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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

QSAR estimation, negative.

OECD 476, mammalian cells, the substance is non mutagenic.

OECD 486, micronucleus, the substance is non mutagenic.

OECD 471, ames, negative.

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Source study has reliability 2. Details on the read across are attached in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 97, TA 98, TA 1535, TA 100

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

The sample was dissolved in water. The highest possible concentration is 1500 μg per 0.1 ml.
The test was performed with the range of concentrations from 0.5 to 5000 μg/plate.

Vehicle / solvent:

Water

Untreated negative controls:

yes

Positive controls:

yes

Evaluation criteria:

Results were evaluated according to the modified two-fold increase rule; this is comparable with using statistical methods. The result is considered as positive, when reproducible dose-effect and/or doubling of ratio Rt/Rc is reached.
Rt/Rc is ratio of number of revertants at tested dose to number of revertants at negative control.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

The effect of test material on strain TA 97.

Dose (µg per plate)	TA 97 A
	-S9		+S9
	x ± s	Rt/Rc	x ±  s	Rt/Rc
0.0	137 ± 8	1.0	155 ± 10	1.0
0.5	141 ± 19	1.0	156 ± 18	1.0
5.0	144 ± 4	1.0	158 ± 6	1.0
50.0	148 ± 7	1.1	174 ± 6	1.1
500.0	153 ± 4	1.1	163 ± 6	1.0
1500.0	146 ± 10	1.1	150 ± 17	1.0
5000.0	152 ± 34	1.1	144 ± 11	0.9
NPD	636 ± 18	4.6	---
2 -AF	---		604 ± 64	3.9

The effect of test material on strain TA 98.

Dose (μg per plate)	TA 98
	-S9		+S9
	x ± s	Rt/Rc	x ± s	Rt/Rc
0.0	18 ±  3	1.0	22 ± 2	1.0
0.5	16 ± 3	0.9	21 ± 1	0.9
5.0	21 ± 3	1.2	19 ± 2	0.9
50.0	22 ± 6	1.3	21 ± 3	1.0
500.0	23 ± 3	1.3	17 ± 4	0.8
1500.0	20 ± 2	1.1	14 ± 2	0.6
5000.0	20 ± 2	1.1	15 ± 6	0.7
NPD	1776 ± 42	100.5	---
2 -AF	---		1350 ± 47	61.4

The effect of test material on strain TA 100

Dose (μg per plate)	TA 100
	-S9		+S9
	x ± s	Rt/Rc	x ± s	Rt/Rc
0.0	141 ± 20	1.0	129 ± 10	1.0
0.5	143 ± 16	1.0	126 ± 10	1.0
5.0	154 ± 9	1.1	126 ± 9	1.0
50.0	146 ± 5	1.0	141 ± 12	1.1
500.0	129 ± 11	0.9	149 ± 6	1.2
1500.0	135 ± 14	1.0	129 ± 15	1.0
5000.0	139 ± 9	1.0	144 ± 12	1.1
AS	1209 ± 40	8.6	---
2 -AF	---		846 ± 60	6.5

The effect of test material on strain TA 1535.

Dose (μg per plate)	TA 1535
	-S9		+S9
	x ± s	Rt/Rc	x ± s	Rt/Rc
0.0	14 ± 3	1.0	15 ± 4	1.0
0.5	16 ± 4	1.1	12 ± 2	0.8
5.0	15 ± 4	1.1	14 ± 3	1.0
50.0	12 ± 3	0.9	20 ± 3	1.4
500.0	11 ± 3	0.8	11 ± 3	0.8
1500.0	13 ± 1	1.0	13 ± 3	0.9
5000.0	17 ± 2	1.2	12 ± 4	0.8
AS	882 ± 33	63.0	---
2 -AA	---		54 ± 8	3.6

Conclusions:

The substance was tested according to the guideline OECD 471.
On the base of results, the substance was considered non mutagenic with as well as without metabolic ctivation.

Executive summary:

Method

Mutagenicity was assessed in 4 Salmonella typhimurium strains, i.e. TA 97, TA 98, TA 100 and TA 1535, performing an Ames test according to OECD guideline 471. The range of concentration was 0.5 -5.000 g per plate. Metabolic activation experiments were performed with supernatant of rat liver and mixture of cofactors.

Results

Test material was unable to revert any of above mentioned strains, either with or without metabolic activation.

Based on these results, test sample was considered as non mutagenic with as well as without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Source study has reliability 1. Details on the read across are attached in section 13.

Qualifier:

according to guideline

Guideline:

other: OECD 487 (“In vitro Mammalian Cell Micronucleus Test”, adopted July 22, 2010)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Stocks of the V79 cells (obtained from LMP; Technical University Darmstadt, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR. This allows the repeated use of the same cell culture batch in experiments. Before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar on time because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. About 5 x 105 cells per flask ere seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine, Hepes (25 mM) and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL). The cells were sub-cultured twice a week. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

mammalian microsomal fraction S9 mix

Test concentrations with justification for top dose:

Dose selection was performed following OECD guideline 487. In general, test item should be tested up to a maximum concentration of 5 mg/ml, 5 µl/ml or 10 mM if test item is not toxic. The highest treatment concentration chosen for evaluation of genotoxicity should reduce the proliferation index to 55 ± 5 % of the solvent control. The solubility of test item and changes in the pH value and osmolarity influence the dose selection.
With regard of purity (85 %) of test item, 5882.0 µg/ml of test material was applied as top concentration for the treatment in the pre-experiment. Test item concentrations between 11.5 and 5882.0 µg/ml (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of test item was observed at 1470.5 µg/ml and above without S9 mix and at 2941.0 µg/ml and above with S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated as Experiment I.
Dose selection of Experiment II was influenced by the results obtained in Experiment I. In Experiment I, cytotoxicity was observed at 1470.5 µg/ml and above. Therefore, concentrations between 5.9 and 3000.0 µg/ml in the absence of S9 mix as well as concentrations between 75.0 and 3000.0 µg/ml in the presence of S9 mix were applied.

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

mitomycin C

other: Griseofulvin

Details on test system and experimental conditions:

Experimental Performance
Culture medium and conditions: for seeding and treatment of cell cultures, culture medium was MEM (minimal essential medium) containing Hank’s salts, glutamine, Hepes (25 mM) and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/ml/100 mg/ml). All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
Seeding of the cultures: exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/l NaCl, 200 mg/l KCl, 200 mg/l KH2PO4 and 150 mg/l Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes, which contained microscopic slides. The cells were seeded into Quadriperm dishes, which contained microscopic slides. Into each chamber 1.0E5 – 1.5E5 cells were seeded with regard to the preparation time.

Treatment
Exposure period 4 hours: culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 ml S9 mix per ml medium was added.
Concurrent solvent and positive controls were performed. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/l NaCl, 400 mg/l KCl, 1100 mg/l glucose × H2O, 192 mg/l Na2HPO4 × 2 H2O and 150 mg/l KH2PO4. Then the cells were cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.
Exposure period 24 hours: culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item without S9 mix. The medium was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.
Preparation of cultures: for the micronucleus analysis, 24 hours after the start of the exposure, the cells were treated on the slides in the chambers of the quadriperm dishes with deionised water for 1 to 1.5 min at 37 °C. Afterwards the cells were fixed twice with a solution containing 3 parts ethanol, 1 part acetic acid and 1.25 % (v/v) formaldehyde. After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.
Analysis of micronuclei and cytotoxicity: evaluation of cultures was performed manually using NIKON microscopes with 40× objectives. Micronuclei were counted in cells showing a clearly visible cytoplasm area. Briefly micronuclei were stained in the same way as the main nucleus.
The area of the micronucleus did not extend the third part of the area of the main nucleus. 1000 cells in two parallel cultures were scored for micronuclei, so that at least 2000 cells from clones with 2 - 8 cells were analysed per test group. The frequency of micronucleated cells was reported as % micronucleated cells. Cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), and 5 - 8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represented the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated. Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee for a sufficient cell proliferation during treatment and recovery.

Evaluation criteria:

Many experiments with Chinese Hamster V79 cells have established a range of micronucleus frequencies acceptable for control cultures. The current historical data range together with the statistical significance, confirmed by the Chi square test (α < 0.05), should be considered for classification of the test item.
The micronucleus assay will be considered acceptable if it meets the following criteria:
a) micronucleus frequency in the solvent controls falls within the historical laboratory control data range,
b) micronucleus frequency in the positive controls is statistically significantly increased,
c) quality of the slides must allow the evaluation of a sufficient number of analyzable cells.

A test item can be classified as mutagenic if:
a) the number of micronucleated cells is not in the range of the historical control data and
b) either a statistically significant concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.
A test item can be classified as non-mutagenic if:
a) the number of micronucleated cells in all evaluated test groups is in the range of the historical control data and
b) no statistically significant concentration-related increase in the number of micronucleated cells is observed.
Statistical significance was confirmed by means of the Chi square test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1470.5 µg/ml and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Summary of results of micronucleus test

exp	preparation interval	test item concentration in µg/ml	proliferation index	micronucleated cells (*) in %
exposure period 4 hours without S9 mix
I	24h	solvent control (1)	2.85	0.35
		positive control (2)	2.28	14.95 (s)
		183.8	2.81	1.05 (s)
		367.6	2.86	0.50
		735.3	2.81	1.10
exposure period 24 hours without S9 mix
II	24h	solvent control (1)	2.87	0.40
		positive control (3)	2.40	80.55 (s)
		93.8	2.43	0.60
		187.5 (P)	2.33	0.65
		375.0 (P)	2.46	0.85
		750 .0 (P)	2.23	0.70
exposure period 4hours with S9 mix
I	24h	solvent control (1)	1.68	1.05
		positive control (4)	1.49	10.50 (s)
		183.8	2.04	1.25
		367.6	2.31	1.58 (**)
		735.3	2.44	1.50 (**)
II	24h	solvent control (1)	2.06	0.60
		positive control (5)	1.46	3.95 (s)
		700.0	2.41	0.65
		900.0	2.50	1.20 (s)
		1100.0	2.56	0.90

*     number of micronucleated cells was determined in a sample of 2000 cells

**   number of micronucleated cells was determined in a sample of 4000 cells

^S     number of micronucleated cells statistically significantly higher than corresponding control values

¹       deionised water   10.0 % (v/v)

²           Mitomycin C          0.3 µg/ml

³           Griseofulvin          8.0 µg/ml

⁴           CPA                    20.0 µg/ml

⁵           CPA                    15.0 µg/ml

Conclusions:

The substance was tested according to the guideline OECD 487.
Under the experimental conditions, no mutagenicity was observed in the absence and presence of metabolic activation.

Executive summary:

Method

Test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix according to OECD guideline 487. The following study design was performed:

	experiment I		experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
exposure period	4 h	4 h	24 h	4 h
recovery	20 h	20 h	0 h	20 h
preparation interval	24 h	24 h	24 h	24 h

In each experimental group, two parallel cultures were analyzed and at least 1000 cells per culture were scored for micronuclei. Appropriate mutagens were used as positive controls.

Results

In the absence and presence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

No mutagenicity was observed with or without S9 mix. In the absence and presence of S9 mix, a single statistically significant increase being clearly in the range of the laboratory historical control data was observed after treatment with test item.

Positive controls induced statistically significant increases (p < 0.05) in the percentage of micronucleated cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Source study has reliability 1. Details on the read across are attached in section 13.

Qualifier:

according to guideline

Guideline:

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

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:

other: gene mutation in mammalian cells

Target gene:

Thymidine Kinase locus (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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 5.0E-3 M
Aminopterin 2.0E-5 M
Thymidine 1.6E-3 M
Glycin 5.0E-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 1.0E-4 M
Thymidine 1.6E-3 M
After this incubation, the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium)

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 test item and in 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

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 experiment (immediately before treatment), test item was diluted with deionised water (10 %). The final concentration of deionised water in the culture medium was 10 % (v/v).
Osmolarity and 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 test substance at 6000 µg/ml
osmolarity [mOsm] 270 304
pH-value 7.51 7.70

Experimental performance
In the mutation experiment 1.0E7 (3.0E6 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 test item either in presence or absence of metabolic activation. Positive and solvent controls were performed in parallel. After 4 h (24 h in the second experiment), test item was removed by centrifugation (425 × 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 3.0E5 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 4.0E3 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):
NaCl 8000 mg; KCl 400 mg; glucose 1100 mg; Na2HPO4×7H2ONa2HPO4×2H2O 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 1.0E6 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 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 1.0E6 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.

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

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:

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 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 a 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 1.0E6 cells; the range of the groups treated with the test item was from 33 up to 148 mutant colonies per 1.0E6 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 %). 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.

Summary of results, experiment I and II

	conc. µg/ml	S9 mix	relative total growth	mutant colonies 10^6 cells	treshold	relative total growth	mutant colonies 10^6cells	treshold
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

Conclusions:

The substance was tested according to the guideline OECD 476.
On the base of results, the substance was considered non-mutagenic in mouse lymphoma assay both with and without metabolic activation.

Executive summary:

Method

The potential of test substance to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y was investigated according to OECD guideline 476.

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.

Appropriate reference mutagens were used as positive controls.

 

Results 

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 test item.

Positive controls showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

QSAR Estimation

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

According to Annex VII of the REACH Regulation (EC 1907/2006) and ECHA Guidance Chapter R.7a: Endpoint specific guidance, Version 5.0 – December 2016, a preliminary assessment of mutagenicity should normally include data from a gene mutation test in bacteria unless existing data for analogous substances indicates that this would be inappropriate. When the result of the bacterial test is positive, it is important to consider the possibility of the substance being genotoxic in mammalian cells, whereas a negative response would generally not require any further testing. However, if results from further studies of genotoxicity are available, it is recommended to take them into account in the assessement.

 

No experimental data on genotoxic potential of target substance was available, thus the assessment was based on QSAR Estimation and on data on Similar Substance 01. A detailed description of the read across approach is attached in section 13.

The QSAR estimation was performed according to the prediction Tool Developed by: Milano Chemometrics and QSAR Research Group, Dept. Earth and Environmental Sciences, University Milano-Bicocca, Italy. The estimation resulted in the applicability domain and Negative.

 

In order to support this estimation the following studies were considered for the assessment.

 

Genotoxic potential of Similar Substance 01 was examined in a standard battery of tests, which allowed to trace its genotoxic profile: in vitro gene mutation assays in bacteria (OECD 471), in vitro mouse lymphoma assay (OECD 476) and in vitro micronucleus assay (OECD 487).

 

A gene mutation assay at the thymidine kinase locus  (TK) in mouse lymphoma L5178Y cells was performed with and without metabolic activation at concentrations up to 6000 µg/ml. Cytotoxicity was not seen in the preliminary toxicity test, while it was detected at 6000 µg/ml in the second experiment after 24 hour of exposure with metabolic activation. Mutagenic effects were not noted, thus test substance was considered as not mutagenic in this assay.

 

A micronucleus test in Chinese hamster V79 cells was performed with and without metabolic activation. In a preliminary test (exp. I), precipitation was noted starting at 1470.5 and 2941 µg/ml without and with metabolic activation, respectively. Toxicity was noted starting at 1470.5 µg/plate both with and without metabolic activation. Based on results in exp. I, concentrations up to 3000 µg/ml were used in exp. II. No increase in micronucleated cells was noted, thus test substance was considered as not mutagenic in this assay.

 

As for bacterial gene mutation, in a study on Salmonella typhimurium strains TA 97, TA 98, TA 100 and TA 1535, no mutagenic effects were noted up to the highest concentration of 5000 µg/plate both with and without metabolic activation. Purity of test sample was 63 %.

 

 

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), Annex I, Part 3, substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans are classified in Category 2. This classification is based on positive evidence obtained in:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Note: substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

In vitro mutagenicity tests are the following:

— in vitro mammalian chromosome aberration test;

— in vitro mammalian cell gene mutation test;

— bacterial reverse mutation tests.

Based on the responses in the available experimental studies and estimation, a genotoxic potential for target substance is excluded. Therefore, it is not classified according to the CPL Regulation (EC 1272/2008).