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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available results from the read across studies, C12-14 TMAC can be considered to be non-genotoxic.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Ames test
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 18 April, 1989 to 08 May, 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
S. Typhimurium TA102 and/or E.coli WP2 strains not included; conducted according to old version of Guideline
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
the version of the OECD guideline should be specified: OECD 471 (1983).
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
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
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
1, 3, 10, 32 and 100 µg/plate for both with and without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
Untreated negative controls:
yes
Remarks:
Solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Tested with TA 1535, TA 100 in the absence of S-9 mix
Untreated negative controls:
yes
Remarks:
Solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Tested for TA 1535 in the presence and absence of S9 mix
Untreated negative controls:
yes
Remarks:
Solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Tested for TA 1537 in the absence of S9-mix
Untreated negative controls:
yes
Remarks:
Solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Tested for TA 98 in the absence of S9-mix
Untreated negative controls:
yes
Remarks:
Solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Tested for TA 1537, TA 100 and TA 98 in the presence and absence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (pour-plate method)

DURATION
- Incubation period: 48 h at approximately 37°C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertant colonies were counted, either manually or with a Biotran II automatic colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the background lawn of non-revertant cells on minimal plates were verified.

Evaluation criteria:
The number of the revertants per plate of the tester strain is compared to the control.
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:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test: Yes

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Based on the results of the read across study, the test substance, C12 -14 TMAC, can also be considered to be not mutagenic with and without metabolic activation in the Ames test.
Executive summary:

A study was conducted to determine the mutagenic potential of read across substance, Coco TMAC (active ingredient 33%), according to OECD 471 and EPA OPPTS 870.5265 Guidelines, in compliance with GLP. Using pour-plate assays, the test substance was examined for mutagenic activity in four strains of Salmonella typhimurium: TA 100, TA 1535, TA 1537 and TA 98 in the absence and in the presence of S9-mix. The test concentrations included 0, 1, 3, 10, 32 and 100 µg/plate (i.e., equivalent to 0, 0.33, 0.99, 3.3, 10.56 and 33 µg/plate). No increase in reversion to prototrophy was obtained with any of the bacterial strain at any concentration level either in the presence or absence of S9-mix. Further, inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test substance at 100 µg/plate. Appropriate positive control chemicals (with S-9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S-9 mix. Under the test conditions, the substance was not mutagenic with and without metabolic activation (May, 1989). Based on the results of the read across study, the test substance, C12 -14 TMAC, can also be considered to be not mutagenic with and without metabolic activation in the Ames test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
chromosome aberration assay
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 18 April, 1989 to 09 May, 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Batch no.: D1263LA
- Description: Clear colourless liquid
Supplied sample was identified as 33% w/v solution
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Cultured peripheral human lymphocytes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
- without S9-mix: 0.4, 2 and 10 µg/L
- With S9-mix: 2, 10 and 50 µg/L
Vehicle / solvent:
Sterile distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: chlorambucil
Remarks:
without S9-mix
Details on test system and experimental conditions:
Study Type: Other: Chromosomal aberration test
Organism/cell type: Cultured peripheral human lymphocytes
Metabolic activation system: S9 mix
Positive control : Chlorambucil (without S9 mix), cyclophosphamide (with S9-mix)
Statistics:
Data from each treatment was compared with the respective solvent control group using Fisher Exact Probability test. A one-sided test was applied and p <0.05 was used as the lowest level of significance.
Key result
Species / strain:
lymphocytes: cultured human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
An increased incidences in polyploidy would normally be associated with spindle dysfunction; such a event would also be expected to increase the incidence of aneuploidy and if this occurred in vivo, micronuclei would be produced in the target cell population. However, a mouse bone marrow micronuclei test, performed elsewhere has no induction following oral administration of the test substance.
Conclusions:
Based on the results of the read across study, the test substance, C12 -14 TMAC, can also be considered to be non-clastogenic with and without metabolic activation in the chromosomal aberration assay.
Executive summary:

A study was conducted to determine the clastogenic potential of the read across substance, Coco TMAC (active ingredient 33%), in cultured human lymphocytes according to OECD Guideline 473, in compliance with GLP. After a concentration range finding test, two independent tests were performed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (S9-mix). With S9-mix cells were exposed for 24 hours. The test substance produced a reduction in mitotic activity (i.e., approximately 48%) at 10 µg/L in the absence of S9-mix and at 10 (i.e., 20%) and 50 (i.e., 37%) µg/L in the presence of S9-mix. Further, consideration of mean aberrant cell frequencies showed no real increases in culture tested with the test substance, when compared to control. Statistical analysis confirmed these observations. However, there was an apparent increase in the number of polyploid cells, compared to concurrent control values in all tested cultures, although a marked effect was seen only in activated cultures exposed to 50 µg/mL the author concluded that the test substance is not clastogenic in human lymphocytes under the experimental conditions. Under the conditions of the study, the test substance was not found to be clastrogenic in human lymphocytes (Richardson, 1989). Based on the results of the read across study, the test substance, C12 -14 TMAC, can also be considered to be non-clastogenic with and without metabolic activation in the chromosomal aberration assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Mouse lymphoma assay
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 15 October, 2001 to 05 March, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission directive 2002/32/EC and U.K Environmental Mutagen Society
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase gene
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:
For Experiment 1: 0.625, 1.25, 2.5, 5, 10 and 20 µg/mL for both with and without S9-mix
For Experiment 2: 0.313, 0.625, 0.938, 1.25, 2.5 and 5 µg/mL (without S9 mix) and 2.5, 5, 10, 15, 20 and 30 µg/mL (with S9-mix)
Vehicle / solvent:
RPMI 1640 without serum
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
in the absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in the presence of S9-mix
Details on test system and experimental conditions:
After a preliminary toxicity test, 0.625, 1.25, 2.5, 5, 10 and 20 µg/L was selected for the first experiment. The entire experiment was repeated to confirm the results of the first experiment. 3 h exposure were used both with and without S9 mix while for the second experiment, the exposure time was increased to 24 h. For the second experiment, the dose range was 2.5 to 30 µg/L with S9 mix and 0.313 to 5 µg/L without S9 mix.
Evaluation criteria:
For a test substance to give a significant result then two or more of the following criteria should be met:
a) A greater than three-fold increase in the mutant frequency/survivor over the negative control value.
b) A dose-related increase in the mutant frequency per survivor.
c) An increase in the absolute number of mutants.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
RANGE-FINDING/SCREENING STUDIES: Yes

There was evidence of toxicity with the test substance in both the presence and absence of S9-mix as indicated by relative suspension growth (RSG), this was confirmed by the decrease in relative total growth (RTG). There was no evidence of a reduction in Day 2 viability, therefore indicating that no residual toxicity occurred, in either the presence or absence of S9-mix. Optimum level of toxicity was achieved, in both the presence and absence of S9-mix. The toxicity observed at 30 µg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%; therefore it was excluded from the statistical analysis.

Conclusions:
Based on the results of the read across study, the test substance, C12 -14 TMAC, can also be considered to be not mutagenic with and without metabolic activation in the mouse lymphoma assay.
Executive summary:

A study was conducted to determine the potential of the read across substance, Coco TMAC (active ingredient 35%), to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells. Study was performed accoding to OECD 476 Guideline, EU Method B.17 and Commission directive 2002/32/EC and U.K Environmental Mutagen Society, in compliance with GLP. After a preliminary toxicity test, 0.625, 1.25, 2.5, 5, 10 and 20 µg/L was selected as the test concentrations for the first experiment. The experiment was repeated to confirm the results of the first experiment. Three hour exposures were used both with and without S9-mix while for the second experiment, the exposure time was increased to 24 h. For the second experiment, the concentration range was 2.5 to 30 µg/L with S9-mix and 0.313 to 5 µg/L without S9-mix. The test substance did not induce a statistically significant or concentration-related increase in the mutant frequency at any concentration level, either with or without metabolic activation. Adequate levels of toxicity were achieved in all exposure groups. Under the conditions of the study, the test substance was not found to show mutagenic activity in mouse lymphoma assay (Nolan, 2002). Based on the results of the read across study, the test substance, C12 -14 TMAC, can also be considered to be not mutagenic with and without metabolic activation in the mouse lymphoma assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Study 1:A study was conducted to determine the mutagenic potential of read across substance, Coco TMAC (active ingredient 33%), according to OECD 471 and EPA OPPTS 870.5265 Guidelines, in compliance with GLP. Using pour-plate assays, the test substance was examined for mutagenic activity in four strains of Salmonella typhimurium: TA 100, TA 1535, TA 1537 and TA 98 in the absence and in the presence of S9-mix. The test concentrations included 0, 1, 3, 10, 32 and 100 µg/plate (i.e., equivalent to 0, 0.33, 0.99, 3.3, 10.56 and 33 µg/plate). No increase in reversion to prototrophy was obtained with any of the bacterial strain at any concentration level either in the presence or absence of S9-mix. Further, inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test substance at 100 µg/plate. Appropriate positive control chemicals (with S-9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S-9 mix. Under the test conditions, the substance was not mutagenic with and without metabolic activation (May, 1989). Based on the results of the read across study, the test substance, C12 -14 TMAC, can also be considered to be not mutagenic with and without metabolic activation in the Ames test.

Study 2:A study was conducted to determine the clastogenic potential of the read across substance, Coco TMAC (active ingredient 33%), in cultured human lymphocytes according to OECD Guideline 473, in compliance with GLP. After a concentration range finding test, two independent tests were performed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (S9-mix). With S9-mix cells were exposed for 24 hours. The test substance produced a reduction in mitotic activity (i.e., approximately 48%) at 10 µg/L in the absence of S9-mix and at 10 (i.e., 20%) and 50 (i.e., 37%) µg/L in the presence of S9-mix. Further, consideration of mean aberrant cell frequencies showed no real increases in culture tested with the test substance, when compared to control. Statistical analysis confirmed these observations. However, there was an apparent increase in the number of polyploid cells, compared to concurrent control values in all tested cultures, although a marked effect was seen only in activated cultures exposed to 50 µg/mL the author concluded that the test substance is not clastogenic in human lymphocytes under the experimental conditions. Under the conditions of the study, the test substance was not found to be clastrogenic in human lymphocytes (Richardson, 1989). Based on the results of the read across study, the test substance, C12 -14 TMAC, can also be considered to be non-clastogenic with and without metabolic activation in the chromosomal aberration assay.

Study 3:A study was conducted to determine the potential of the read across substance, Coco TMAC (active ingredient 35%), to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells. Study was performed accoding to OECD 476 Guideline, EU Method B.17 and Commission directive 2002/32/EC and U.K Environmental Mutagen Society, in compliance with GLP. After a preliminary toxicity test, 0.625, 1.25, 2.5, 5, 10 and 20 µg/L was selected as the test concentrations for the first experiment. The experiment was repeated to confirm the results of the first experiment. Three hour exposures were used both with and without S9-mix while for the second experiment, the exposure time was increased to 24 h. For the second experiment, the concentration range was 2.5 to 30 µg/L with S9-mix and 0.313 to 5 µg/L without S9-mix. The test substance did not induce a statistically significant or concentration-related increase in the mutant frequency at any concentration level, either with or without metabolic activation. Adequate levels of toxicity were achieved in all exposure groups. Under the conditions of the study, the test substance was not found to show mutagenic activity in mouse lymphoma assay (Nolan, 2002). Based on the results of the read across study, the test substance, C12 -14 TMAC can also be considered to be not mutagenic with and without metabolic activation in the mouse lymphoma assay.

Justification for classification or non-classification

Based on the available negative results from read across in vitro genotoxicity assays with Coco TMAC, it can be concluded that C12-14 TMAC does not require classification for genotoxicity according to EU CLP criteria (Regulation EC 1272/2008).