Tetrasodium 2-[[8-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]benzoyl]amino]-1-hydroxy-3,6-disulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is expected to be non-mutagenic in Salmonella typhimurium reverse mutation assay.

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:

key study

Study period:

From September 13 to November 10, 1999

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 to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

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 TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: bacterial strains TA 1535, TA 98, TA 100, and TA 102 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),

PERIODICAL CHECK
Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in testing facility.

PRECULTURES
From the thawed ampoules of the strains 0.5 ml bacterial suspension were transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin (25 µg/ml) was added to the strains TA 98, TA 100, and TA 102.20 µl tetracycline (2 µg/ml) was added to strain TA 102. This nutrient medium contains per litre.
8 g Merck Nutrient Broth
5 g NaCl
The bacterial culture was incubated in a shaking water bath for 8 hours at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle: on the day of experiment, the test item was dissolved in deionised water.
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties.

Untreated negative controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

congo red

methylmethanesulfonate

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation. According to the pre-incubation method 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer, and 100 µl bacteria 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.

EXPERIMENTAL PERFORMANCE
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µl Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µl Overlay agar; the overlay agar contains per litre: 6.0 g MERCK Agar Agar; 6.0 g NaCl; 10.5 mg L-Histidine x HCl x H2O; 12.2 mg Biotin. Sterilisations were performed at 121 °C in an autoclave.

NUMBER OF REPLICATIONS: for each strain and dose level, including the controls three plates were used.

PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test item 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 experiment I.
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

RAT LIVER S9
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (BRL, CH-4414 Füllinsdorf; weight approx. 220 - 320 g) which received three applications of 80 mg/kg b.w. Phenobarbital i.p. dissolved in aqua deionised (Desitin; D-22335 Hamburg) and β-Naphthoflavone orally dissolved in corn oil (Aldrich, D-89555 Steinheim). The livers were prepared 24 hours after the last treatment.
After decapitation of the anaesthetised animals the livers of the animals are removed, washed in 150 mM KCI and homogenised. The homogenate, diluted 1+3 in KCl, is centrifuged at 9000 g for 10 minutes at 4 °C.

Rat liver mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with 39 cofactor solution. The amount of S9 supernatant was 15 % vlv in the cultures. The concentrated cofactor solution yields the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCI, 5 mM glucose-6-phosphate and 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH7.4.
During the experiment the S9 mix are stored in an ice bath. The S9 mix preparation is performed according to Ames et al.

HAMSTER LIVER S9
The S9 liver microsomal fraction was obtained from the liver of 7 - 8 weeks old male Syrian golden hamsters (BRL, CH-4414 Füllinsdorf).
After decapitation of the anaesthetised animals the livers of the animals were removed, washed in 0.1 M sodium phosphate buffer pH7.4,0.25 M sucrose and 1 mM disodium EDTA in deionised 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 supernatants containing the microsomes was frozen in ampoules and stored at - 80 °C. Small numbers of the ampoules were kept at -20 °C for up to one week before use.
The protein content is determined using an analysis kit of Bio-Rad Laboratories, D-80939 München (Bio-Rad protein assay, Catalogue No. 5000006). The protein concentration in the S9 preparations was 32.5 (lot no. 110699) in the preexperiment and in experiment I (strains TA 98 and TA 100), 28.4 mglml, lot no 020999, (rat liver) in experiment I and 27.4 mglml, lot no. 270899 (hamster liver) in experiment II.

Hamster liver mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant is 30 % v/v. The concentrated cofactor solution yields the following concentrations in the S9 mix: 8.0 mM MgCl2, 33.0 mM KCI, 20 0 mM Glucose-6-phosphate, 2.8 units/ml Glucose-6-phosphate-dehydrogenase, 4.0 mM NADP, 2.0 mM NADH and 2.0 mM FMN in 100 mM Sodium-Ortho-Phosphate-buffer, pH7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. and Prival and Mitchell.

ACCEPTABILITY PF THE ASSAY
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- normal background growth in the negative and solvent control
- normal range of spontaneous reversion rates in the negative and solvent control
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation criteria:

A test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced.
A test item producing neither a dose related increase in the number of revertants nor a biologically relevant 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 item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100, and TA 102 or thrice in strains 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 item regardless whether the highest dose induced the criteria described above or not.

Species / strain:

S. typhimurium, other: TA 1535, TA1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No relevant 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 item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance 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 acknowledged border of biological relevance.

POSITIVE CONTROL
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

PRE-EXPERIMENT FOR TOXICITY
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The preexperiment is reported as part of experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.

Summary of Results

S9 mix from: Hamster liver

S9 mix from: Rat liver

without S9 mix

Concentrationµg/plate	Revertants/plate mean from three plates
	TA 1535		TA 1537		TA 98		TA 100		TA 102
	I	II	I	II	I	II	I	II	I	II
Negative control	14	13	14	20	18	22	101	115	220	289
Solvent control	16	8	10	24	23	19	102	129	219	251
Positive control*	1509	457	61	156	517	215	1424	949	1330	1091
33	18	12	15	27	17	21	104	125	232	352
100	20	13	14	16	20	29	117	121	221	376
333	18	12	13	21	23	23	116	132	229	358
1000	21	13	12	20	23	19	110	121	245	344
2500	16	11	18	24	23	22	102	147	233	272
5000	17	8	11	11	23	23	89	130	187	225

* Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diarnine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate); Methyl methane sulfonate (5 µg/plate) strain TA 102

with S9 mix

Concentrationµg/plate	Revertants/plate mean from three plates
	TA 1535		TA 1537		TA 98		TA 100		TA 102
	I	II	I	II	I	II	I	II	I	II
Negative control	14	8	23	20	36	36	124	120	343	351
Solvent control	13	8	17	23	34	29	119	133	316	376
Positive control**	264	210	91	262	484	1811	674	942	1006	1237
33	9	8	16	18	29	30	118	113	336	326
100	12	10	18	25	28	40	99	140	320	411
333	13	10	17	26	33	32	101	168	343	401
1000	15	7	18	22	31	45	114	140	324	439
2500	8	7	17	24	30	40	97	159	313	411
5000	13	11	13	20	31	22	98	136	217	204

**2-arninoantlrracene (2.5 µg/plate) strains TA 1535, TA 1537 and TA 100; 2-aminoanthracene (10.0 µg/plate) strain TA 102 congo red (500 µg/plate) strain TA 98

Conclusions:

The test item was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 µg/plate.

No relevant 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 item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five 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

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, test item was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There is no information about the in vitro gene mutation potential in bacteria of Reactive Red 147, thus the available information on the structural analogous Similar Substance 01 has been taken into consideration; 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 section 13).

The study was performed to investigate the potential of Similar Substance 01 to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I) and the pre-incubation test with hamster liver S9 (experiment II), using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments, both with and without liver microsomal activation. The test item was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 µg/plate. No relevant 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 item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increase in revertant colony numbers of any of the five 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.

Therefore, during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and test item was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

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.