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Genetic toxicity in vitro

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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:
supporting study
Study period:
11 July 1994 - 21 September 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to Japanese Guideline and in compliance with Japanese Good Laboratory Practise. The details provided in this study report were not sufficient to assess the full property of the test material.
Qualifier:
according to
Guideline:
other: Japanese Industrial Safety and Health Law (Notification No.77, 1988)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, DW
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-finyl)actylamide (AF-2)
Remarks:
without S9 Mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, DW
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 Mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, DW
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 Mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, DW
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 Mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, DW
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with S9 Mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, DW
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 Mix
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative

Based on these results, it was concluded that MTF was not mutagenic under the test conditions of this study.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 July 2002 - 21 February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD, EU, US FDA and Japanese (EPA, MHLW and METI) test guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(Commission Directive 2000/32/EC; L136, 57-64, Annex 4D)
Deviations:
no
Qualifier:
according to
Guideline:
other: US FDA Administration Center for Food Safey & Applied Nutrition Office of Premarket Approval, Redbook 2000, Toxicological Principles for the Safety of Food Ingredients, IV.C.1.a Bacterial Reverse Mutation Test, July 07, 2000.
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
"Kanpoan 287 - EPA", "Eisei 127 - MHLW", and "Heisei 09/10/31 Kikyoku 2 - METI"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat-liver post mitochondrial supernatant (S9 fraction)
Test concentrations with justification for top dose:
Range finding test:
strains S. typhimurium TA 100 and E. coli WP2 uvrA (with and without methabolic activation): 20.6, 61.8, 185.2, 555.6, 1666.7 and 5000 µg/plate


without metabolic activation:
strains TA 100, TA 102: 1.0, 3.3, 10, 33 and 100 µg/plate
strain TA 1537: 10, 33, 100, 333 and 1000 µg/plate
strains TA 1535, TA 98, WP2 uvrA: 33, 100, 333, 1000 and 2500 µg/plate

with metabolic activation (pre-incubation assay):
strains TA 100, TA 1537, TA 102: 10, 33, 100, 333 and 1000 µg/plate
strains TA 1535, TA 98, WP2 uvrA: 33, 100, 333, 1000 and 2500 µg/plate

Due to a wide range of toxicity in the pre-incubation assay with microsomal activation, additional concentrations were tested with metabolic activation:
strains TA 100, TA 1537: 0.3, 1.0, 3.3, 10 and 33 µg/plate
strains T A 1535, T A 98, T A 102: 3.3, 10, 33, 100 and 333 µg/plate.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-AA
Remarks:
With metabolic activation
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative

During the described mutagenicity test and under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used.


Therefore, MTF is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
11 September 1996- 18 March 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted according to Japanese Guideline and in compliance with GLP.
Qualifier:
according to
Guideline:
other: Japanese Guidelines on Industrial Chemicals (1987)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: CHL/IU
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Cell growth inhibition test:
Without methabolic activation (24- hour treatment) (µg/ml): 20, 40, 60, 80, 100*, 120*, 140*, 160*;
Without methabolic activation (48- hour treatment) (µg/ml): 20, 40, 60, 80, 100*, 120*, 140*, 160*;
With methabolic activation (µg/ml): 10, 20, 39, 78, 156*, 313*, 625*, 1250*, 2500*, 5000*.
*: Precipitation of the test substance was recognized at the treatment period .

Chromosomal aberration test:
Without methabolic activation (24- hour treatment) (µg/ml): 10.6, 21.3, 42.5, 85
Without methabolic activation (48- hour treatment) (µg/ml): 10.6, 21.3, 42.5, 85
With metabolic activation (µg/ml): 18.8, 37.5, 75, 150(C) (Toxic)
Without metabolic activation (µg/ml): 18.8, 37.5, 75, 150(C)
C: Precipitation of the test substance was recognized at the treatment period.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With metabolic activation
Key result
Species / strain:
mammalian cell line, other:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations

In the chromosomal aberration test, the incidence of cells with structural and numerical aberration was less than 5%.

Based on this result, the test substance was judged to be negative for the ability to induce chromosomal aberration.

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results, the test substance was judged to be negative for the ability to induce chromosomal aberration.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 2011 - 08 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to official OECD, EU, and US test guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1996) Guideline S2A, and ICH (1998) Guideline S2B
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 µg/mL streptomycin and 2.0 mM glutamine.
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
First Test (3 hours treatment, with and without S9 mix): 18.37, 30.62, 51.03, 81.05, 141.76, 236.26, 393.77, 656.28, 1093.8, and 1823 µg/mL.
Second test (21 hours treatment in the absence of S9 mix): 15, 20, 25, 30, 35, 40, 42.5, 45, 47.5, 50, and 55 µg/mL.
Second test (3 hours treatment in the presence of S9 mix): 60, 70, 80, 90, 100, 110, 115, 120, 125, 130, and 140 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: MTF was found to be miscible in DMSO.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Used in the absence of S9 mix.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Used in the presence of S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours (first test, and second test in the presence of S9 mix), or 21 hours (second test in the absence of S9 mix).
- Expression time (cells in growth medium): 18 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Cultures were prepared in duplicate.

NUMBER OF CELLS EVALUATED: One hundred metaphase figures examined per culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Noted when seen
- Determination of endoreplication: Noted when seen
Evaluation criteria:
Cochran-Armitage test for trend.
Key result
Species / strain:
lymphocytes: Human Lymphocytes
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

First test:

In the absence of S9 mix following 3 hour treatment, MTF caused a reduction in the mitotic index to 53% of the vehicle control value at 60 µg/mL. In the presence of S9 mix (2% v/v) following 3 hour treatment, MTF caused a reduction in the mitotic index to 51% of the vehicle control value at 100 µg/mL. In both the absence and presence of S9 mix, MTF caused no statistically significant increase in the proportion of cells with chromosomal aberrations at any concentration, when compared with the vehicle control.

Second test:

Conclusions:
Interpretation of results (migrated information):
positive

It was concluded that MTF had shown evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix following 21 hour continuous treatment, and in th presence of S9 mix following 3 hours treatment, in this in vitro cytogenetic test system, under the experimental conditions described.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
16 July 2002-13 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to the OECD Guideline and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4A: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test", dated May 19, 2000.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Qualifier:
according to
Guideline:
other: Japanese Guidelines: "Kanpoan No. 287 - Environmental Protection Agency", "Eisei No. 127 - Ministry of Health & Welfare", "Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry"
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
without S9 mix:
Experiment I: 1.3, 2.5, 5.0, 10.0, 15.0, 25.0
Experiment II: 2.5, 5.0, 10.0, 20.0, 30.0, 40.0

with S9 mix:
Experiment I: 3.1, 6.3, 12.5, 25.0, 50.0, 75.0
Experiment II: 3.1, 6.3, 12.5, 25.0, 50.0, 100.00
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid

In both experiments, in the absence and presence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.

The aberration rates of the cells after treatment with the test item (0.0- 2.0% aberrant cells, exclusive gaps) were

within the range of the solvent control values (0.0- 3.5% aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item MTF did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).
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
Study period:
07 June 2002 - 17 December 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study conducted according to OECD test guideline and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L 1362000, Annex 4E, dated May 19, 2000.
Qualifier:
according to
Guideline:
other: Japanese Ministry of Health and Welfare, 1988, Guidelines for Toxicity Studies of Drugs, Chapter 4, "Mutagenicity Study".
Qualifier:
according to
Guideline:
other: "U.S. Food and Drug Administration Center , Toxicological Principles for the Safety of Food Ingredients, IV.C.1.c. Mouse Lymphoma Thymidine Kinase Gene Mutation Assay", July 07, 2000.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Pre-test: 3500µg/ml - the highest applied concentration

Main test Experiment 1:
without S9 mix: 3.4, 6.9, 13.8, 27.5, 41.3 and 55.0
with S9 mix: 1.8, 3.4, 6.9, 13.8, 20.7 and 27.5

Experiment II:
without S9 mix: 1.6, 3.1, 6.3, 12.5, 25.0 and 50.0
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with metabolic activation
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, MTF is considered to be non-mutagenic in this mouse lymphoma assay.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Justification for classification or non-classification