1-amino-4-hydroxy-2-phenoxyanthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Results of available in vitro studies

- AMES test is in progress and expected to be negative

- chromosome aberration test in Chimese hamster V79 cells: negative,

- mammalian cell gene mutation test (HPRT-locus): negative.

Link to relevant study records

Reference

Reference 1

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:

key study

Study period:

2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The complete read across justification is detailed in section 13. Source study has reliability 1.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment for experiment I (without metabolic activation):
0.001, 0.0025, 0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
0.001, 0.0025, 0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM
Experiment I
without and with metabolic activation: 0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM
Experiment II
without metabolic activation: 0.0025, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.15 and 0.20 mM
and with metabolic activation: 0.025, 0.035, 0.045, 0.055, 0.065, 0.075, 0.085, 0.095, 0.12 and 0.25 mM

Vehicle / solvent:

Vehicle (solvent) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 0.8 and 1.0 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in DMSO
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/ml 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: based on relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥ 0.05 mM; Experiment II without S9: ≥ 0.04 mM; Experiment II with S9: 0.25 mM

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

Under the experimental conditions reported, the test item is non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to test substance at concentrations up to 0.005, 0.25 mM (without and with metabolic activation).

In exp. I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.54 was found at a concentration of 0.09 mM with a relative growth of 59.0 %.

In exp. I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.03 was found at a concentration of 0.06 mM with a relative growth of 92.8 %. 

In exp. II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.30 was found at a concentration of 0.01 mM with a relative growth of 75.7 %. 

In exp. II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.28 was found at a concentration of 0.085 mM with a relative growth of 78.2 %.

The positive controls induced the appropriate response.  There was no evidence of a concentration related positive response of induced mutant colonies over background.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The complete read across justification is detailed in section 13. Source study has reliability 1.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1.3, 2.5, 5 µg/mL

Vehicle / solvent:

Minimal essential medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

minimal essential medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

The V79 cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate (doubling time of clone V79/T5 (sub-clone V79/D3) in stock cultures: 12 hrs) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study. Cells have a stable karyotype with a modal chromosome number of 22.
Metabolic activation of chemicals partially achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher's exact test (10) (p < 0.05).
However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications.The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the experimental conditions reported, test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the substance is considered to be non-clastogenic in this chromosome aberration test in the absence and the presence of S9 mix.

Executive summary:

The test item was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments.

The highest applied concentrations in the pre-test on toxicity (2000 µg/ml without S9 mix and 1800 µg/ml with S9 mix) were chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent. Since no relevant toxicity was observed in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD guideline 473. No toxic effects indicated by clearly reduced mitotic indices or cell numbers were observed after treatment with the test item. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No biologically increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with chromosome aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver metabolic activation system

Test concentrations with justification for top dose:

Highest dose tested: 5000 μg/plate unless limited by cytotoxicity or solubility

Species / strain:

other: all strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Mutagenicity testing strategy is reported in ECHA Guidance Chapter R.7a: Endpoint specific guidance, Version 5.0 – December 2016.

A preliminary assessment of mutagenicity should normally includes data from a gene mutation test in bacteria unless existing data for analogous substances indicates 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.

In order to ensure the necessary minimum level of information is provided, at least a further test is required in addition to the gene mutation test in bacteria. This should be an in vitro mammalian cell test capable of detecting both structural and numerical chromosome aberrations. A suitable option is in vitro chromosome aberration test (OECD guideline 473), i.e. a cytogenetic assay for structural chromosome aberrations using metaphase analysis.

An in vitro gene mutation study in mammalian cells (OECD guideline 476) is the second part of the standard information set usually required, when the results of the bacterial gene mutation test and the first study in mammalian cells (i.e. an in vitro chromosome aberration test or an in vitro micronucleus test) are negative. This is to detect in vitro mutagens that give negative results in the other tests.

Substances for which mammalian cell tests are negative while bacterial test is positive should be evaluated for any further testing on a case-by-case basis. If available, an in vivo test may be used in the assessment.

In general, substances that are positive in the gene mutation test in bacteria, negative in in vitro tests of chromosome aberration or micronucleus and negative in an in vivo test, e.g. micronucleus test, are considered to be non-genotoxic.

As no data on genotoxic potential of Disperse Red 060 was available, except for a bacterial reverse mutation assay which is in progress, a read across approach was followed, in order to complete the assessment. Details on the read across procedure are reported in section 13 .

The following in vitro tests were conducted to assess genotoxicity of the substance.

Bacterial reverse mutation test (AMES test) is in progress and expected to be negative.

Chromosome aberration test was performed on Similar Substance 03 according to OECD guideline 473 (1997) by using V79 Chinese hamster lung fibroblasts.

In the pre-test, concentrations up to 2000 µg/ml without S9 mix and 1800 µg/l with S9 mix were tested and no relevant toxicity, in terms of reduced cell numbers, was noted. Consequently, test item was tested up to concentrations exhibiting clear precipitation in the main study.

In the main study, two independent experiments (I and II) were performed, differing in chromosome preparation interval and exposure duration. Two parallel cultures were set up in each experimental group. Top concentrations of 40 µg/ml and 10 µg/ml where used in exp. I and exp. II, respectively. In both experiments, it was noted that: mitotic indices and cell numbers were not substantially reduced; there was no biologically relevant increase in the rate of polyploid methaphases; there were no biologically nor statistically relevant increase in cells with structural aberrations, based on 200 cells scored per concentration. Notably, current version of OECD guideline 473 (2014) is more detailed than version followed in the study (1997). In particular, it would require to score 300 cells per concentration for structural chromosomal aberrations. Moreover, the recommended cytotoxicity parameters for cell lines, as V79 cell line, would be the relative population doubling (RPD) or the relative increase in cell count (RICC). In the present study, 200 metaphases were examined and the mitotic index and polyploidy were used as indicators of cytotoxicity in the main test, while the number of cells was taken in the pre-test. However, since aberration rates of treated cells were within the solvent control and the historical control data and no indications of cytotoxicity were found with respect to the observed parameters, the result is considered as indicative of a lack of mutagenic potential of the substance in this test.

A further in vitro study on mammalian cells was available. HPRT assay was conducted on Similar Substance 02 following OECD guideline 476 (1997). A pre-test on cytotoxicity was run to select the concentrations. Two experiments were run in the main assay using Chinese Hamster V79 cells. In particular:

1. exp. I with and without metabolic activation, concentration up to 0.25 mM and 4 -hour shorth-term exposure

2. exp. II with metabolic activation, concentration up to 0.25 mM and 4 -hour short-term exposure;

exp. II without metabolic activation, concentration up to 0.20 mM and 20 -hour long-term exposure.

Toxicity was evaluated in terms of number of cells per flask and cloning efficiency. A biologically relevant growth inhibition (reduction of relative growth below 70 %) was noted in exp. I without and in exp. II without and with metabolic activation at the highest concentrations adopted. Mutagenicity was evaluated by considering the number of mutant colonies. No significant differences were found with respect to the controls, thus the substance was considered as non mutagenic in this assay. As for the HPRT assay, current version of OECD guideline 476 (2015) contains more details on duration of exposure and post-exposure treatment, number of cells; moreover, it lowers the highest concentration useable if there is no cytotoxicity or precipitation. Such differences do not affect the validity of the available study.

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.

The overall assessement on the genotoxic potential of test substance was based on: expected negative outcome in bacterial reverse mutation assay (AMES test), negative outcome in in vitro chromosome aberration assay, negative result in in vitro HPRT assay.

All studies were conducted according to OECD guidelines and have a high reliability. Differences with respect to current versions of guidelines, do not significantly influence the overall evaluation, which was thus taken as valid and reliable.

Based on these results, which fulfil the mutagenicity testing strategy required by Annex VII and VIII of the REACH Regulation (EC 1907/2006), the test susbstance was considered as non genotoxic and it was not classified within the CLP Regulation (EC 1272/2008).