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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The Ames test with Solvent Green 28 was negative.

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. Category Justification document attached.

A read across for chromosome aberration and a HPRT assay was conducted with Reinblau RLW (CAS 41611-76-1). All in-vitro tests were negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Macrolexgrün G was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37°C. Other conditions remained unchanged.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
name of test substance: MACROLEX Grün G
appearance: dark-green powder
chemical names: 9,10-Anthracenedione, 1,4-bis{[4- (1,1-dimethylethyl)phenyl]amino}- 5,8-dihydroxy
molecular weight: 534
molecular formula: C34H34N2O4
CAS No.: 4851-50-7
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 mix
Test concentrations with justification for top dose:
The following doses per plate were evaluated [µg per plate]:
Negative control 0
MACROLEX Grün G 5000
MACROLEX Grün G 1581
MACROLEX Grün G 500
MACROLEX Grün G 158
MACROLEX Grün G 50
MACROLEX Grün G 16
Vehicle / solvent:
MACROLEX Grün G was mixed with DMSO and formed a dark-green suspension.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
other: nitrofurantoin, 4-nitro-1,2-phenylene diamine, 2-amino­ anthracene
Details on test system and experimental conditions:
The initial plate incorporation test followed the directions of Ames. For the mutant count, one plate was used, both with and without S9 mix, for each strain and dose. The independent repeat was performed as preincubation in a water bath at 37°C for minutes. At the end of the preincubation period 2 ml of molten soft agar were added to the tubes, the content mixed and plated.
One plate, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained one plate per strain. The amount of solvent for the test substance and for the controls was 0.1 ml/plate.
Evaluation criteria:
The following criteria determined the acceptance of an assay:
a) The negative controls had tobe within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/ or the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

Only trials which complied with all three of the above crite­ ria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.

Assessment Criteria
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There was no indication of a bacteriotoxic effect of MACROLEX Grün G at doses of up to and including 5000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. At 500 µg per plate, the substance started to precipitate.
Conclusions:
Negative. Evidence of mutagenic activity of MACROLEX Grün G was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.
Executive summary:

MACROLEX Grün G was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the  histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37°C. Other conditions remained unchanged.

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 500 µg per plate and above.

Evidence of mutagenic activity of MACROLEX Grün G was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-amino­anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, MACROLEX Grün G was considered to be negative (non-mutagenic) without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
Chromosome aberration test with CHL/IU cells
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: CHL/IU
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
concentration [mg/ml] : 0.010, 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.250, 2.500, 5.000
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: MMC treatment group and B[a]P treatment group
Key result
Species / strain:
mammalian cell line, other: 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:
valid
Positive controls validity:
valid

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 and B[a]P treatment group except for the treatment without S9Mix with metabolic activation) and, therefore, these results showed that the test was conducted appropriately.

Conclusions:
Interpretation of results: negative
Executive summary:

A Chromosome aberration test with CHL/IU cells was conducted with Macrolex RLW Tr.in concentrations up to 5 mg/l.

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 and B[a]P treatment group except for the treatment without S9Mix with metabolic activation) 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.Macrolex RLW Tr. was negative in the CA Test.

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 section 13).
Key result
Species / strain:
mammalian cell line, other: 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:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
Executive summary:

A Chromosome aberration test with CHL/IU cells was conducted with Macrolex RLW Tr.in concentrations up to 5 mg/l.

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 and B[a]P treatment group except for the treatment without S9Mix with metabolic activation) 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.Macrolex RLW Tr. was negative in the CA Test.

Endpoint:
in vitro gene mutation study in mammalian cells
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
This in vitro assay was performed to assess the potential of the test item to induce gene mutations by means of two independent HPRT experiments using the Chinese hamster cell line V79. The treatment time was 4 hours in the first experiment with and without metabolic activation. In the second experiment the cells were exposed to the test item for 24 hours without metabolic activation and 4 hours with metabolic activation.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
other: the cells have a stable karyothype with a modal chromosome number of 22
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
exposure S9 concentrations in µg/mL
period mix
Experiment I
4 hours - 9.8 19.5 39.0 78.0p 156.0p 312.0p
4 hours + 9.8 19.5 39.0p 78.0p 156.0p 312.0p
Experiment II
4 hours - 4.9 9.8 19.5 39.0 78.0 p 156.0 p
4 hours + 2.5 4.9 9.8 19.5 39.0 p 78.0 p
p = precipitation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: No relevant cytotoxic effect indicated by a relative cloning efficiency I and/or relative cell density below 50% in both parallel cultures occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

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:
Interpretation of results: negative
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:
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 section 13).
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: No relevant cytotoxic effect indicated by a relative cloning efficiency I and/or relative cell density below 50% in both parallel cultures occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
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

Justification for classification or non-classification

The Ames test with Solvent Green 28, the Chromosome aberration and HPRT assay with Reinblau RLW (CAS 41611-76-1) (structural analogue or surrogate) were negative. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.