2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27-11-2016 to 19-12-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

EC Number:

287-719-5

EC Name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Cas Number:

85567-07-3

Molecular formula:

C36H42Cl2KN6NaO28S8

IUPAC Name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Test material form:

solid

Details on test material:

Batch: 1283067001

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Experiment I: S9 mix from rat liver; Experiment II: S9 mix from hamster liver

Test concentrations with justification for top dose:

Pre-experiment (plate incorporation):
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades.

Experiment I (plate incorporation test with and without rat liver S9):
31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Experiment II (pre-incubation test with and without hamster liver S9):
10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s): Aqua dest.
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

solvent control is negative control

Negative solvent / vehicle controls:

yes

Remarks:

Aqua dest.

True negative controls:

no

Positive controls:

yes

Remarks:

S. typhimurium: TA 100, TA 1535 without metabolic activation (dissolved in A. dest.)

Positive control substance:

sodium azide

Controls 2

Positive controls:

yes

Remarks:

S. typhimurium: TA 98, TA 1537 without metabolic activation (dissolved in DMSO)

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD) 10 μg/plate for TA 98, 40 μg/plate for TA 1537

Controls 3

Positive controls:

yes

Remarks:

S. typhimurium: TA 102 without metabolic activation (dissolved in A. dest).

Positive control substance:

methylmethanesulfonate

Controls 4

Positive controls:

yes

Remarks:

typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102 with metabolic activation (Rat Liver) (dissolved in DMSO)

Positive control substance:

other: 2-aminoanthracene (2-AA) 2.5 μg/plate; 10 μg/plate for TA 102

Controls 5

Positive controls:

yes

Remarks:

S. typhimurium: TA 1535, TA 1537, TA 102 with metabolic activation (Hamster Liver) (dissolved in DMSO)

Positive control substance:

other: 2-aminoanthracene (2-AA)

Controls 6

Positive controls:

yes

Remarks:

S. typhimurium: TA 98, TA 100 with metabolic activation (Hamster Liver) (dissolved in A. Dest.)

Positive control substance:

congo red

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation)
Experiment II: preincubation with hamster liver S9

DURATION
Experiment I: in agar (plate incorporation)
- Preincubation period: No
- Exposure duration: at least 48 h in the dark at 37 °C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

Experiment II: preincubation with hamster liver S9
- Preincubation period: 30 min at 30 °C
- Exposure duration: 30 min at 30°C and at least 48 h in the dark at 37 °C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): histidine (overlay agar)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

Eperiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment I

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment I

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 2500 μg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

RANGE-FINDING STUDY:

Substance	Dose (μg/plate)	TA 98 Mutation Factor [toxicity]*		TA 100 Mutation Factor [toxicity]*
		without S9	with S9	without S9	with S9
Solvent Control (A. dest)		1.0	1.0	1.0	1.0
4-NOPD	10.0	16.6	-	-	-
NaN3	10.0	-	-	5.7	-
2-AA	2.50	-	83.4	-	12.8
Test Item	3.16	1.2	1.2	1.0	1.0
	10.0	1.2	1.0	1.0	1.0
	31.6	1.0	0.9	1.1	1.0
	100	1.1	1.0	1.1	1.0
	316	1.3	0.8	1.2	1.0
	1000	1.7	1.2	1.0	0.9
	2500	0.9	0.9	1.0	1.1
	5000	1.1	0.9	1.2	1.1

* [toxicity parameter]: B = Background lawn reduced; N = No background lawn

MAIN STUDY:

Experiment I (rat liver S9):

Mean revertant colonies per plate

		TA 98		TA 100		TA 1535		TA 1537		TA 102
Treatment	Dose/plate (µg)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Aq. Dest		20	29	129	136	20	13	13	11	422	490
Test item	10	20	25	148	133	22	16	13	9	388	432
	100	23	28	139	139	22	12	10	12	396	473
	316	26	25	151	142	19	13	11	14	367	432
	1000	34	34	135	121	21	13	10	12	371	408
	2500	19	25	136	154	14	11	6	7	296	335
	5000	23	26	158	155	16	12	6	7	321	296
4-NOPD	10	337	-	733	-	1112	-	70	-	2274	-
2-AA	2.5	-	2445	-	1743	-	203	-	396	-	1024

Mutation factor

		TA98		TA100		TA 1535		TA 1537		TA 102
Treatment	Dose/plate	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Aq. Dest		1	1	1	1	1	1	1	1	1	1
Test item	31.6 µg	1	0.9	1.1	1	1.1	1.2	1	0.9	0.9	0.9
	100 µg	1.1	1	1.1	1	1.1	0.9	0.8	1.2	0.9	1
	316 µg	1.3	0.8	1.2	1	0.9	1	0.8	1.3	0.9	0.9
	1000 µg	1.7	1.2	1	0.9	1	1	0.8	1.1	0.9	0.8
	2500 µg	0.9	0.9	1	1.1	0.7	0.9	0.5	0.7	0.7	0.7
	5000 µg	1.1	0.9	1.2	1.1	0.8	0.9	0.5	0.7	0.8	0.6
4-NOPD	10 µg	16.6	-	5.7	-	54.7	-	5.3	-	5.4	-
2-AA	2.5 µg	-	83.4	-	12.8	-	15.3	-	37.1	-	2.1

Experiment II (hamster liver S9):

Mean revertant colonies per plate

		TA 98		TA 100		TA 1535		TA 1537		TA 102
Treatment	Dose/plate (µg)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Aq. Dest		17	36	89	97	21	15	7	19	270	444
Test item	10	15	38	88	82	25	11	9	20	328	388
	31.6	16	35	109	95	27	11	9	18	290	430
	100	17	43	106	95	22	16	7	18	229	382
	316	16	39	110	106	25	14	9	17	235	365
	1000	18	42	104	108	22	16	6	18	255	400
	2500	17	27	125	107	19	13	6	13	241	441
	5000	14	25	137	131	18	16	5	11	281	260
Congo Red	700	-	242	-	301	1104	-	120	-	1227	-
NaN3	10	425	-	748	-	-	230	-	294	-	1513

Mutation factor

		TA 98		TA 100		TA 1535		TA 1537		TA 102
Treatment	Dose/plate (µg)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Aq. Dest		1	1	1	1	1	1	1	1	1	1
Test item	10	0.9	1.1	1	0.8	1.2	0.8	1.2	1.1	1.2	0.9
	31.6	0.9	1	1.2	1	1.3	0.7	1.2	0.9	1.1	1
	100	1	1.2	1.2	1	1	1	1	0.9	0.8	0.9
	316	0.9	1.1	1.2	1.1	1.2	0.9	1.3	0.9	0.9	0.8
	1000	1	1.2	1.2	1.1	1	1	0.8	0.9	0.9	0.9
	2500	1	0.8	1.4	1.1	0.9	0.8	0.8	0.7	0.9	1
	5000	0.8	0.7	1.5	1.4	0.9	1.1	0.8	0.6	1	0.6
Congo Red	700	-	6.8	-	3.1	53.4	-	17.1	-	4.5	-
NaN3	10	24.5	-	8.4	-	-	15.4	-	15.5	-	3.4

Applicant's summary and conclusion

Conclusions:

The test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item was assessed for its ability to induce gene mutations in a study performed according to OECD 471, a bacterial reverse mutation test. The plate incorporation test with rat liver S9 (experiment I) and the pre-incubation test with hamster liver S9 (experiment II), were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in experiment I and 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in experiment II. The concentrations, including the controls, were tested in triplicate.

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II, with one exception: In experiment I toxic effects of the test item were noted in tester strain TA 1537 at concentrations of 2500 μg/plate and higher (without metabolic activation). No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met.

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