial background lawn was observed. No precipitation of the test item was visible.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any concentration level, neither in the presence nor in the absence of an metabolic activation system.
In the first mutagenicity test without metabolic activation on strain E. coliWP2 uvrA, a concentration-dependent increase in the number of revertant colonies was observed at higher concentrations. Since this effect did not reach the threshold of twice of the corresponding solvent control and could not be repeated in the second experiment, it is attributed to occasionally occurring fluctuations in bacterial growth.
ln both experiments on strain TA 98 without metabolic activation, the number of revertant colonies in the positive control were somewhat below the expected value. This effect is considered to be based upon biological irrelevant fluctuations in the number of revertant colonies and has no impact on the outcome of the study.

CONTROLS
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

PRE-EXPERIMENT FOR TOTOXICITY - range finding test
Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.
From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 3000.0 µg/plate with and without metabolic activation.

Summary of the First Mutagenicity Test - Experiment with metabolic activation

Concentration (µg/plate)	Mean
	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
Bidist. Water	143	13	32	39	14
Test item 187.5	154	24	34	34	't7
Test item 375	167	18	37	34	15
Test item 750	161	22	33	29	18
Test item 1500	160	17	41	34	15
Test item 3000	150	19	32	38	16
2-A-anthracene 1.5	1803	-	-	1373	281
2-A-anthracene 20.0	-	-	947	-	-
CPA 200.0	-	272	-	-	-

Summary of the First Mutagenicity Test - Experiment without metabolic activation

Concentration (µg/plate)	Mean
	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
Bidist. Water	135	14	24	17	15
Test item 187.5	141	14	26	30	12
Test item 375	161	17	27	29	10
Test item 750	165	21	29	24	15
Test item 1500	167	15	39	22	14
Test item 3000	158	17	41	24	12
2-Nitrofluorene 5.0	-	-	-	261	-
4-Nitroquinoline 2.0	-	-	446	-	-
9-A-acridine 80.0	-	-	-	-	962
Sodium azide 2.0	676	427	-	-	-

Summary of the Second Mutagenicity Test - Experiment with metabolic activation

Concentration (µg/plate)	Mean
	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
Bidist. Water	142	21	50	33	15
Test item 187.5	163	21	50	40	16
Test item 375	155	18	58	42	27
Test item 750	137	20	55	32	26
Test item 1500	149	20	40	36	19
Test item 3000	134	18	49	28	13
2-A-anthracene 1.5	1992	-	-	834	203
2-A-anthracene 20.0	-	-	587	-	-
CPA 200.0	-	477	-	-	-

Summary of the Second Mutagenicity Test - Experiment without metabolic activation

Concentration (µg/plate)	Mean
	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
Bidist. Water	132	17	38	22	13
Test item 187.5	143	21	46	29	13
Test item 375	125	17	38	34	14
Test item 750	140	19	43	31	18
Test item 1500	156	19	45	21	12
Test item 3000	151	20	47	21	12
2-Nitrofluorene 5.0	-	-	-	335	-
4-Nitroquinoline 2.0	-	-	314	-	-
9-A-acridine 80.0	-	-	-	-	900
Sodium azide 2.0	793	482	-	-	-

Range-finding test

Strain	TA 100				WP2 uvrA
Concentration (µg/plate)	Revertants per plate		Factor		Revertants per plate		Factor
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Bidist. Water	118	178	1.0	1.0	38	35	1.0	1.0
12.3	125	125	1.1	0.7	30	36	0.8	1.0
37.0	128	148	1.1	0.8	34	41	0.9	1.2
111.1	143	151	1.2	0.9	49	46	1.3	1.3
333.3	150	151	1.3	0.9	43	35	1.1	1.0
1000.0	144	163	1.2	0.9	32	35	0.8	1.0
3000.0	149	154	1.3	0.9	43	53	1.1	1.5

Conclusions:

Under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used.
Therefore, test substance is considered to be non-mutagenic in Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 100, TA 1535, TA 98, TA 1537 and E. coliWP2 uvrA. The test was performed in two independent experiments both with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The test item was dissolved in bidistilled water and tested at five concentrations: 187.5, 375.0, 750.0, 1500.0 and 3000.0 µg/plate.

In order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations used in the first experiment. The test with metabolic activation was carried out as pre-incubation assay.

Bidistilled water was selected to be the best solvent compared with the usual organic solvents. However, the limit of solubility was at the concentration of 3000 µg/plate.

Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation with the concentrations of 12.3, 37.0,1 1 1 .1 , 333.3, 1000.0 and 3000.0 µg/plate.

Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.

From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 3000.0 µg/plate with and without metabolic activation.

In the first mutagenicity test performed with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with test item at any concentrations. In the second mutagenicity test carried out with and without metabolic activation, again treatment of the tester strains with the test item did not lead to an increase in the number of revertant colonies at any concentrations. In both mutagenicity tests normal background growth was observed with all strains at all concentrations. The number of revertant colonies was not reduced.

The test item did not precipitate on the surface of the agar plates.

Negative (solvent) and positive controls were found to be within acceptable ranges.

Conclusion

Under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used.

Therefore, test substance is considered to be non-mutagenic in Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Acid Green 025 was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 100, TA 1535, TA 98, TA 1537 and E. coli WP2 uvrA. The test was performed in two independent experiments both with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system.

A pre-experiment for toxicity was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA: normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.

In the first mutagenicity test performed, no substantial increase in the number of revertant colonies was observed after treatment with test item at any concentrations. In the second mutagenicity test, again treatment of the tester strains with the test item did not lead to an increase in the number of revertant colonies at any concentrations. In both mutagenicity tests normal background growth was observed with all strains at all concentrations. The number of revertant colonies was not reduced. The test item did not precipitate on the surface of the agar plates.

A second experiment on Acid Green 025 is available; however, it was not taken into consideration for the classification assessment. The AMES test was run with the strains of Salmonella typhimurium TA 98, TA 100, TA 1535 TA 1537 and TA 1538, with and without metabolic activation. The test consists in two experiments, i.e. range-finding experiment and experiment 1, conducted at the following concentrions: 1.6, 8, 40, 200, 1000 and 5000 μg/plate. Statistically significant increases in revertant numbers were observed following test item treatment of strain TA98 in the presence of S-9 and TA 1538 treatment in the absence and presence of S-9. These increases showed some evidence of a dose-related response and were considered sufficient to be indicative of test item mutagenic activity in these strains under the treatment conditions. No statistically significant increases in revertant numbers were observed following any other treatments. No precipitation of test agent was observed and no evidence of toxicity was observed following any of the dose treatments in any of the test strains in either the presence or the absence of S-9.

In was concluded that the test item induced mutation in Salmonella typhimurium strain TA 98 in the presence of S-9 and strain TA 1538 in both the absence and presence of S-9.

It should be noted that, althought it was indicated that the increses of revertants was dose-related, the review of the tables of results shows that, in all cases, a significant increase of the number of revertants was recorded at 1000 μg/plate, while at 5000 μg/plate the number of revertants was appreciably lower than that recorded at 1000 μg/plate.

Considering that this second experiment was indicated as a screening assay, it is likely that the complete experiment, here used as key study, was performed in order to deeper assess the gene mutation potential in bacteria and in order to verify the screening test outcomes.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the criteria to classify the substance for genetic toxicity are not met, according to the CLP Regulation (EC) No 1272/2008.