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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Cyclamen Aldehyde Extra: Ames - Negative

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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
Study period:
The experimental phase of this study was performed between 03 September 2002 and 26 September 2002.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
Dose range finding test 1: 3,10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Dose range finding test 2: 3, 10, 33, 100, 333, 1000,3330 and 5000 µg/plate

Main Test:
Based on the results of the dose range finding study the following dose range was selected for the mutation assay:

Experiment 1: 3, 10,33,100, 200 and 300 µg/plate.

Experiment 2: 100, 300, 600 and 1000 ~g/plate.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
dimethyl sulfoxide

- Justification for choice of solvent/vehicle:
Not specified in the report
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO = dimethyl sulfoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO = dimethyl sulfoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2.5 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO = dimethyl sulfoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates ofTA100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO = dimethyl sulfoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA102
Negative solvent / vehicle controls:
yes
Remarks:
DMSO = dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8 dihydroxy anthraquinone: 30 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix Migrated to IUCLID6: sodium azide: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 60 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9 mix Migrated to IUCLID6: Nirofluorene
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Saline
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Saline
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Daunomycin: 4 µg/plate
Remarks:
Without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 30 minutes
- Exposure duration: 48hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:
DATA EVALUATION AND STATISTICAL PROCEDURES

No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:

a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.

b) The negative response should be reproducible in at least one repeated experiment.

A test substance is considered positive (mutagenic) in the test if:

a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation.

However, any mean plate count of less than 20 is considered to be not significant.

b) The positive response should be reproducible in at least one repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Standard deviation
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
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose range finding test 1
CYCLAMEN ALDEHYDE EXTRA was tested in tester strain TA100 with concentrations of 3,10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix in the direct plate assay.

Precipitate:
The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation of CYCLAMEN ALDEHYDE EXTRA on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate.

Toxicity:
To determine the toxicity of CYCLAMEN ALDEHYDE EXTRA, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

A slight reduction in the number of revertant colonies was observed at the test substance concentration of 100 µg/plate in the presence of S9-mix only. An extreme reduction of the bacterial background lawn and an increase in the size of the microcolonies was observed at the test substance concentration of 333 µg/plate. A complete lack of any microcolony background lawn was observed at test substance concentrations 1000, 3330 and 5000 µg/plate.

Mutagenicity
No increase in the number of revertants was observed upon treatment with CYCLAMEN ALDEHYDE EXTRA under all conditions tested.

Dose range finding test 2
CYCLAMEN ALDEHYDE EXTRA was tested in tester strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000,3330 and 5000 µg/plate in the absence and presence of S9-mix in the preincubation assay.

Precipitate:
The test substance precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of CYCLAMEN ALDEHYDE EXTRA on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate.

Mutagenicity
No increase in the number of revertants was observed upon treatment with CYCLAMEN ALDEHYDE EXTRA under all conditions tested.

MUTATION ASSAY
Direct plate assay 1
Based on the results of the first dose range finding test the following dose range was selected for the mutation assay with the tester strains TA1535, TA1537, TA98 and TA102: 3, 10,33,100, 200 and 300 µg/plate.

Precipitate:
CYCLAMEN ALDEHYDE EXTRA precipitated in the top agar at concentrations of 200 and 300 µg/plate. Precipitation of CYCLAMEN ALDEHYDE EXTRA on the plates was not observed at the start or at the end of the incubation period at all dose levels tested.

Toxicity
In the presence of S9-mix, no reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed.

Mutagenicity
No increase in the number of revertants was observed upon treatment with CYCLAMEN ALDEHYDE EXTRA under all conditions tested.

Direct plate assay 2
Since not enough toxicity was observed in the tester strains TA1535, TA1537, TA98 and TA102 in the presence of S9-mix in the direct plate assay, a second mutation experiment was performed. The following dose range was selected for this mutation experiment: 100, 300, 600 and 1000 µg/plate.

Precipitate:
CYCLAMEN ALDEHYDE EXTRA precipitated in the top agar at concentrations of 300 µg/plate and upwards. Precipitation of CYCLAMEN ALDEHYDE EXTRA on the plates was not observed at the start or at the end of the incubation period.

Mutagenicity
No increase in the number of revertants was observed upon treatment with CYCLAMEN ALDEHYDE EXTRA under all conditions tested.

PRE-INCUBATION ASSAY
To obtain more information about the mutagenicity of CYCLAMEN ALDEHYDE EXTRA, a preincubation assay was performed with the strains TA1535, TA1537, TA98 and TA102. Based on the results of the dose range finding study the following dose range was selected for the mutation assay:

Without S9-mix: 1,3,10,33,100 and 333 µg/plate
With S9-mix: 3, 10, 33, 100, 333 and 1000 µg/plate

Precipitate:
CYCLAMEN ALDEHYDE EXTRA precipitated in the top agar at concentrations of 333 and 1000 µg/plate. Precipitation of CYCLAMEN ALDEHYDE EXTRA on the plates was only observed at the end of the incubation period at the concentration of 1000 µg/plate.

Mutagenicity:
No increase in the number of revertants was observed upon treatment with CYCLAMEN ALDEHYDE EXTRA under all conditions tested.

Conclusions:
Interpretation of results:negative.
Based on the results of this study it is concluded that CYCLAMEN ALDEHYDE EXTRA is not mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains.

The study procedures described in this report were based on the following guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (adopted July 21, 1997).

- European Economic Community (EEC). Adapting to technical progress for the twenty-sixth time Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.13/14: "Mutagenicity: "Reverse Mutation Assay using bacteria". EEC Publication Commission Directive (Brussels May 19, 2000).

CYCLAMEN ALDEHYDE EXTRA was tested in the Salmonella typhimurium reverse mutation assay with five histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102). The test was performed in two separate experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). To obtain more information about the mutagenicity of CYCLAMEN ALDEHYDE EXTRA, an additional experiment was performed with the strains TA1535, TA1537, TA98 and TA102 (presence of S9).

In the direct plate assay, at first CYCLAMEN ALDEHYDE EXTRA was tested in a dose range finding study up to concentrations of 5000 µg/plate in strain TA100. CYCLAMEN ALDEHYDE EXTRA precipitated on the plates at dose levels of 3330 and 5000 µg/plate. Toxicity was observed at dose levels of 333 µg/plate and upwards in the absence of S9-mix and at 100 µg/plate and upwards in the presence of S9-mix.

Secondly, CYCLAMEN ALDEHYDE EXTRA was tested up to concentrations of 300 µg/plate in the strains TA1535, TA1537, TA98 and TA102. CYCLAMEN ALDEHYDE EXTRA did not precipitate on the plates at this dose level. Toxicity was only observed in the absence of S9-mix.

In an additional direct plate assay, CYCLAMEN ALDEHYDE EXTRA was tested up to concentrations of 1000 µg/plate in the presence of S9-mix in the tester strains TA1535, TA1537 and TA98 and TA102. Toxicity was observed in all tester strains.

In the preincubation assay, at first CYCLAMEN ALDEHYDE EXTRA was tested in a dose range finding study up to concentrations of 5000 µg/plate in the strain TA100. CYCLAMEN ALDEHYDE EXTRA precipitated on the plates at dose levels of 3330 and 5000 µg/plate. Toxicity was observed at dose levels of 33 1J9/plate and upwards in the absence of S9-mix and at 100 µg/plate and upwards in the presence of S9-mix.

Subsequently, CYCLAMEN ALDEHYDE EXTRA was tested up to concentrations of 333 and 1000 µg/plate in the absence and presence of S9-mix, respectively in the strains TA1535, TA1537, TA98 and TA102. Toxicity was observed in all tester strains.

CYCLAMEN ALDEHYDE EXTRA did not induce a dose-related increase in the number of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA98, TA100 and TA102) both in the absence and presence of S9-metabolic activation. These results were confirmed in separate experiments.

Based on the results of this study it is concluded that CYCLAMEN ALDEHYDE EXTRA is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
not specified
Remarks:
study ongoing
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Following ECHA decision (CCH-D-2114361700-57-01/F) on3-p-cumenyl-2-methylpropionaldehyde, EC No 203-161-7, it was requested to conduct additional toxicological studies:
1. In vitro cytogenicity study in mammalian cells (Annex VIII, Section 8.4.2., test method: OECD TG 473) or in vitro micronucleus study (Annex VIII, Section 8.4.2, test method: OECD TG 487
2. In vitro gene mutation study in mammalian cells (Annex VIII, Section 8.4.3.; test method: OECD TG 476 or TG 490) with the registered substance provided that the study requested under 1. has negative results;
3. Sub-chronic toxicity study (90-day), oral route (Annex IX, Section 8.6.2.; test method: EU B.26./OECD TG 408) in rats with the registered substance;
4. Pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: EU B.31./OECD TG 414) in a first species (rat or rabbit), oral route with the registered substance;
5. Extended one-generation reproductive toxicity study (Annex IX, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance after providing the OECD 408 outcome.

The OECD 487, OECD 476 and OECD 414 are actually ongoing, the attached contract with the labs justify this fact. The studies will be submitted as soon as available.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Colorless to pale yellow liquid
Batch: VE00661246
Expiry date: 29 Aug 2021
Purity: 98,6%
Target gene:
HPRT locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Study ongoing
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
study ongoing
Metabolic activation:
with and without
Genotoxicity:
other: study ongoing
Remarks:
study ongoing
Additional information on results:
Study ongoing
Remarks on result:
other: Study ongoing
Conclusions:
Study ongoing
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
other: study ongoing
Remarks:
study ongoing
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Following ECHA decision (CCH-D-2114361700-57-01/F) on3-p-cumenyl-2-methylpropionaldehyde, EC No 203-161-7, it was requested to conduct additional toxicological studies:
1. In vitro cytogenicity study in mammalian cells (Annex VIII, Section 8.4.2., test method: OECD TG 473) or in vitro micronucleus study (Annex VIII, Section 8.4.2, test method: OECD TG 487
2. In vitro gene mutation study in mammalian cells (Annex VIII, Section 8.4.3.; test method: OECD TG 476 or TG 490) with the registered substance provided that the study requested under 1. has negative results;
3. Sub-chronic toxicity study (90-day), oral route (Annex IX, Section 8.6.2.; test method: EU B.26./OECD TG 408) in rats with the registered substance;
4. Pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: EU B.31./OECD TG 414) in a first species (rat or rabbit), oral route with the registered substance;
5. Extended one-generation reproductive toxicity study (Annex IX, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance after providing the OECD 408 outcome.

The OECD 487, OECD 476 and OECD 414 are actually ongoing, the attached contract with the labs justify this fact. The studies will be submitted as soon as available.
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
updated and adopted 29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Colorless to pale yellow liquid
Batch: VE00661246
Expiry date: 29 Aug 2021
Purity: 98,6%
Target gene:
In vitro Micronucleus in Mammalian cells
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Key result
Species / strain:
lymphocytes: Study ongoing
Remarks:
Study ongoing
Metabolic activation:
with and without
Genotoxicity:
other: test ongoing
Remarks:
Study ongoing
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Study ongoing
Conclusions:
Study ongoing
Executive summary:

Study ongoing

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

Genetic toxicity in vivo

Description of key information

There is no available in vivo genotoxicity study on the registered substance

Additional information

Justification for classification or non-classification

Based on the data available on Cyclamen Aldehyde, no classification for mutagenicity is necessary according to the (EC) No 1272/2008 Regulation (CLP).