Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, disodium salt, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, disodium salt, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid, sodium salts

EC Number:

215-397-8

EC Name:

Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, disodium salt, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid, sodium salts

Cas Number:

1325-54-8

Molecular formula:

Molecular formula of the main constituents: (1) C38H24N8Na4O12S4 (2) C52H32N10Na6O18S6

IUPAC Name:

decasodium 2-[(1E)-2-{2-sulfonato-4-[(1E)-2-{3-sulfonato-4-[(1E)-2-{2-sulfonato-4-[(1E)-2-{4-[(1E)-2-(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]phenyl}ethenyl]phenyl}diazen-1-yl]phenyl}ethenyl]-5-[(1E)-2-{4-[(1E)-2-(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzene-1-sulfonate 2-[(1E)-2-{2-sulfonato-4-[(1E)-2-{4-[(1E)-2-(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]phenyl}ethenyl]-5-[(1E)-2-{4-[(1E)-2-(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzene-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Batch number: PAL-022588

Method

Target gene:

Thymidine Kinase locus (TK)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Informatio on cell cultures:
Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with:
Hypoxanthine 5.0 * 10-3 M
Aminopterin 2.0 * 10-5 M
Thymidine 1.6 * 10-3 M
Glycin 5.0 * 10-3 M
The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium containing:
Hypoxanthine 1.0* 10-4 M
Thymidine 1.6 * 10-3 M
After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal concentration of the test item and in the solvent control without metabolic activation.

According to the results of the pre-test at least four adequate concentrations were chosen for the mutation experiment.

Experiment I
Exposure period 4h (S9 mix -): 187.5, 375, 750, 1500, 3000, 6000 µg/mL
Exposure period 4h (S9 mix +): 187.5, 375, 750, 1500, 3000, 6000 µg/mL

Experiment II
Exposure period 24h (S9 mix -): 375, 750, 1500, 3000, 4500, 6000 µg/mL
Exposure period 4h (S9 mix +): 375, 750, 1500, 3000, 4500, 6000 µg/mL

Following the expression phase of 48 hours the cultures at the lowest concentrations (187.5, 375 ) in experiment I and II were not continued since a minimum of only four concentrations is required by the guidelines.

Vehicle / solvent:

Water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Local tap water deionised at Harlan CCR

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

On the day of the experiment (immediately before treatment), the test item was diluted with deionised water (10 %). The final concentration of deionised water in the culture medium was 10% (v/v).

The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation:

Solvent control DIRECT ORANGE 39 (C.I. 40215)
6000 µg/mL
Osmolarity [mOsm] 270 304
pH-value 7.51 7.70


Experimental Performance
In the mutation experiment 1*10E7 (3x10E6 during 24 h exposure) cells/flask (80 cm2 flasks) suspended in 10 mL RPMI medium with 3 % horse serum (15 % horse serum during 24 h exposure) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. Positive and solvent controls were performed in parallel. After 4 h (24 h in the second experiment) the test item was removed by centrifugation (425 x g, 10 min) and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium and incubated for an expression and growth period of 48 h.

The cell density was determined each day and adjusted to 3*10E5 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector.

After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4*10E3 cells in selective medium (see below) with TFT (Serva, 69042 Heidelberg, Germany). The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 370°+- 1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated. The relative total growth (RTG) is calculated by the RSG multiplied by the viability.

Complete Culture Medium
RPMI 1640 medium supplemented with 15 % horse serum (HS) (3 % HS during 4 hour treatment), 1% of 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 % Amphotericin used as antifungal agent.

Selective Medium
RPMI 1640 (complete culture medium) by addition of 5 µg/mL TFT.

Saline G Solution: The "saline G" solution was composed as follows (per litre):
NaCl8000 mg; KCl 400 mg; Glucose 1100 mg; Na2HPO4x7H2ONa2HPO4x2H2O 192 mg; KH2PO4 150 mg; pH: 7.2

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression (least squares) will be performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT(R)11 (SYSTAT Software, Inc.) statistics software. The number of mutant colonies obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance will be considered together

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

6000 µg/mL (24h)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test medium was checked for precipitation visible to the naked eye at the end of the 4 hours treatment just before the test item was removed. No precipitation meeting the criteria mentioned above was noted in the pre-experiment and in both main experiments.

A relevant cytotoxic effect as indicated by a relative total growth of 50% or below at in both parallel cultures was solely observed in experiment II following 24 hours treatment at the maximum concentration of 6000 µg/mL without metabolic activation.

No substantial and reproducible increase of the mutation frequency was noted in both experiments with and without metabolic activation. The threshold of 126 above the corresponding solvent control was not reached.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups.

In this study the range of the solvent controls was from 51 up to 88 mutant colonies per 106 cells; the range of the groups treated with the test item was from 33 up to 148 mutant colonies per 106 cells. The viability of the solvent control of the first experiment, culture II without metabolic activation slightly exceeded the upper limit of the acceptance criteria (121 versus 120%, c.f. table 8, column 8). This deviation was judged as irrelevant as it was very minor and the viability of the parallel culture remained within the acceptable range.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 µg/mL and 4.5 µg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity at with at least one of the concentrations of the controls. The positive controls remained within the range of the historical positive control data throughout the study.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results, Experiment I and II

			relative	mutant		relative	mutant
	conc. ug/mL	S9 mix	total growth	colonies 10^6 cells	treshold	total growth	colonies 10^6cells	treshold
column	1	2	3	4	5	6	7	8
experiment I / 4 h treatment			culture I			culture II
Solvent control with water		-	100.0	88	214	100.0	86	212
Pos. control with MMS	19.5	-	38.8	312	214	19.4	346	212
Test item	187.5	-	culture was not continued			culture was not continued
Test item	375.0	-	132.3	68	214	78.1	81	212
Test item	750.0	-	160.3	59	214	106.8	62	212
Test item	1500.0	-	119.2	58	214	45.2	142	212
Test item	3000.0	-	73.2	81	214	60.8	77	212
Test item	6000.0	-	92.6	90	214	29.6	127	212
Solvent control with water		+	100.0	59	185	100.0	54	180
Pos. control with CPA	3.0	+	52.7	216	185	46.5	231	180
Pos. control with CPA	4.5	+	18.8	417	185	17.9	481	180
Test item	187.5	+	culture was not continued			culture was not continued
Test item	375.0	+	92.4	50	185	137.1	39	180
Test item	750.0	+	91.0	46	185	150.7	33	180
Test item	1500.0	+	106.0	66	185	108.7	35	180
Test item	3000.0	+	92.0	56	185	91.4	41	180
Test item	6000	+	98.2	73	185	110.7	37	180
Experiment II / 24 h treatment			culture I			culture II
Solv. control with water		-	100.0	51	177	100.0	51	177
Pos. control with MMS	13.0	-	23.2	461	177	19.0	439	177
Test item	375.0	-	culture was not continued			culture was not continued
Test item	750.0	-	125.1	47	177	115.2	66	177
Test item	1500.0	-	57.0	92	177	50.7	148	177
Test item	3000.0	-	59.8	57	177	52.4	77	177
Test item	4500.0	-	50.6	75	177	33.5	77	177
Test item	6000.0	-	22.5	112	177	18.5	76	177
Experiment II / 4h treatment			culture I			culture II
Solvent control with water		+	100.0	68	194	100.0	81	207
Pos. control with CPA	3.0	+	23.8	379	194	24.0	495	207
Pos. Control with CPA	4.5	+	6.7	648	194	11.3	527	207
Test item	375.0	+	culture was not continued			culture was not continued
Test item	750.0	+	98.0	51	194	109.0	99	207
Test item	1500.0	+	135.3	76	194	188.0	93	207
Test item	3000.0	+	223.6	59	194	208.5	79	207
Test item	4500.0	+	151.5	47	194	182.3	63	207
Test item	6000	+	116.2	52	194	144.4	89	207

threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

#   culture was not continued since a minimum of only four concentrations is required by the guidelines

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, DIRECT ORANGE 39 (C.I. 40215) is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of DIRECT ORANGE 39 (C.I. 40215) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

 

The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The treatment period of the second experiment was 4 hours with and 24 hours without metabolic activation.

 

The maximum test item concentration of 6000 µg/mL used in the pre-experiment and in the main experiments with and without metabolic activation was equal to approximately 5000 µg/mL of the pure substance. The test item was dissolved in deionised water.

 

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.