Disodium 7-[[2-(acetylamino)-4-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]phenyl]azo]naphthalene-1,3-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item resulted to be non-mutagenic in the Salmonella typhimurium reverse mutation assay performed.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From November 27th to December 10th, 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

The read across approach is detailed into the document attached below.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

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 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: the bacterial strains TA 1535, TA 98, and TA 100 were obtained from Dr. B.N. Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

PRECULTURES
From the thawed ampoules of the strains 0.5 ml suspension was transferred to 250 ml Erlenmeyer flasks containing 20 rnl nutrient medium. A solution of 20 pl ampicillin was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl.
The bacterial cultures were incubated in a shaking water bath for 8 hours at 37 °C.

CHARACTERISTICS CHECK
Regular checking of the properties of the Salmonella strains with regard to membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in testing facility according to Ames et al. (l).

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 and hamster liver S9

Test concentrations with justification for top dose:

33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate

Vehicle / solvent:

- Vehicle used: deionised water.
- Justification for choice of solvent/vehicle: solvent was chosen according to its solubility properties. No precipitation of the test article occurred up to the highest investigated dose.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

congo red

other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
Experiment I: plate incorporation test
Experiment II and IIa: pre-incubation test

In the pre-incubation assay 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and incubated at 30 °C for 30 minutes.
After pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

OVERLAY AGAR
The overlay agar contains per litre: 6.0 g MERCK Agar Agar, 6.0 g NaCl, 10. 5 mg L-Histidin x HCl x H2O, 12.2 mg Biotin.
Sterilizations were performed at 121 °C in an autoclave.

NUMBER OF REPLICATIONS: concentration, including the controls, was tested in triplicate.

PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in the pre-experiment were the same as described for the main experiments.

MAMMALIAN MICROSOMAL FRACTION
The protein concentration in the S9 preparations was 24.4 mg/ml (rat liver) in the pre-experiment and in experiment I and 38.2 mg/ml (hamster liver) in experiments II and IIa.

Rat liver S9
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (weight approx. 22O - 320 g) which received daily applications of 80 mg/kg b.w. Phenobarbital i.p. dissolved in aqua deionised and p-Naphthoflavone orally dissolved in corn oil on three subsequent days. The livers were prepared 24 hours after the last treatment.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCI and homogenised. The homogenate, was diluted 1+3 in KCI and centrifuged at 9000 g for 10 minutes at 4 °C.

Rat S9 Mix: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the cofactor solution was chosen to yield the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCI, 5 mM, glucose-6-phosphate, 5mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

Hamster liver S9
The S9 liver microsomal fraction was obtained from the livers of 7 - 8 weeks old male Syrian golden hamsters. After cervical dislocation the livers of the animals were removed, washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and I mM disodium EDTA in bidistilled water and homogenised. The homogenate, diluted 1 + 3 in sodium phosphate buffer was centrifuged at 9000 g for 10 minutes at 4 °C.
A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to several weeks before use.

Hamster S9 Mix: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 22.5 %, v/v. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix: 33 mM KCI, 8 mM MgCl2, 20 mM glucose 6-phosphate, 2.8 units/ml glucose 6-phosphate dehydrogenase, 4 mM NADP, 2 mM NADH, 2 mM FMN in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

Evaluation criteria:

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is ihduced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows: a test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

toxic effects observed in strain TA 100 in the second experiment were judged as biologically irrelevant

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 article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with test item at any concentration level, neither in the
presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledlged border of biological relevance.

CYTOTOXICITY
No toxic effects evident as a reduction in the number of revertants occurred in the test groups with and without metabolic activation up to the highest investigated dose except in strain TA 100 in the second experiment.
Strain TA 100 showed a very low frequency of revertant colonies at all concentrations in the absence of metabolic activation and at concentrations above 333.3 µg/plate in the presence of metabolic activation. Since these striking toxic effects did not occur in strain TA 100 in the first experiment or in any of the other strains in both experiments, an additional experiment following the same procedure (experiment IIa) was performed with strain TA 100 to verify the toxic effects observed in the second experiment. This additional experiment did not show any toxic effects up to the maximal concentration with or without metabolic activation. Therefore, the toxic effects observed in strain TA 100 in the second experiment were judged as biologically irrelevant.

CONTROLS
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental
conditions reported, the test article did not induce geni mutations by base pair changes
or frameshifts in the genome of the strains used.

Overview of the results obtained in the plate incorporation tests run with/without rat liver microsomal activation system

	TA 1535		TA 1537		TA 98		TA 100
	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
Negative control	24	23	14	20	15	20	118	131
Solvent control	16	19	14	20	15	17	106	120
Test item 33.3 µg/plate	22	24	14	17	13	20	110	118
Test item 100.0 µg/plate	18	21	12	20	13	21	102	143
Test item 333.3 µg/plate	23	25	14	20	17	18	118	140
Test item 1000.0 µg/plate	18	23	12	21	13	17	111	135
Test item 2500.0 µg/plate	18	19	14	17	13	16	118	126
Test item 5000.0 µg/plate	16	19	11	18	14	14	126	146
Sodium azide 10 µg/plate	1576	-	-	-	-	-	757	-
2-aminoanthracene 2.5 µg/plate	-	311	-	129	-	120	-	629
4-nitro-o-phenylene-diamine 50 µg/plate	-	-	164	-	711	-	-	-

Overview of the results obtained in the pre-incubation test run on SCARLET RN 1165 with/without hamster liver microsomal activation system

	TA 1535		TA 1537		TA 98		TA 100		TA 100
	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
Negative control	24	21	12	15	16	20	126	107	114	148
Solvent control	22	21	9	14	20	22	117	90	102	157
Test item 33.3 µg/plate	23	21	13	10	18	19	22	104	103	146
Test item 100.0 µg/plate	22	22	12	14	21	23	59	122	115	162
Test item 333.3 µg/plate	19	18	7	18	20	18	36	50	120	157
Test item 1000.0 µg/plate	19	23	9	14	12	15	5	14	100	141
Test item 2500.0 µg/plate	19	21	10	16	21	23	4	9	108	156
Test item 5000.0 µg/plate	20	19	12	16	19	32	3	7	105	162
Sodium azide 10 µg/plate	1334	-	-	-	-	-	646	-	938	-
2-aminoanthracene 2.5 µg/plate	-	178	-	163	-	-	-	694	-	740
4-nitro-o-phenylene-diamine 10 - 50 µg/plate	-	-	129	-	309	-	-	-	-	-
Congo red 500 µg/plate	-	-	-	-	-	294	-	-	-	-

Conclusions:

The test item resulted to be non-mutagenic in the Salmonella typhimurium reverse mutation assay for azo dyes performed.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test for azo dyes (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. An additional experiment (experiment IIa) was performed with strain TA 100 only according to the procedure of the second experiment to verifu erratic toxic effects observed in strain TA 100 in the second experiment. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate.

No reproducible toxic effects evident as a reduction in the number of revertants occurred in the test groups with and without metabolic activation up to the highest investigated dose.

The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

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

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay for azo dyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN VITRO GENE MUTATION ASSAY IN BACTERIA

Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the Reactive Yellow 125 using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The method used conforms with the OECD guidelines 471. The test material caused no visible reduction in the growth of the bacterial lawn at any of the dose levels to any of the strains of Salmonella tested. The test material was, therefore, tested up to the maximum recommended dose of 5000 µg/plate. A yellow/orange colouration was observed at and above 1500 µg/plate, this did not interfere with the scoring of revertant colonies. 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.

Although the test was conducted following procedures in accordance with generally accepted scientific standards and was described in sufficient detail, the purity degree of the lot tests cannot be confirmed.

Thus, the available data on the structural analogous Similar Substance 01 has been taken into consideration, in order to support the study outcomes on the target substance; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID endpoint record).

The in vitro gene mutation assay was performed according to the plate incorporation test (experiment I) and the pre-incubation test for azo dyes (experiment II and IIa), using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation; an additional experiment (experiment IIa) was performed with strain TA 100 only according to the procedure of the second experiment to verify erratic toxic effects observed in strain TA 100 in the second experiment.

No reproducible toxic effects evident as a reduction in the number of revertants occurred in the test groups with and without metabolic activation up to the highest investigated dose. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used. No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay for azo dyes.

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 substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC) No 1272/2008.