Reaction Mass of disodium [(E)-7-nitro-3-oxido-4-((2-oxidonaphthalen-1-yl)diazenyl)naphthalene-1-sulfonate) (E)-1-((3,5-dinitro-2-oxidophenyl)diazenyl)naphthalen-2-olate) chromate(2-)] and Hydrogen [(E)-7-nitro-3-oxido-4-((2-oxidonaphthalen-1-yl)diazenyl)naphthalene-1-sulfonate)(E)-1-((3,5-dinitro-2-oxidophenyl)diazenyl)naphthalen-2-olate) chromate(2-)]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames - OECD TG 471 - nitroreductase deficient strains - key study - read-across - with and without S9 - positive.

Ames - OECD TG 471 - supporting study - read-across - with and without S9 - positive. 

OECD TG 476 - key study - read-across - 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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Section 13: see the attached document containing the justification for the read-across.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

clone 65/3Origin: Dr. D. Wild, Freiburg, Germany

Metabolic activation:

with

Metabolic activation system:

Post mitochondrial supernatant (S9 fraction) from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Cytotoxicity test: range with metabolic activation: 0.24 to 500.0 µg/ml; range without metabolic activation: 0.24 to 500.0 µg/ml.
Mutagenicity test original experiment: range with metabolic activation: 18.52 to 500.0 µg/ml; range without metabolic activation: 14.81 to 400.0 µg/ml.
Confirmatory experiment: range with metabolic activation: 18.52 to 500.0 µg/ml; range without metabolic activation: 12.96 to 350.0 µg/ml.

Vehicle / solvent:

Dimethylsulfoxide (suspension): since the test substance was insoluble in all common solvents, it had to be prepared as a suspension in DMSO. The highest suitable concentration of test substance in DMSO was determined in a preliminary solubilisation test to be 50.0 mg/ml. Lower concentrations of the test substance were obtained by appropriate dilution of the stock suspension with DMSO. The respective suspensions were added 1:100 to the cell culture medium. The final concentration of DMSO in the culture medium was 1%. The highest concentration caused a homogenious turbidity after 100-fold dilution with culture medium as determined in the preliminary solubility test by microscopic evaluation. Due to the dense black staining by the test compound, observation of the cultures with the naked eye did not allow todecide whether the compound precipitated or not. It was therefore not determined which concentrations caused the formation of precipitates in the toxicity and mutagenicity tests. The test substance suspensions were prepared immediately before the start of the test.

Untreated negative controls:

yes

Remarks:

dimethylsulfoxide

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Remarks:

N-dimethylnitrosamine with metabolic activation, ethylmetanesulphonate without metabolic activation

Evaluation criteria:

All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of theresult. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated. Criteria for a positive responseThe test substance will be considered to be mutagenic if: the assay is valid, the mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20, there is a significant dose-relationship as indicated by the linear trend analysis and the effects described above are reproducible.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Negative. The test substance was tested for in vitro gene mutation in mammalian cells following OECD 476. Under the experimental conditions the test substance did not show any mutagenic potential.

Executive summary:

The test substance was tested for in vitro gene mutation in mammalian cells following OECD 476. Chinese Hamster lung fibroblasts V79 were exposed to the test substance with and without metabolic activation at dose ranging up to 500 µg/l and 400 µg/l respectively. After the expression period, the mutant frequency was determined by 6-thioguanine screening and the substance resulted not having mutagenic potential.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

From 06 January 2021 to 19 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD, 1997, as corrected in 2020

Deviations:

yes

Remarks:

Due to the nature of the test article, a modified (reductive Prival) metabolic activation system was employed, and only a single experiment was performed. Use of NR-deficient strains.

Principles of method if other than guideline:

The traditional strains used for OECD 471 will be checked in parallel with the same strains deficient in the nitro-reductase enzyme (present only in bacteria) as to avoid the NO2 group reduction present in the test substance. Reduction of the nitro groups in fact produce in the substance aromatic amines that typically give false positive for the traditional tested strains.

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

Additional strain / cell type characteristics:

nitroreductase deficient

Remarks:

TA 98NR, TA100NR

Metabolic activation:

with and without

Metabolic activation system:

- Type and composition of metabolic activation system: reductive (Prival) metabolic activation system.
- Source of S9: the metabolic activation system was prepared from uninduced male Golden Syrian hamsters.
- Method of preparation of S9 mix: the S-9 was stored frozen at <-50°C, and thawed prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities). Treatments were carried out both in the absence and presence of S-9 by addition of either buffer solution or 30% reductive (Prival) S-9 mix respectively. The composition of the mix and buffer solutions are reported in the section "Any other information on material and methods incl. tables".
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of 30% reductive S-9 mix or buffer solution.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

Mutation Experiment treatments of all the tester strains were performed using a
pre-incubation methodology in the absence and presence of a modified (reductive)
S-9 mix using final concentrations of Acid Black 107 at 5, 16, 50, 160, 500, 1600 and
5000 μg/plate.

Highest dose tested: 5000 μg/plate unless limited by cytotoxicity or solubility. This is the maximum recommended concentration according to current regulatory guidelines.

Vehicle / solvent:

Purified water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

mitomycin C

other: metronidazole (MTZ), 2-aminoanthracene (AAN)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate.
- Number of independent experiments: one indipendent experiment.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable.
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: pre-incubation methodology.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: quantities of test article or control suspension, bacteria and S-9 mix or buffer solution detailed above, were mixed together and placed in an orbital incubator set to either 37°C (for the treatments in the absence of S-9) or 30°C (for treatments in the presence of S-9) for 30 minutes.
- Exposure duration/duration of treatment: 3 days.
- Harvest time after the end of treatment (sampling/recovery times): not reported.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition.
- Any supplementary information relevant to cytotoxicity: the background lawns of the plates were examined for signs of toxicity. Revertant plate count data were also assessed, as a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response was also considered as evidence of toxicity.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate). However, adequate interpretation of biological relevance was of critical importance.

Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as intensely coloured agar, precipitation, contaminated plate or absence of background lawn affected the accuracy of the automated counter.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if: a concentration related increase in revertant numbers was ≥ 1.5-fold (in strain TA 102), ≥2-fold (in strains TA 98, TA 98NR, TA 100 or TA 100NR) or ≥3-fold (in strains TA 1535 or TA 1537) respect to the concurrent vehicle control values.
The test article was considered positive in this assay if the above criterion was met. The test article was considered negative in this assay if the above criterion was not met.
Results which only partially satisfied the above criteria were dealt with on a case-by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations. Data from strain TA98 were compared (non-statistically) with that from TA98NR, and data from strain TA100 were compared with that from TA100NR. Where a mutagenic response was seen in one or both parent strains but was absent or much reduced in the corresponding NR variant strain(s), this was considered to be indicative that bacterial nitroreduction enzymes play a significant role in the mutagenicity of the test compound as observed in this study.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

bacteria, other: TA 100NR

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity, Solubility and Concentration Selection: mutation Experiment treatments of all the tester strains were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S-9 mix using final concentrations of Acid Black 107 at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity ranging from a thinning of the background bacterial lawn with or without a concurrent marked reduction in revertant numbers to a complete killing of the test bacteria was observed in all strains in the absence of S-9 and in most strains in the presence of S-9, with the toxic effects (where observed) extending down to either 500, 1600 or 5000 μg/plate in each case. Further evidence of toxicity in the form of a tailing off of the mutagenic response was also seen at the higher concentration(s) in all strains where a clear mutagenic response was observed. Precipitation of test article was observed on all the test plates treated at 1600 μg/plate and above in the absence of S-9 and at 500 μg/plate and above in the presence of S-9.

Data Acceptability and Validity: from the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges, with the exception of a few isolated vehicle control counts that fell slightly outside the laboratory control ranges. In each case, these counts were comparable to the other vehicle control replicate counts and the laboratory historical control ranges, and therefore these data were accepted as characteristic and valid. The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle controls, and ≥2-fold with NQO, B[a]P and CR in strain TA98NR and NaN3, AAN and CR in strain TA100NR. The positive control treatments with 2NF in TA98NR and with MTZ in TA100NR each demonstrated a reduced response compared to the equivalent treatments in the parent strains TA98 and TA100, confirming the nitroreductase deficient status of strain TA98NR and TA100NR. The control treatments therefore confirmed discrimination between different strains, and an active S-9 preparation, and the correct strain and assay functioning was demonstrated. The study data were therefore accepted as valid.

Mutation: following Acid Black 107 treatments of all the test strains in the absence and presence of S-9, notable and concentration-related (up to the lower limit of toxicity and/or precipitation) increases in revertant numbers were observed in all strains except TA98NR in the absence of S-9 and TA102 in the presence of S-9. Where observed, these increases were all ≥1.5-fold (in strain TA102 in the absence of S-9), ≥2-fold (in strains TA98, TA98NR (in the presence of S-9 only), TA100 or TA100NR) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values, although it should be noted that in strain TA100NR in the absence of S-9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 5000 μg/plate. These increases were therefore all sufficient to be considered as clear evidence of Acid Black 107 mutagenic activity in these strains in this assay system.
The mutagenic responses in the nitroreductase deficient strain TA100NR in the absence and presence of S-9 were much reduced in magnitude compared to those with the corresponding treatments in the parent (nitroreductase proficient) strain TA100. An even clearer difference in mutagenic activity was evident for the strain TA98 and TA98NR treatments in the absence of S-9, where a clear mutagenic response was observed in the parent (nitroreductase proficient) strain TA98, but no mutagenic response was observed in the nitroreductase deficient strain TA98NR. In relation to the strain TA100 and TA100NR data in particular, it should also be noted that strains TA98NR and TA100NR still retain some low level residual nitroreductase activity (Rosenkranz and Mermelstein, 1983), and therefore complete elimination of any mutagenic response was not expected in these strains. Therefore, overall the relative mutagenic responses in each of these cases were considered as a clear indication that nitroreduction plays a significant role in the mutagenic activity of Acid Black 107 observed in this assay system. When the mutagenic responses in strain TA98 and TA98NR in the presence of S-9 were compared, there appeared to be a greater magnitude of response (in terms of fold increase over the concurrent vehicle control level) in the nitroreductase deficient strain TA98NR than in the parent (nitroreductase proficient) strain TA98. However, the vehicle control counts in strain TA98NR in the presence of S-9 were much lower than those in strain TA98 (although the counts in both strains were all within the laboratory historical range), which serves to exaggerate the magnitude of response in the strain with the lower vehicle control level. When the increases in revertants are assessed in terms of number of induced revertants (above the vehicle control level), the maximum increase in the parent (nitroreductase proficient) strain TA98 is approximately 1.75 times that seen in the nitroreductase deficient strain TA98NR (177.3 revertants/plate in strain TA98 compared to 101.3 revertants/plate in strain TA98NR), which therefore provides further evidence that nitroreduction plays a significant role in the mutagenic activity of Acid Black 107.

Conclusions:

It was concluded that Acid Black 107 induced mutation in histidine-requiring strains TA98, TA100, TA100NR, TA1535 and TA1537 of Salmonella typhimurium when tested in the absence and in the presence of a reductive (Prival) hamster liver metabolic activation system (S-9) under the conditions of this study, and also in Salmonella typhimurium strain TA98NR when tested in the presence of S-9 and in Salmonella typhimurium strain TA102 when tested in the absence of S-9. The conditions employed in this study included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines and a precipitating, and in most cases toxic, concentration). It may be noted that an increase in revertant numbers achieving the threshold level for an increase to be considered as clear evidence of mutagenic activity in strain TA100NR in the absence of S-9 was only achieved at the maximum treatment concentration of 5000 μg/plate. The relative mutagenic responses between the nitroreductase proficient and nitroreductase deficient strains used in this study indicated that nitroreduction plays a significant role in the observed mutagenic activity of Acid Black 107.

Executive summary:

Acid Black 107 was assayed for mutation in seven histidine-requiring strains (TA98, TA100, TA1535, TA1537, TA102, TA98NR and TA100NR) of Salmonella typhimurium, both in the absence and in the presence of a reductive hamster liver metabolising system (S-9), in a single experiment. All Acid Black 107 treatments in this study were performed using formulations prepared in purified water. As Acid Black 107 is an azo compound, testing in the presence of S-9 in this study was performed using a modified reductive (Prival) S-9 pre-incubation methodology, as it is known that azo compounds can be reduced to free aromatic amines, which can be mutagenic. Mutation Experiment treatments of all the tester strains were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S-9 mix using final concentrations of Acid Black 107 at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following these treatments, evidence of toxicity was observed in all strains in the absence of S-9 and in most strains in the presence of S-9, with the toxic effects (where observed) extending down to either 500, 1600 or 5000 μg/plate in each case. Precipitation of test article was observed on all the test plates treated at 1600 μg/plate and above in the absence of S-9 and at 500 μg/plate and above in the presence of S-9.

 

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control
treatments, and were elevated by positive control treatments.

Following Acid Black 107 treatments of all the test strains in the absence and presence of S-9, notable and concentration-related (up to the lower limit of toxicity and/or precipitation) increases in revertant numbers were observed in all strains except TA98NR in the absence of S-9 and TA102 in the presence of S-9. Where observed, these increases were all ≥1.5-fold (in strain TA102 in the absence of S-9), ≥2-fold (in strains TA98, TA98NR (in the presence of S-9 only), TA100 or TA100NR) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values, although it should be noted that in strain TA100NR in the absence of S-9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 5000 μg/plate. These increases were therefore all sufficient to be considered as clear evidence of Acid Black 107 mutagenic activity in these strains in this assay system.

The mutagenic responses in the nitroreductase deficient strain TA100NR in the absence and presence of S-9 were much reduced in magnitude compared to those with the corresponding treatments in the parent (nitroreductase proficient) strain TA100. An even clearer difference in mutagenic activity was evident for the strain TA98 and TA98NR treatments in the absence of S-9, where a clear mutagenic response was observed in the parent (nitroreductase proficient) strain TA98, but no mutagenic response was observed in the nitroreductase deficient strain TA98NR. In relation to the strain TA100 and TA100NR data in particular, it should also be noted that strains TA98NR and TA100NR still retain some low level residual nitroreductase activity (Rosenkranz and Mermelstein, 1983), and therefore complete elimination of any mutagenic response was not expected in these strains. Therefore, overall the relative mutagenic responses in each of these cases were considered as a clear indication that nitroreduction plays a significant role in the mutagenic activity of Acid Black 107 observed in this assay system. When the mutagenic responses in strain TA98 and TA98NR in the presence of S-9 were compared, there appeared to be a greater magnitude of response (in terms of fold increase over the concurrent vehicle control level) in the nitroreductase deficient strain TA98NR than in the parent (nitroreductase proficient) strain TA98. However, the vehicle control counts in strain TA98NR in the presence of S-9 were much lower than those in strain TA98 (although the counts in both strains were all within the laboratory historical range), which serves to exaggerate the magnitude of response in the strain with the lower vehicle control level. When the increases in revertants are assessed in terms of number of induced revertants (above the vehicle control level), the maximum increase in the parent (nitroreductase proficient) strain TA98 is approximately 1.75 times that seen in the nitroreductase deficient strain TA98NR, which therefore provides further evidence that nitroreduction plays a significant role in the mutagenic activity of Acid Black 107.

It was concluded that Acid Black 107 induced mutation in histidine-requiring strains TA98, TA100, TA100NR, TA1535 and TA1537 of Salmonella typhimurium when tested in the absence and in the presence of a reductive (Prival) hamster liver metabolic activation system (S-9) under the conditions of this study, and also in Salmonella typhimurium strain TA98NR when tested in the presence of S-9 and in Salmonella typhimurium strain TA102 when tested in the absence of S-9. The conditions employed in this study included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines and a precipitating, and in most cases toxic, concentration). It may be noted that an increase in revertant numbers achieving the threshold level for an increase to be considered as clear evidence of mutagenic activity in strain TA100NR in the absence of S-9 was only achieved at the maximum treatment concentration of 5000 μg/plate. The relative mutagenic responses between the nitroreductase proficient and nitroreductase deficient strains used in this study indicated that nitroreduction plays a significant role in the observed mutagenic activity of Acid Black 107.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

supporting study

Study period:

From March 08, 1993 to June 28, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Section 13: see the attached document containing the justification for the read-across.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no statistical method applied to the results

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:

Rat liver S9 mix

Test concentrations with justification for top dose:

61.7 to 5000 µg/plate

Vehicle / solvent:

The test substance was dissolved in dimethylsulfoxide at the concentration of 50 mg/ml. The three highest concentrations were weighed out separately. Lower concentrations were obtained by appropriate dilution of the third highest concentration with dimethylsulfoxide. At the concentrations of 555.6 µg/plate and above the test material precipitated on the agar plates.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

mitomycin C

other: 2-aminoanthracene

Remarks:

Cyclophosphamide and 2-aminoanthracene were used with S9 mix, while the other positive control substances were used without S9 mix.

Details on test system and experimental conditions:

Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment. Control of the genotype of the strains: the characteristics of the strains were checked monthly. Histidine-auxotrophy of the strains was demonstrated by the requirement for 1-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene (strains TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Strain TA 102 additionally was checked for tetracycline resistence (presence ofmulticopy plasmid pAQl). The presence of the uvr+ gene was demonstrated by the reslstence of strain TA 102 against UV-light. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).

Evaluation criteria:

A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director. Criteria for a positive response:
The test substance is considered to be mutagenic in this test system if the following conditions are met: at least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains = S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. Generally a concentration-related effect should be demonstrable.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Positive. The test substance was tested for gene mutation in bacteria. Under the experimental conditions the test substance show a potential to be mutagenic on bacteria.

Executive summary:

The test substance was tested for gene mutation in bacteria. Different strains of Salmonella T. were tested at 5 different concentrations up to 5000 µg/plate of the test substance with and without metabolic activation (S9 mix). The concentrations were chosed after a dose range finding tests. Precipitation of the test substance from the tested suspension occured over 500 µg/plate and slight cytotoxicity was observed at 5000 µg/plate. All strains except TA102 showed mutagenic effect that were dose related.

Under the experimental conditions the test substance show a potential to be mutagenic on bacteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

OECD TG 474 - key study - read-across - negative

Comet Assay - OECD TG 489 - Read-Across in progress

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

Not yet defined

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: analogue substance 03

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
This part refers to the available studies on the target substance Acid Black 107:1 (EC: 943-640-4):
- Available GLP studies: not available
- Available non-GLP studies: not available.
- Historical human data: not available.
- (Q)SAR: not available.
- Weight of evidence: not available.
- Grouping and read-across:
✓ In vitro gene mutation study in bacteria (OECD TG 471) – NR-deficient strains – Read-Across on analogue substance 02.
✓ In vitro gene mutation study in bacteria (OECD TG 471) – Read-Across on analogue substance 01.
✓ In vitro gene mutation in mammalian cells (OECD TG 476) – Read-Across on analogue substance 01.
✓ In vivo mammalian erythrocyte micronucleus Test (OECD TG 474) – Read-Across on analogue substance 01.

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:
Under Annex VIII Section 8.4., column 2 of REACH, further mutagenicity studies must be considered in case of a positive result in an in vitro gene mutation study in bacteria.
Guidance on information requirements R7a, section 7.7.6 (2017), states that regarding Annex VIII, when both the mammalian cell tests are negative but there was a positive result in the bacterial test, it will be necessary to decide whether any further testing is needed on a case-by-case basis. For example, suspicion that a unique positive response observed in the bacterial test was due to a specific bacterial metabolism of the test substance could be explored further by investigation in vitro. Alternatively, an in vivo test may be required.

The submitted dossier contains results for the in vitro gene mutation study in bacteria, following the OECD TG 471 with nitro-reductase deficient strains, conducted on the analogue substance 02. This test raises the concern for in vivo gene mutation, since the influence of the nitro-reductase is demonstrated but needs further evidences.

In particular, annex VIII, Column 2 requires the registrant to consider appropriate mutagenicity in vivo studies already at the Annex VIII tonnage level, which involves studies mentioned in Annex IX (among OECD TG 474 Mammalian Erythrocyte micronucleus test, OECD TG 488 Transgenic Rodent Mutation Assay, OECD TG 489 In vivo mammalian Alkaline Comet Assay and OECD TG 486 Unscheduled DNA Synthesis).

CONSIDERATIONS ON THE IN VIVO STUDIES INSERTED IN THE DOSSIER AND EXPERT ASSESSMENT ON TESTING PROPOSAL:
There are no in vivo studies conducted on the target substance submitted with the present dossier.
An OECD TG 474 (mammalian erythrocyte micronucleus test) in vivo study, conducted on the analogue substance 01 (see the read-across justification), is available and showed a negative result.
Therefore, in order to further and completely assess its gene mutation properties in different tissues of the animal, a Comet Assay, OECD TG 489, on analogue substance 03 was presented as testing proposal and it will be also used in read across for assessing the in vivo potential gene mutation properties of the target substance Acid Black 107:1 (EC: 943-640-4).

Analogue substance 03 is, in fact, considered as representative of the mutagenic behavior of Acid Black 107:1 (EC: 943-640-4) as specified in the read-across section.
OECD TG 489 allows to measure DNA strand breaks, that may result from direct interactions with DNA, alkali labile sites or as a consequence of incomplete excision repair. Therefore, the alkaline comet assay recognizes primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but will also detect DNA damage that may be effectively repaired or lead to cell death. The comet assay can be applied to almost every tissue of an animal from which single cell or nuclei suspensions can be made, including specific site of contact tissues.

Therefore, a confirmation by the Comet assay (for azo dyes the intestinal tract is the site of major metabolism and dye/metabolites absorption) would be sufficient to assess the genotoxic potential of the substance.
Finally, as reported in literature, from the analysis of 91 chemicals with published data from Comet Assay and Transgenic rodent mutation assay (TGR), the comet assay appears to yield similar results to the TGR assay in liver and gastrointestinal tract (predominantly stomach and colon data) and, hence, can be confidently performed to confirm in vivo gene mutation activity in terms of genotoxicity in general.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Sex:

not specified

Genotoxicity:

other: to be performed

Remarks on result:

other: the test is in read-across from a submitted testing proposal still under evaluation.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

In deviation to the protocol for a short period the relative humidity was higher than 70% (highest value 88%). Three of the males exceeded the upper limit of the body weight range slightly 40.1 g, 40.4 g and 42.0 g instead of 40 g)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: BRL, CH-4414 Füllinsdorf.

Number of Animals: 60 (30 males/30 females).

Initial age at start of acclimatization: 8-12 weeks.

Acclimatization: minimum 5 days.

Initial body weight at start of treatment: males mean value 36.5 g (SD ± 2.4 g), females mean value 28.8 g (SD ± 2.2 g).

According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of RCC - CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.

Husbandry: the animals were kept conventionally. The experiment was conducted under standard laboratory conditions.

Housing: single Cage, Makrolon Type I, with wire mesh top, (EHRET GmbH, D-79302 Emmendingen).

Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe).

Feed: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe).

Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)

Environment: temperature 21 ± 3°C

Relative humidity: 30-88 %

Artificial light: 6.00 a.m. - 6.00 p.m.

Vehicle:

On the day of the experiment, the test article was formulated in deionised water. The vehicle was chosen to its non-toxicity for the animals. All animals received a single standard volume of 10 ml/kg body weight orally.

Details on exposure:

Gavage. At the start of the experiment the animals were weighed and the administed volume was adjusted to the weight of the animal

Frequency of treatment:

once

Remarks:

Doses/Concentrations: 0, 200, 67 and 2000 mg/kg bw - Basis: nominal in water

No. of animals per sex per dose:

5 male and 5 females per dose

Positive control(s):

cyclophosphamide administed at 40 mg/kg bw

Tissues and cell types examined:

Bone marrow polychromatic erytrocytes

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.Analysis of Cells: Evaluation of the slides was performed using NIKON microscopes with 1OOx oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points. A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.This can be confirmed by means of the nonparametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: qualitative result

 

Test group	dose (mg/kg bw)	sampling time (h)	PCEs with micronuclei (%)	PCE/NCE
Vehicle	0	24	0.09	2000/1508
Test article	200	24	0.06	2000/1533
Test article	670	24	0.120	2000/1488
Test article	2000	24	0.110	2000/1571
Cyclophosphamide	40	24	1.29	2000/2108
Test article	2000	48	0.045	2000/2252

Conclusions:

The test substance was tested for in vivo chromosome aberration test following OECD 474. Under the experimental conditions the substance did not show any mutagenic potential.

Executive summary:

The test article was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670, and 2000 mg/kg b.w.. 48 h preparation interval: 2000 mg/kg b.w.. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. The mean number of normochromatic erythrocytes was increased after treatment with the test article as compared to the mean value of NCEs of the vehicle controls at preparation interval 48 hours, indicating that the test item had cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with test item were near to the range of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For further details refer to the attached document on genotoxicity assessment.

Justification for classification or non-classification

Classification for mutagenicity is warranted for substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The classification in Category 2 is based on:

— Positive evidence obtained from experiments in mammals and/or in some cases from "In vitro" experiments, obtained from:

— 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.

 

A new evaluation of the genotoxic potential will be performed once the results of the "In vivo" COMET Assay on the Analogue Substance #3 will be available.