Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagen

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: experimental study on similar substance
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Colipa report n. C174
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: Mouse Lymphoma
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
uninduced Syrian golden hamster liver homogenate
Test concentrations with justification for top dose:
156.3, 312.5, 625, 1250, 2500, 5000 μg/ml with and without S9
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: DMNA
Details on test system and experimental conditions:
4 hours treatment
Species / strain:
other: all strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
Not mutagenic
Executive summary:

There is no indication of increase in mutation frequency induced by the substance in both experiments in the absence of metabolic activation and in the second experiment in the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: experimental study on similar substance
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Source: Dr. Bruce N.Amex (University of Berkeley, California).
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Source: Dr. Bruce N.Amex (University of Berkeley, California).
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
50, 250 and 1000 µg/plate
Vehicle / solvent:
water or DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: nitroquinoline-N-oxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: anthragallol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-antrhamine
Details on test system and experimental conditions:
Plate tests. Plates containing various concentrations of the test agents incorporated with the top agar and in some cases 100--200 pg of the test agent contained in a 0.25-inch sterile concentration disk applied to the top agar were incubated 3 days at 35 ° .
In addition many of the azo dyes dissolved and plated as described above were incubated for 16 h at 37 ° in an anaerobic glove box similar in most respects to that described by
Aranki et al. [3]. These were subsequently incubated for 3 days in the normal {aerobic) fashion.
Species / strain:
other: all strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Not mutagenic
Executive summary:

The results of this study indicate that the tested dye is not mutagenic in the Salmonella / microsome test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: experimental study on similar substance
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Primary reference with few details on methods and results.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Source: B.N. Ames, Biochemistry Department, University of California, Berkeley.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
3.3, 1.65 and 0.165 mg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
Two of the histidine requiring mutants (TA98 and TA1537) were developed for the detection of frame-shift mutagens and the other two (TA100 and TA1535) base-pair substituted mutants. Strains TA98 and 100 contained the resistance-transfer factor which made them more sensitive to a wide range of mutagens [1]. Upon receipt of the strains they were cultured
and checked for histidine requirements; crystal violet and ampicillin sensitivity; and mutagenicity with 9-aminoacridine, 2- aminofluorine, and Nmethyl- N-nitro-N'-nitrosoguanidine.
At regular' intervals during the tests the cultures were checked to determine if they displayed the original mutant phenotype. Permanent stock cultures of each strain were made by adding
dimethyl sulfoxide (DMSO) to nutrient broth cultures; the cultures ere quickly frozen in alcohol-dry ice bath and stored at -74°C in a Revco freezer.
Fresh cultures were obtained as described by Ames et al. except the cultures were grown at 37°C on a roller drum at a speed of 50 r.p.m.
Statistics:
The statistical sampling used in this study was a twostage, double sampling scheme for estimating binomial (yes/no) data with misclassifications (Tenenbein, 1970).
Species / strain:
other: TA98, TA100, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Not mutagenic
Executive summary:

The results of this study indicate that the tested dye is not mutagenic in all tested strains in the Ames mutagenicity test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: experimental study on similar substance
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Primary reference with few details on methods and results.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA98, TA100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
50, 250 and 1000 µg/plate
Vehicle / solvent:
water or DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
Chemicals were tested in a preincubation procedure in strains TA98 and TA100 without metabolic activation and with activation provided by Aroclorinduced rat and hamster liver homogenates (S9).
If a positive response was seen in one of these two strains, the strain/metabolic activation combination producing that response was repeated, and no further testing was performed. If no positive responses were seen, the chemical was tested in strains TA97 and TA1535
Statistics:
The statistical sampling used in this study was a twostage, double sampling scheme for estimating binomial (yes/no) data with misclassifications (Tenenbein, 1970).
Species / strain:
other: TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Not mutagenic
Executive summary:

The results of this study indicate that the tested dye is not mutagenic in the Ames mutagenicity test.

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

Genetic toxicity in vivo

Description of key information

Not mutagen

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
other: experimental study on similar substance
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: MNT
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
30 mice were used for the experiment (6 mice per treatment, positive control, and negative control group).
Animals were purchased at 6 weeks of age from Charles River Laboratories, Japan and were acclimatized for 7 days before treatments. Food (CRF-1 pellet feed, Oriental Yeast, Japan) and water were provided ad libitum.

Route of administration:
oral: gavage
Vehicle:
0.5% carboxymethylcellulose sodium
Details on exposure:
oral gavage
Frequency of treatment:
the test article and the negative control were orally administered daily for 2 days with a 24-h interval using stomach tubes and plastic syringes.
Dosing formulations of thempositive control substance were administered in a single intraperitoneal injection using a disposable syringe fitted with a 25-G needle.
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
30 mice were used in experiments:
6 mice in each of the 3 dose groups
6 mice in the positive control groups
6 mice in the negative control groups.
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitocymin C dissolved in water for injection.
Statistics:
Frequencies of MNPCEs in treatment and positive control groups were compared with those in the negative control group using conditional binomial tests (Kastenbaum and Bowman test, uppertailed significance level of 0.05).
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No deaths and no clinical signs of toxicity were observed in any of the treatment groups.
No differences in body weight were observed between control and treatment groups at the end of treatment.
In micronucleus tests, no significant differences in MNPCEs were found between the treated groups and the negative control group.
The frequency of PCEs, which offers an index of the influence of the test substance on bone marrow cells, did not differ between any of the treatment groups and the negative control group.
In contrast, the frequency of MNPCEs in the positive control group was markedly increased in comparison with that in the negative control group.
Conclusions:
Not mutagen
Executive summary:

The tested substance did not show any mutagenic effect in the in vivo micronucleous test performed according OECD 474.

Endpoint:
genetic toxicity in vivo, other
Remarks:
TRG assay
Type of information:
other: experimental study on similar substance
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: MNT
Species:
mouse
Strain:
other: Muta Mice
Sex:
male
Details on test animals and environmental conditions:
In total, 28 mice were used for the experiment (6 mice per treatment and negative control groups and 4 mice in the positive control group).
Male Muta™ Mice (CD2-LacZ80/HazfBR) were purchased at 9 weeks of age from Japan Laboratory Animals, Inc., Japan and were acclimatized for 11 days before treatments. Food (CRF-1 pellet feed, Oriental Yeast, Japan) and water were provided ad libitum.
Route of administration:
oral: gavage
Vehicle:
water for injection
Details on exposure:
oral gavage
Frequency of treatment:
the test article and the negative control were orally administered daily for 2 days with a 24-h interval using stomach tubes and plastic syringes.
Dosing formulations of them positive control substance were administered in a single intraperitoneal injection using a disposable syringe fitted with a 25-G needle.
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
28 mice were used in experiments:
6 mice in each of the 3 dose groups
6 mice in the positive control groups
4 mice in the negative control groups.
Control animals:
yes, concurrent vehicle
Positive control(s):
7, 12-dimethylbenz[a]anthracene was mixed with olive oil
Tissues and cell types examined:
Mice were sacrificed by CO2 asphyxiation 3 days after the last treatment. Subsequently, liver and stomach tissues were collected, stomach tissues were divided into forestomach and glandular stomach, and genomic DNA was extracted from liver and glandular stomach samples according to the reported methods.
Statistics:
Differences in MFs were analyzed for significance by the Conditional Binomial test (Kastenbaum and Bowman method: upper tailed significance level of 0.05).
The results were evaluated as positive when the mutant frequency in the test substance treated group was significantly different from that in the negative control group.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No deaths and no clinical signs of toxicity were recorded in any of the treatment groups. In addition, body weight gains during treatment were the same in all treatment groups.
MFs of cII genes in liver and glandular stomach tissues from the treated animals were not significantly higher than those in respective negative control animals.
In contrast, the positive control DMBA significantly increased MFs in the liver and MFs were slightly but insignificantly induced in stomach tissues.
However, these weak response may be adequate as positive control in TGR assay.
Conclusions:
Not mutagen
Executive summary:

The tested substance did not show any mutagenic effect in the in vivo mutagenicity test performed according OECD 488.

Endpoint:
genetic toxicity in vivo, other
Remarks:
Comet assay
Type of information:
other: experimental study on similar substance
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: comet assay
Species:
mouse
Strain:
other: [CD2F1/Crlj (CDF1), SPF/VAF]
Sex:
male
Details on test animals and environmental conditions:
In total, 25 mice were used in experiments, including 5 mice in each of the 3 dose groups and 5 each in the positive and negative control groups.
Animals were purchased at the age of 8 weeks from Charles River Laboratories, Japan. Animals were acclimatized for 8 days before the start of the treatments.
Food (CE-2 pellet feed, CLEA Japan) and water were provided ad libitum.
Route of administration:
oral: gavage
Vehicle:
physiological saline
Details on exposure:
oral gavage
Frequency of treatment:
2 application
The test chemical and the negative control substance were orally administered 2 times using stomach tubes and plastic syringes at 24 h and 3 h before tissue sampling.
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 mice were used in experiments:
5 mice in each of the 3 dose groups
5 mice in the positive control groups
5 mice in the negative control groups.
Control animals:
yes, concurrent no treatment
Positive control(s):
Ethylmethanesulphonate dissolved in physisological saline.
Statistics:
Mean percentages of tail DNA were compared between each treatment group and the negative control group using Dunnett's multiple comparison test (1-tailed), and differences were considered significant when p < 0.05 and p < 0.01.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No deaths and no clinical signs of toxicity were observed in any of the treatment groups.
No significant differences in numbers of hedgehogs or DNA damages were observed in either organ between the treated animals and negative control animals.
In contrast, the positive control (EMS) group showed significant increases in percentages of DNA in Comet tails from both organs.
Conclusions:
Not mutagen
Executive summary:

The tested substance does not show any significant differences in numbers of hedgehogs and no DNA damages were observed both in treated and negative control animals.

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

Additional information

Justification for classification or non-classification

There are different in vitro and in vivo studies performed on similar substance:

In vitro Ames test (OECD 471) - negative in different studies and different strains both with and without metabolic activation
In vitro Moude Lymphoma test (OECD 476) - negative

In vivo Comet assay (OECD 489) - negative

In vivo MNT (OECD 474) - negative

In vivo TRG assay (OECD 488) - negative

Based on the in vitro and in vivo studies results, it could be concluded that the tested substance is not mutagen.