1,4-bis[[4-(1,1-dimethylethyl)phenyl]amino]-5,8-dihydroxyanthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genetic toxicity: Rel 1, Key, Ames Test, eq. OECD 471, 1997; non mutagenic.

In vitro genetic toxicity: Read-accross, Rel 2, Key, CAT, OECD 473, 1997; non clastogenic.

In vitro genetic toxicity: Read-across, Rel 1, Key, HPRT test, OECD 476, 2012; non mutagenic.

 

Based on the similar chemical structure and biological activity a category has been defined. The category consists of substances all having the diamino-anthraquinone structure as a common moiety which is linked to phenyl groups via the amino groups. Differences within the category are described by various alkyl groups bound to the phenyl groups. The main properties of all members are the chemical and biochemical stability, the extremely low water solubilities and the high water-octanol partition coefficients. These properties lead to a reduced bioavailability for organisms. Therefore the substances do no exert any toxic effects and thus they are not classified. A justification document is attached to the Category dossier.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Principles of method if other than guideline:

The experiments ere performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella thphimurium and Escherichia coli reverse mutation assays. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.

GLP compliance:

yes

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Key result

Species / strain:

S. typhimurium TA 1535

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

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

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Negative. No substantial increase in revertant colony numbers of any of the five fester strains was observed following treatment with SANDOPLAST GREEN G at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

Executive summary:

This study was performe to investigate the potential of SANDOPLAST GREEN G to induce gene mutations according to t e plate incorporation test (experiment I) and the preincubation

test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, induding the controls, was tested in triplicate.

The test item was tested at the following concentrations:

33; 100; 333; 1000; 2500; and 5000 pg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five fester strains was observed following treatment with SANDOPLAST GREEN G at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive Controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, SANDOPLAST GREEN G is considered to be negative (non-mutagenic) in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Justification Document for the Category of Six Anthraquinone Dyes

LANXESS Deutschland GmbH has registered five mono-constituent anthraquinone dyes of similar chemical structure using a category approach: Solvent Violet 36 (CAS No 82-16-6), Solvent Green 3 (CAS No 128-80-3), Reinblau RLW (CAS No 41611-76-1), Reinblau BLW (CAS No 32724-62-2) and Solvent Green 28 (CAS No 4851-50-7). Additional data were taken from another registered anthraquinone dye, Solvent Blue 104 (CAS No 116-75-6), leading to a category consisting of six members (see attached justification in Category dossier).

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

cytotoxicity was not recognized in the treatment with and without metabolic activation, 5 mg/mL was set as the highest concentration in each treatment condition

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Data on osmolality: not reported
- Possibility of evaporation from medium: not reported
- Water solubility: not soluble
- Precipitation and time of the determination: not reported
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: not reported

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Number of chromosomal aberrant cells increased in the positive control group (MMC treatment group without S9 and B[a]P treatment with S9 group) and, therefore, these results showed that the test was conducted appropriately.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index. Not available
- Genotoxicity results (for both cell lines and lymphocytes): Number of chromosomal aberrant cells (structural aberrant cells and polyploid cells) did not increase in the treatment without or with metabolic activation. (cf. table of results attached to this ESR)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: not reported
- Negative (solvent/vehicle) historical control data: not reported

Conclusions:

The test substance did not have the ability to induce chromosomal aberration under the test conditions.

Executive summary:

A Chromosome aberration test with CHL/IU cells was conducted with the test substance suspended in 1% CMC-Na at concentrations up to 5 mg/mL .

The number of chromosomal aberrant cells (structural aberrant cells and polyploid cells) did not increase in the treatment without or with metabolic activation. Number of chromosomal aberrant cells increased in the positive control group (MMC treatment group without S9Mix and B[a]P treatment group with S9Mix) and, therefore, these results showed that the test was conducted appropriately.

Based on the above results, it was judged that the test substance did not have the ability to induce chromosomal aberration.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Justification Document for the Category of Six Anthraquinone Dyes

LANXESS Deutschland GmbH has registered five mono-constituent anthraquinone dyes of similar chemical structure using a category approach: Solvent Violet 36 (CAS No 82-16-6), Solvent Green 3 (CAS No 128-80-3), Reinblau RLW (CAS No 41611-76-1), Reinblau BLW (CAS No 32724-62-2) and Solvent Green 28 (CAS No 4851-50-7). Additional data were taken from another registered anthraquinone dye, Solvent Blue 104 (CAS No 116-75-6), leading to a category consisting of six members (see attached justification in Category dossier).

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.35 vs 7.37 in solvent control
- Data on osmolality: 376 mOsm vs 387 in solvent control
- Possibility of evaporation from medium: not reported
- Water solubility: not reported. The highest concentration (1250 μg/mL) used in the range finding pre-experiment was limited by the solubility of the test item in DMSO and aqueous media
- Precipitation and time of the determination: The test medium was checked for precipitation or phase separation at the end of each treatment period prior to removal to the test item. Precipitation was observed at 39.1 µg/mL and above following 4 and 24 hour treatment with and without metabolic activation.
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: No relevant cytotoxic effect, indicated by a relative cloning efficiency below 50% was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: cf attached Table of Results
The highest solvent control value (37.0 colonies per 10E6 cells in experiment I, culture II with metabolic activation) slightly exceeded the range of historical solvent control data (3.4 - 36.6 mutant colonies/10E6 cells). The mean value of both parallel cultures however, (23.7 and 37.0 equal to 30.4 colonies per 10E6 cells) is fully acceptable. The cloning efficiency II of the solvent controls of the second culture with and without metabolic activation did not exceed 50% as required by the acceptance criteria. The data are valid however, as the solvent controls of the parallel cultures and the mean values of both parallel cultures (60%) are acceptable.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 10.6 up to 37.0 mutants per 10E6 cells; the range of the groups treated with the test item was from 4.9 up to 53.0 mutants per 10E6 cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: cf Tables of Results
- Genotoxicity results: cf. Tables of Results
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration.
In experiment I with metabolic activation the absolute value of the mutation frequency exceeded the range of the historical solvent control data (3.4 - 36.6 mutant colonies/10E6 cells) at 19.5 jg/mL (39.1 mutant colonies per 106 cells in culture I) and at 156.0 µg/mL (53.0 mutant colonies/10E6 cells in culture II). However, the threshold of three times the mutation frequency of the corresponding solvent control was neither reached nor exceeded at these data points.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: cf Tables of Results
- Negative (solvent/vehicle) historical control data: cf Tables of Results

The highest concentration (1250 μg/mL) used in the range finding pre-experiment was limited by the solubility of the test item in DMSO and aqueous media. The dose range of the main experiments was limited by precipitation of the test item.

The tested concentrations are 9.8 - 312 µg/ml in experiment I and 2.5 - 168 µg/ml in experiment II.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

The study was performed to investigate the potential of Macrolex Blau 3R to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

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

The highest concentration (1250 μg/mL) used in the range finding pre-experiment was limited by the solubility of the test item in DMSO and aqueous media. The dose range of the main experiments was limited by precipitation of the test item.

The tested concentrations are 9.8 - 312 µg/ml in experiment I and 2.5 - 168 µg/ml in experiment II.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Macrolex Blau 3R was negative in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6.1/1 : Summary of genotoxicity tests:

 

Test n°	Test Guideline / Reliability	Focus	Strains  / cells tested	Metabolic activation	Test concentration	Statement
1 (1997) Solvent Green 28	Ames Test (eq. OECD 471, GLP) K, rel.1	Gene mutation	S. thyphimurium TA 1535, TA 1537, TA 98, TA 100, TA 102	-S9 +S9	Tested up to 5000 µg/plate in DMSO	-S9: not mutagenic + S9: not mutagenic
2 (2002) Solvent Green 28	Ames Test (OECD 471, GLP) S, rel.4	Gene mutation	S. thyphimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2	-S9 +S9	Tested up to 5000 µg/plate in DMSO	-S9: not mutagenic + S9: not mutagenic
3 (1997) Read-across Reinblau RLW	CAT (OECD 473, GLP) K, rel.2	Chromosomal aberration	Chinese Hamster lung CHL/IU	-S9 +S9	Tested up to 5 mg/mL in 1% CMC-Na solutions	-S9: not clastogenic + S9: not clastogenic
4 Read-across Solvent Blue 104	CAT (OECD 473, GLP) Source : ECHA disseminated dossier	Chromosomal aberration	Human Lymphocytes	-S9 +S9	Tested up to 32 µg/mL in THF (limited by precipitation)	-S9: not clastogenic +S9: not clastogenic
5 (2012) Read-across Reinblau RLW	HPRT (OECD 476, GLP) K, rel.1	Gene mutation	Chinese Hamster lung fibroblast (V79)	-S9 +S9	Tested up to 312 µg/mL in DMSO (limited by precipitation)	-S9: not mutagenic + S9: not mutagenic
6 Read-across Solvent Blue 104	HPRT (OECD 476, GLP) Source : ECHA disseminated website	Gene mutation	Chine Hamster lung fibroblast (V79)	-S9 +S9	Tested up to 280 µg/mL in THF (limited by precipitation)	-S9: not mutagenic +S9: not mutagenic

Gene mutation (Test n° 1, 2, 5 & 6)

A bacterial reverse mutation assay (Ames test) was performed with the substance (Test n°1). This study was used to conclude on the potential of the substance to induce gene mutation in bacteria.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains with any dose of test material, either in the presence or absence of metabolic activation, up to limit concentration. The test indicate that the substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test. 

Another Ames test on the substance confirms this conclusion (Test n°2).

No study was located on the substance, but inability to produce gene mutation was confirmed using an in vitro forward mutation assay in Chinese Hamster V79 cells on Reinblau RLW, a member of the category (Test n°5). None of the dose levels up to the precipitating concentration, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. Reinblau RLW does not induce forward mutations at the hprt locus in V79 mouse cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Reinblau RLW, and by analogy Solvent Green 28, are therefore considered as negative for inducing forward mutations at the hprt locus in V79 mouse cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames tests and extends the non-mutagenic effect of these substances to mammalian cells.

Another in vitro mammalian cell gene mutation (hprt / V79 cells) on Solvent Blue 104 – a member of the category – confirms this conclusion (test n°6).

 

Chromosomal aberration (Test n°3 & 4)

No study was located on the substance but the clastogenic potential of the substance was determined on Reinblau RLW, a member of the category, using an in vitro chromosome aberration test in Chinese hamster lung cells, which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured Chinese hamster lung cells (Test n°3). None of the dose levels up to the limit concentration with the substance, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. Reinblau RLW, and by analogy Solvent Green 28, are therefore considered as negative for inducing chromosomal mutations in Chinese hamster lung cells under activation and non-activation conditions used in this assay.

Another in vitro chromosome aberration test (CAT / HL) on Solvent Blue 104 – a member of the category – confirms this conclusion (test n°4).

Justification for classification or non-classification

Harmonised classification: 

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self-classification: 

The Ames test with Solvent Green 28, the Chromosome aberration and HPRT assay with two members of the category (Reinblau RLW and Solvent Blue 104) were negative. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.