Propanenitrile, 3-[[4-[[5,6(or 6,7)-dichloro-2-benzothiazolyl]azo]phenyl]ethylamino]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance showed an increase in revertant colony numbers  in strain TA 100 in the presence of S9 mix.

As the relevance of this effect for humans cannot be evaluated from this result, an in-vivo Comet Assay was proposed as follow-up of the positive effect.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-09-14 till 2021-09-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

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 E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Since a positive result was obtained in experiment I, a second experiment with non-induced hamster liver S9 mix as exogenous metabolic activation system was not performed.

Vehicle / solvent:

Solvent used: DMSO
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation

DURATION:


NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

up to 2.2-fold at 2500 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without S9 mix, except of strain TA 1535 in the presence of S9 mix.

Summary of Experiment I

Study Name: 2183806	Study Code: ICCR 2183806
Experiment: 2183806 HV1 Plate	Date Plated: 14.09.2021
Assay Conditions:	Date Counted: 17.09.2021

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD) TA 1535	Revertant Colony Counts (Mean ±SD) TA 1537	Revertant Colony Counts (Mean ±SD) TA 98	Revertant Colony Counts (Mean ±SD) TA 100	Revertant Colony Counts (Mean ±SD) WP2 uvrA
Without Activation	DMSO			10 ± 1	12 ± 0	33 ± 7	130 ± 24	41 ± 11
	Untreated			10 ± 5	10 ± 3	36 ± 10	117 ± 19	46 ± 7
	Disperse Red	3 µg		10 ± 4	11 ± 1	39 ± 3	125 ± 6	47 ± 7
	153	10 µg		14 ± 3	13 ± 6	28 ± 5	131 ± 13	55 ± 8
		33 µg		12 ± 2	9 ± 2	28 ± 6	133 ± 24	50 ± 7
		100 µg		12 ± 3	7 ± 2	28 ± 9	128 ± 15	49 ± 1
		333 µg		14 ± 3	10 ± 3	26 ± 9	108 ± 12	30 ± 3
		1000 µg		9 ± 3^P M	7 ± 2^P M	17 ± 2^P M	98 ± 8^P M	22 ± 3^P
		2500 µg		7 ± 0^P M	5 ± 1^P M	18 ± 3^P M	61 ± 7^P M	13 ± 1^P M
		5000 µg		4 ± 2^P M	2 ± 1^P M	6 ± 1^P M	7 ± 2^P M	9 ± 1^P M
	NaN3	10 µg		1096 ± 105			1448 ± 68
	4-NOPD	10 µg				714 ± 70
	4-NOPD	50 µg			96 ± 12
	MMS	2.0 µL						813 ± 81
With Activation	DMSO			11 ± 1	13 ± 3	50 ± 9	111 ± 10	57 ± 6
	Untreated			15 ± 6	15 ± 1	53 ± 12	120 ± 20	70 ± 7
	Disperse Red	3 µg		12 ± 2	11 ± 3	54 ± 6	112 ± 9	51 ± 10
	153	10 µg		13 ± 2	14 ± 2	52 ± 8	133 ± 13	69 ± 2
		33 µg		13 ± 4	11 ± 3	58 ± 14	137 ± 13	64 ± 6
		100 µg		11 ± 4	11 ± 3	57 ± 8	185 ± 34	66 ± 13
		333 µg		9 ± 2	12 ± 2	69 ± 9	209 ± 26	60 ± 3
		1000 µg		11 ± 0^P	10 ± 4^P	62 ± 11^P	234 ± 15^P	42 ± 5^P
		2500 µg		7 ± 3^P M	8 ± 2^P M	45 ± 7^P M	245 ± 26^P M	20 ± 6^P M
		5000 µg		9 ± 3^P M	6 ± 2^P M	17 ± 5^P M	46 ± 8^P M	5 ± 1^P M
	2-AA	2.5 µg		348 ± 12	539 ± 24	2543 ± 167	3126 ± 68
	2-AA	10.0 µg						290 ± 9

 

 

 

 

 

 

 

 

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

 

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes in the genome of strain TA 100 in the presence of S9 mix.

Executive summary:

This study was performed to investigate the potential of Disperse Red 153 to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation (S9 mix). The experiment was performed with induced rat liver S9 mix as an exogenous metabolic activation system. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

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.

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I
	without S9 mix	with S9 mix
TA 1535	5000	/
TA 1537	2500 – 5000	5000
TA 98	5000	5000
TA 100	5000	5000
WP2 uvrA	2500 – 5000	2500 – 5000

/ = no toxic effects (induction factor ≥ 0.5)

A substantial and dose depended increase in revertant colony numbers was observed following treatment with Disperse Red 153 in strain TA 100 in the presence of S9 mix. The relevant threshold of two-fold the revertant colony count of the corresponding solvent control was exceeded at a concentration from 1000 to 2500 µg/plate. Based on overlapping toxic effects the number in revertant colony count decreased below the threshold of toxicity at the highest concentration.

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

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Disperse Red 153
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: no in vivo studies are available to adequately address this endpoint.
- Available non-GLP studies: no in vivo studies are available to adequately address this endpoint.
- Historical human/control data: There is no historical human data available for this substance on ‘genetic toxicity in vivo’. Hence no evidence of human adverse effect.
- (Q)SAR: (Q)SARs are not considered adequate to address this endpoint.
- In vitro methods: there are no adequate in-vitro methods to address this endpoint.
- Weight of evidence: in absence of additional data no WoE can be done.
- Grouping and read-across: no analogues were identified with data on genotoxicity that were considered relevant to read across.
- Substance-tailored exposure driven testing: not applicable.
CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The ECHA guidance R.7a states that following a positive result in an in vitro test, “adequately conducted somatic cell in vivo testing is required to ascertain if this potential can be expressed in vivo.”
FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design: According to OECD 489 in liver, glandular stomach and duodenum of one sex only, as no toxicologically relevant differences were observed between males and females.
No germ cells will be collected, as the assay is not validated for germ cells, the TG states "this guideline is not considered appropriate to measure DNA strand breaks in mature germ cells. Since high and variable background levels in DNA damage were reported in a literature review on the use of the comet assay for germ cell genotoxicity".

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

rat

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

2 treatments (24 h and 4 h prior to preparation)

Frequency of treatment:

2 treatments

Post exposure period:

24 h and 4 h

No. of animals per sex per dose:

6 (one sex, as there is no gender difference)

Control animals:

yes, concurrent vehicle

Positive control(s):

yes

Tissues and cell types examined:

A) Analysis of hepatocytes
Isolation of hepatocytes by mincing
Determination of dead cells in 500 cells per slide (1500 per animal)
Performance of the alkaline comet assay
Analysis of 50 cells per slide (150 cells per animal)
B) Analysis of cells from the stomach
Isolation of cells from the stomach by scraping
Determination of dead cells in 500 cells per slide (1500 per animal)
Performance of the alkaline comet assay
Analysis of 50 cells per slide (150 cells per animal)
C) Analysis of cells from small intestine
Isolation of cell nuclei from the small intestine by mincing
Determination of dead cells in 500 cells per slide (1500 per animal)
Performance of the alkaline comet assay
Analysis of 50 cells per slide (150 cells per animal)

Additional information

This study was performed to investigate the potential of Disperse Red 153 to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation (S9 mix). The experiment was performed with induced rat liver S9 mix as an exogenous metabolic activation system. Each concentration, including the controls, was tested in triplicate. The test item was tested at concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate. 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.

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at concentrations of 2500 and/or 5000 µg/plate in all strains with and without S9, except is TA 1535, were toxicty was only observed without S9.

An increase in revertant colony numbers was observed following treatment with Disperse Red 153 in strain TA 100 in the presence of S9 mix. The relevant threshold of two-fold the revertant colony count of the corresponding solvent control was exceeded at a concentration from 1000 to 2500 µg/plate. Based on overlapping toxic effects the number in revertant colony count decreased below the threshold of toxicity at the highest concentration.