Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: Negative with and without metabolic activation (S9 mix)

Chromosome aberration: no evidence of an increase in the frequency of structural chromosome aberrations.

Mouse Lymphoma Assay: no evidence of mutagenic activity with or without metabolic activation.  

Link to relevant study records

Referenceopen allclose all

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 - 21 January 2003
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)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4E, dated May 19, 2000.
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Health and Welfare, 1988, Guidelines for Toxicity Studies of Drugs, Chapter 4, "Mutagenicity Study".
Deviations:
no
Qualifier:
according to
Guideline:
other: "U.S. FDA Center for Food Safety & Applied Nutrition Office of Premarket Approval, Redbook 2000, Toxicological Principles for the Safety of Food Ingredients, IV.C.1.c. Mouse Lymphoma Thymidine Kinase Gene Mutation Assay", July 07, 2000.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 Medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Experiment I
Without S9 Mix: 3.1, 6.3, 12.5, 25.0, 37.5, and 50 µg/mL.

Experiment II
Without S9 Mix: 6.3, 12.5, 25.0, 50.0, 75.0, and 100.0 µg/mL.
With S9 Mix: 3.1, 6.3, 12.5, 25.0, 37.5, and 50.0 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: This solvent was chosen due to its solubility properties, and its relative non-toxicity to the cell cultures.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Used in the absence of S9 mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
Used in the presence of S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours in the first experiment, 24 hours in the second experiment.
- Expression time (cells in growth medium): 72 hours (48 hours in the second experiment without metabolic activation).
- Selection time (if incubation with a selection agent): 10 - 15 days
- Fixation time (start of exposure up to fixation or harvest of cells): 72 hours

SELECTION AGENT (mutation assays): RPMI 1640 medium containing 5 µg/mL Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Two plates were seeded per culture.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.

A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.


A significant response is described as follows:
The test item is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.

The test item is classified as mutagenic if there is a· reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.

However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.

Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 is less than 10 Ok of the solvent control or the cloning efficiency 2 after the expression period is less than 20 %.

Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations and c1astogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was seen at 50 µg/mL with and without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effect relative to the control
- Effects of osmolality: Osmolarity of solvent control = 275; osmolarity of TCD formulation, 1600 µg/mL = 321
- Precipitation: In the pre-experiment, precipitation occurred at 50 µg/mL and above without metabolic activation, and at 100 µg/mL with metabolic activation durinf pulse treatment, and at 400 µg/mL and above at continuous treatment.
Conclusions:
Interpretation of results: negative

Under the experimental conditions used 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.
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:
guideline study
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)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Kanpoan No 287 - Environment Protection Agency; Eisei No 127 - Ministry of Health, Labour and Welfare; Heisei 09/10/31 Kikyoku No 2 - Ministry of Economy, Trade, and Industry
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:
S9 Fraction
Test concentrations with justification for top dose:
Initial test (plate incorporation)
S. Typhimurium - 33, 100, 333, 1000, 2500, and 5000 µg/plate
E. coli - 100, 333, 1000, 2500, and 5000 µg/plate

Confirmation test
All strains (plate incorporation, without Metabolic activation) - 3.3, 10, 33, 100, 333, 1000, and 5000 µg/plate.
With metabolic activation, pre-incubation assay:
TA100, TA1537, TA102 - 0.1, 0.3, 1.0, 3.3, 10, 33, and 100 µg/plate.
TA1535, TA98 - 0.3, 1.0, 3.3, 10, 33, 100, and 333 µg/plate.
WP2 uvrA - 3.3, 10, 33, 100, 333, 1000, and 5000 µg/plate.


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: DMF was selected with preference to Dimethylsulphoxide (DMSO) as the test material was poorly soluble in DMSO.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: Used for strains TA100 and TA1535 without metabolic activation.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: Used for strains TA98 without metabolic activation.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: Used for strain TA1537 without metabolic activation.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: Used for strain WP2 uvrA without metabolic activation.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: Used for strain TA102 without metabolic activation.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: Used for strain TA1535 with metabolic activation.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; Used for strains TA1537, TA98, TA100, TA102, and WP2 uvrA with metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation), and preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Three plates per concentration in each test.

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the number of revertant colonies and the background lawn (reduction relative to the solvent control).
Key result
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Tests were conducted up to cytotoxic concentrations for all strains except WP2 uvrA (no reduction in the background lawn or reduction in the number of revertant colonies seen in the presence of metabolic activation)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item on the surface of the agar plates was observed at any concentration tested.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results: negative

In the mutagenicity test described and under the experimental conditions reported, TCD did not induce any mutations in the genome of strains of Salmonella typhimurium or Escherichia coli.
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:
07 August 2002 - 21 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (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:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH-Harmonised Tripartite Guidelines S2A and S2B.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal Essential Medium, supplemented with 10% fetal calf serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Without S9 mix: Experiment I - 1.6, 3.1, 6.3, 12.5, 18.8, 25.0 µg/mL.
Experiment II (18 hour exposure) - 1.6, 3.1, 6.3, 12.5, 25.0, 50.0 µg/mL; (28 hour exposure) - 6.3, 12.5, 25.0, 50.0 µg/mL.

With S9 mix: Experiment I - 6.3, 12.5, 18.8, 25.0, 37.5, 50.0 µg/mL.
Experiment II - 6.3, 12.5, 25.0, 50.0, 75.0, 100.0 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative nontoxicity to the cell cultures.
Untreated negative controls:
yes
Remarks:
Culture medium control
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Used concurently to experiments without metabolic activation.
Untreated negative controls:
yes
Remarks:
Culture medium control
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Used concurently to experiments with metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration: 4, 18, or 28 hours
- Selection time (if incubation with a selection agent): 2.5 hours

SELECTION AGENT (mutation assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 slides per group were prepared in each experiment

NUMBER OF CELLS EVALUATED: 100 well spread metaphase cells per culture were scored for cytogenetic damage; 500 metaphase cells per culture were assessed for the number of polyploid cells.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes (% of polyploid metaphases were calculated per 500 metaphase cells were calculated for each slide)
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effect on pH was seen
- Effects of osmolality: No effect on osmolarity was seen
- Precipitation: In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 201.3 µg/ml and above in the absence of S9 mix and 100.6 µg/ml and above in the presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES:
In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 12.6 and 1610 µg/ml were applied. Clear toxic effects were observed after 4 hrs treatment with 25.2µg/ml and above in the absence of S9 mix and with 50.3 µg/ml and above in the presence of S9 mix. In addition, 24 hrs continuous treatment with 25.2 µg/ml and above in the absence of S9 mix induced strong toxic effects.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results: negative

It was concluded that under the experimental conditions applied in this study, TCD did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Bacterial reverse mutation (Ames) assay

In a study conducted according to OECD test guideline 471 (RCC, 2003, study number 843388), five mutant strains of Salmonella Typhimurium (TA1535, TA1537, TA98, TA100 and TA 102) and one strain of Escherichia coli (WP2 uvrA) were treated with TCD at a series of concentrations (33 µg/plate to 5000 µg/plate) with and without metabolic activation (S9 mix). No increase in the number of revertant colonies was seen relative to the concurrent vehicle controls in any of the strains tested at any concentration, either with or without metabolic activation and it was concluded that TCD showed no evidence of mutagenic activity in the bacterial system used, under the conditions employed

Chromosome aberration study

A chromosomal aberration study (RCC, 2003, study number 844249) was conducted according to OECD test guideline 473 to assess the cytogenetic potential of TCD in Chinese hamster V79 cells. Tests were carried out with and without metabolic activation and with short a treatment time (6hr) and with continuous treatment (18 -28 hr). There was no statistically significant or biologically relevant increase in the number of cells carrying structural chromosomal aberrations after treatment with the test item, regardless of the use or non-use of metabolic activation and the length of treatment time. Based on this, it was judged that the test substance does not possess clastogenic properties.

Mouse lymphoma study

A mouse lymphoma assay (RCC, 2003, study number 843385) was conducted to assess the mutagenic potential of TCD. The study was conducted according to OECD guideline 476 and in compliance with GLP. Mouse lymphoma L5178Y cells were treated with a solution of TCD in acetone at concentrations up to 100 µg/mL, with and without metabolic activation (S9 mix). No increase in mutant colony was seen in any of the tests conducted, nor was any relevant shift of the ratio of small versus large colonies up to the maximum concentration of the test. It was concluded that TCD had not shown any mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Overall conclusion

The substance has given negative results in three in vitro tests that cover bacterial gene mutation, gene mutation in mammalian cells and cytogenicity in mammalian cells. In accordance with Table R.7.7 -5 of ECHA Endpoint specific guidance Chapter 7(a) Version 2.0 the substance can therefore be regarded as not genotoxic and no further tests are required.

Justification for selection of genetic toxicity endpoint

Three studies are available all with negative results for TCD. This mouse lymphoma study is selected on the basis that it is a valid test on a mammalian species and is conducted under GLP.

Justification for classification or non-classification

Three in-vitro studies (as described above) examined aspects of the mutagenic potential of TCD. None of these studies indicated any mutagenic potential and so it is not necessary to classify TCD as hazardous on the basis of mutagenic activity according to the CLP Regulation (Regulation (EC) 1272/2008).