Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

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
Study period:
Nov - Dec 2016
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:
(1997)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Physical state/Appearance: Dark brown solid
Sponsor’s description: Solid, dark blue
EC Number: 916-899-6
Empirical formula: UVCB substance
Expiry Date: 28 April 2017
Storage Conditions: Room temperature over silica gel in the dark
Stability under test conditions: The stability of the substance in the vehicle used is analytically verified for a period of at least 24 hours.
Formulated concentrations were adjusted to allow for the stated water/impurity content of the test item.
Target gene:
Histidine locus
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 preparation from the liver of Phenobarbital/ beta-Naphtha flavone induced sprague-Dawley rats.
Test concentrations with justification for top dose:
Exp. 1 (plate incorporation test, with and without metabolic activation): 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate
Exp. 2 (pre-incubation method, with and without metabolic activation): 15, 50, 150, 500, 1500, and 5000 µg/plate
Vehicle / solvent:
sterile distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
The positive controls N-ethyl-N'-nitro-N-nitrosoguanidine, 9-Aminoacridine, and 4-Nitroquinoline-1-oxide were used without S9 mix, the positive controls 2-Aminoanthracene and benzo(a)pyrene with S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 = Plate Incorporation Test; as independent repeat Experiment 2 = preincubation method
Every concentration is assayed in triplicate against each tester strain, all with and without the addition of a rat liver homogenate metabolizing system.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity)
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study: 1. A dose-related increase in mutant frequency over the dose range tested. 2. A reproducible increase at one or more concentrations. 3. Biological relevance against in-house historical control ranges. 4. Statistical analysis of data as determined by UKEMS. 5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, and E. coli WPuvrA
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:
not applicable
Positive controls validity:
valid
Additional information on results:
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) or Experiment 2 (pre incubation method).
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
A mauve test item colouration was initially noted at 50 µg/plate becoming an increasingly intense purple colour as the dose concentrations increased, this observation did not prevent the scoring of revertant colonies.

TEST-SPECIFIC CONFOUNDING FACTORS
None

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) or in the second mutation test (pre-incubation method) up to and including the limit concentration of 5000 µg/plate.
Conclusions:
The test substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:

A bacterial reverse mutation assay (Ames test, OECD TG 471) was conducted with the tester strains S. thyphimurium TA 1535, TA 1537, TA 98, TA 100, and E. coli WP2 with and without metabolic activation. The test was performed as plate incubation (Exp. 1) and, as independent repeat (Exp. 2), with the preincubation method with concentrations up to the maximum recommended dose level (5000 µg/plate).

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Exp. 1 or 2 up to and including the limit concentration of 5000 µg/plate.  

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Exp. 1 or 2.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

A mauve test item colouration was initially noted at 50 µg/plate becoming an increasingly intense purple colour as the dose concentrations increased, this observation did not prevent the scoring of revertant colonies.

In conclusion, the test substance was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
For the assessment of chromosome aberration read across to the structurally similar Bayscript Blaukomponente MDA (EC No 916-916-7; source component) is performed. For further information on the read across see separate document attached to this study endpoint record.

HYPOTHESIS FOR THE ANALOGUE APPROACH
Both, the substance (target) and its read across substance (source), consist of three nearly identical main constituents and further isomers in minor amounts. These are naphthylenesulfonic acid moieties linked with azo groups. Target and source mainly differ in their ammonium counter ion, which is TEA (CAS 102-71-6) for the target and MDA (CAS 105-59-9) for the source.

For the ammonium counter ion TEA itself no genotoxic potential was concluded (references: Substance Evaluation Report for 2,2’,2”-NITRILOTRIETHANOL (TEA), CAS 02-71-6, by UK REACH CA, August 2015).
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4 hour treatment, with S9-mix: 500 µg/ml and above; 4-hour treatment, without S9-Mix: 1000 µg/ml and above; 24 hour treatment, without S9-mix: 25 µg/ml and above
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The assessment for mutagenic activity in the micronucleus test in vitro according to OECD TG 487 in the presence and in th absence of a metabolic activation revealed no biologically relevant increase in the frequencies of micronucleus.
Executive summary:

Based on read across to Bayscript Blaukomponente MDA (source substance, EC No. 916 -916 -7) a study is available that examines the mutagenic activity in the micronucleus test in vitro according to OECD TG 487 in the presence and in the absence of a metabolic activation system. Cytotoxic effects were observed (4 hour treatment, with S9-mix: 500 µg/ml and above; 4-hour treatment, without S9-Mix: 1000 µg/ml and above; 24 hour treatment, without S9-mix: 25 µg/ml and above). No precipitation was observed. No biologically relevant increase in the frequencies of micronucleus containing V79 cells treated with the test item in the absence (4 hours or 24 hours treatment) or in the presence of S9-mix were seen.

Evaluation of the data does not indicate that Bayscript Blaukomponente MDA is a mutagen in the micronucleus test in vitro in the absence or presence of metabolic activation.

The test result can be used for assessment of Bayscript Blaukomponente TEA (target substance), since both, the target and the source substance consist of three nearly identical main constituents of naphthylenesulfonic acid moieties linked with azo groups and further such isomers in minor amounts. Target and source mainly differ in their ammonium counter ion, which is TEA (CAS 102-71-6) for the target and MDA (CAS 105-59-9) for the source.

For the ammonium counter ion TEA itself no genotoxic was concluded (references: Substance Evaluation Report for 2,2’,2”-NITRILOTRIETHANOL (TEA), CAS 02-71-6, by UK REACH CA, August 2015).

For further information on the read across see separate document attached to this record.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
For the assessment of gene mutation read across to the structurally similar Bayscript Blaukomponente MDA (EC No 916-916-7; source component) is performed. For further information on the read across see separate document attached to this study endpoint record.

HYPOTHESIS FOR THE ANALOGUE APPROACH
Both, the substance (target) and its read across substance (source), consist of three nearly identical main constituents and further isomers in minor amounts. These are naphthylenesulfonic acid moieties linked with azo groups. Target and source mainly differ in their ammonium counter ion, which is TEA (CAS 102-71-6) for the target and MDA (CAS 105-59-9) for the source.

For the ammonium counter ion TEA itself no genotoxic potential was concluded (references: Substance Evaluation Report for 2,2’,2”-NITRILOTRIETHANOL (TEA), CAS 02-71-6, by UK REACH CA, August 2015).
Key result
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-mix: at 10650 µg/ml and above; with S9-mix: at 7100 µg/ml and above; in experiment II at 7100 µg/ml and above with and without S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

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:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Bayscript Blaukomponente is considered to be non-mutagenic in this HPRT assay.
Executive summary:

Based on read across a study is available that investigates the potential of Bayscript Blaukomponente MDA (source substance, EC No. 916 -916 -7) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD TG 476. 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. No precipitation of the test item at the end of treatment was noted. Cytotoxicity was observed in experiment I without S9-mix: at 10650 µg/ml and above; with S9-mix: at 7100 µg/ml and above; in experiment II at 7100 µg/ml and above with and without S9-mix.

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.

In conclusion it can be stated that under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Byscript Blaukomponente MDA is considered to be non-mutagenic in this HPRT assay.

The test result can be used for assessment of Bayscript Blaukomponente TEA (target substance), since both, the target and the source substance consist of three nearly identical main constituents of naphthylenesulfonic acid moieties linked with azo groups and further such isomers in minor amounts. Target and source mainly differ in their ammonium counter ion, which is TEA (CAS 102-71-6) for the target and MDA (CAS 105-59-9) for the source.

For the ammonium counter ion TEA itself no genotoxic was concluded (references: Substance Evaluation Report for 2,2’,2”-NITRILOTRIETHANOL (TEA), CAS 02-71-6, by UK REACH CA, August 2015).

For further information on the read across see separate document attached to this record.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bayscript Blaukomponente TEA proved to be no genetic toxicant in the Ames test and, based on read across, also in the HPRT and MNT in vitro. All three tests were performed according to the respective guidelines and GLP.

Justification for classification or non-classification

No classification concluded for Genetic Toxicity according to Regulation (EC) No 1272/2008, Annex I.