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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bayscript Blaukomponente proved to be non-mutagenic in the Ames test, the HPRT-test and in the MNT in vitro. All three tests were performed according to the respective guidelines and GLP conditions and evaluated with Klimisch score 1.

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
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 471 (Bacterial Reverse Mutation 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 TA 102
Additional strain / cell type characteristics:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix was made from the livers of at least 5 adult male Sprague-Dawley rats . For enzyme induction the animals received a single intraperitoneal injection of Aroclor 1254 dissolved in corn oil five days prior to sacrifice
Test concentrations with justification for top dose:
with and without S9-mix:
50, 160, 500, 1600, 5000 µg/plate
Vehicle / solvent:
deionized water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
cumene hydroperoxide
mitomycin C
other: 4-nitro-1,2-phenylend diamine; 2-aminoanthracene
Details on test system and experimental conditions:
2 independent trials :
according to preincubation methodology or according to plate incorporation methodology
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100, TA1537, TA98 this increase should be about twice that of negative controls. For TA102 an increase of about 100 mutants should be reached.
Otherwise the result is evaluated as negative
Statistics:
no data
Key result
Species / strain:
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
Conclusions:
Interpretation of results: negative
According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.
Executive summary:

Bayscript Blaukomponente was initially investigated in the Ames test according to OECD TG 471 and GLP using the Salmonella typhimurium TA 98, TA100, TA102, TA1535, TA1537 and concentrations up to and including 5000 µg/plate. Bacteriotoxicity was not reached. None of the 5 strains used showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls neither in the presence nor in the absence of the metabolic activation system S9 -mix. The positive controls were functional. Therefore, Bayscript Blaukomponente was considered to be 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 / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
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:
other: OECD TG 487, 2010
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
other: Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
liver homogenate (S9: 9000 x g fraction) from livers of Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
4 hour-treatment:
with S9-mix: and without S9-mix:
250, 500, 1000, 2000, 3000, 4000, 5000 µg/ml
24 hour-treatment:
without S9-mix:
25, 50, 100, 250, 500, 1000, 2000 µg/ml
Vehicle / solvent:
deionized water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
cyclophosphamide
mitomycin C
other: 4 hour treatment, with S9-mix Cyclophosphamide; without S9-mix: Mitomycin C; Vinblastine sulfate salt
Details on test system and experimental conditions:
In this study, two independent assays were performed, the first assay conducted with a treatment time of 4 hours (pulse treatment), consisting of one experiment in the absence and one experiment in the presence of an extrinsic metabolizing system (S9 mix). In the second assay, an experiment without S9 mix was performed with treatment time extended to 24 hours (continuous treatment).
Evaluation criteria:
The test item is classified as mutagenic if one of the test substance concentrations induces a micronucleus frequency that is three times higher than the micronucleus frequency of the negative control
Statistics:
no statistical methods available
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:
Interpretation of results: negative
According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.
Executive summary:

Bayscript Blaukomponente was examined 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 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 is a mutagen in the micronucleus test in vitro in the absence or presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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)
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 karyotype with a modal chromosome number of 22
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was prepared from 8-12 weeks old male Wistar rats induced by intraperitoneal applications of phenobarbital and peroral application of ß-naphthoflavone on 3 consecutive days
Test concentrations with justification for top dose:
EXPERIMENT I
without S9-mix (4 hour treatment):
887.5, 1775, 3559, 7100, 10650, 14200 µg/ml
with S9-mix(4 hour treatment)
443.8, 887.5, 1775, 3550, 7100, 10650 µg/ml
EXPERIMENT II
without S9-mix (24 hour treatment)
443.8, 887.5, 1775, 3550, 7100, 10650 µg/ml
with S9-mix (4 hour treatment)
443.8, 887.5, 1775, 3550, 7100, 10650 µg/ml
Vehicle / solvent:
deionised water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: EMS; ethylmethane sulfonate; With metabolic activation: DMBA; 7,12-dimethylbenz(a)anthracene
Details on test system and experimental conditions:
the assay was performed in 2 independent experiments, using 2 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
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
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:
Interpretation of results: negative
According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.
Executive summary:

The study was performed to investigate the potential of Bayscript Blaukomponente 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 is considered to be non-mutagenic 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

Ames test

Bayscript Blaukomponente was initially investigated in the Ames test according to OECD TG 471 and GLP using the Salmonella typhimurium TA 98, TA100, TA102, TA1535, TA1537 and concentrations up to and including 5000 µg/plate. Bacteriotoxicity was not reached. None of the 5 strains used showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls either in the presence nor in the absence of the metabolic activation system S9-mix. The positive controls were functional.

Due to these results Bayscript Blaukomponente has to be regarded as non-mutagenic in this Ames test.

HPRT test

The study was performed to investigate the potential of Bayscript Blaukomponente 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, Bayscript Blaukomponente is considered to be non-mutagenic in this HPRT assay.

MNT in vitro

Bayscript Blaukomponente was examined for 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 is a mutagen in the micronucleus test in vitro in the absence or presence of metabolic activation.


Justification for selection of genetic toxicity endpoint
No endpoint was selected because Bayscript Blaukomponente proved to be non-mutagenic in the Ames test, the HPRT-test and in the MNT in vitro. All
three tests were performed according to the respective guideines and GLP conditions and evaluated with Klimisch score 1

Short description of key information:
Bayscript Blaukomponente proved to be non-mutagenic in the Ames test, the HPRT-test and in the MNT in vitro. All three tests were performed according to the respective guideines and GLP conditions and evaluated with Klimisch score 1

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Bayscript Blaukomponente was negative in the Ames test, the HPRT-test and in the MNT in vitro. All three tests were performed according to the respective guidelines and GLP conditions and evaluated with Klimisch score 1. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.