3-p-cumenyl-2-methylpropionaldehyde

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

3-p-cumenyl-2-methylpropionaldehyde

EC Number:

203-161-7

EC Name:

3-p-cumenyl-2-methylpropionaldehyde

Cas Number:

103-95-7

Molecular formula:

C13H18O

IUPAC Name:

2-methyl-3-[4-(propan-2-yl)phenyl]propanal

Constituent 2

Reference substance name:

3-p-cumenyl-2methylpropionaldehyde

IUPAC Name:

3-p-cumenyl-2methylpropionaldehyde

Test material form:

other: liquid

Details on test material:

Sponsor's identification: CYCLAMEN ALDEHYDE EXTRA
Description: Colourless to pale yellow liquid

Purity: 95.5 %
Stability in vehicle: Dimethyl sulfoxide: Not indicated
Expiry date: 24 July 2003
Storage conditions: room temperature in the dark
Stability under storage conditions: Stable

Method

Target gene:

Histidine for Salmonella typhimurium

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

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

Controls

Controls 1

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:

Remarks:

With S9 mix

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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

Controls 6

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

Controls 7

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

Controls 8

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

Controls 9

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

Controls 10

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.

Rationale for test conditions:

following guideline OECD 471

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

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

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

True negative controls validity:

not applicable

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.

Any other information on results incl. tables

MUTAGENIC RESPONSE OFCYCLAMEN ALDEHYDE EXTRAINTHE

SALMONELLA TYPHIMURIUMREVERSE MUTATION

 

Direct plate assay 1Day of performance:

TA100: 04 September 2002

TA1535, TA1537, TA98 and TA102: 11 September 2002

  

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with  different strains ofSalmonellatyphimurium
	TA1535	TA1537	TA98	TA100	TA102
	Without S9-mix
positive cx:ntml    solvent cx:ntml	895 ±     110   17 ±       3	141 ±     22   6±          0	753 ±     124   17 ±       2	433 ±     54   101 ±     12	437 ±     71   209 ±     28
3	12 ± 3	5 ± 4	14 ±       8	114 ±     15	225 ±     31
10	9±          3	6±          2	12 ±       1	121 ±     9	187 ±     27
33	12 ±       6	6±          1	13 ±       3	108 ±     19	172 ±     12
100	11±        3	5±          2	11 ±       2	100 ±     2	154 ±     13
200	4±          1	2 ±         2	8 ±         1		128 ±     9
300	o±          o'	M:: 2	M:: 2		104 ±     8
333				M::: 2
1000				o±          o'3
3330 SP				o±          o'3
5000 SP				o±          o'3
With S9-mix
positive cx:ntml   solvent cx:ntml	110 ±     15   13 ±       3	546 ±     51   5±          1	971 ±     49   22 ±       7	454 ±     23   117 ±     19	603 ±     38   213 ±     14
3	15 ±      1	8 ±         3	22 ±       3	98 ±       11	289 ±     92
10	15 ±      4	4 ±         2	18 ±       2	94 ±       7	247 ±     7
33	11 ±       3	6±          2	17 ±       3	99 ±       9	246 ±     15
100	12 ±       9	9±          5	15 ±       6	74 ±       3	189 ±     17
200	8 ±         2	2±          2	16 ±       1		186 ±     9
300	11 ±       2	7 ±         1	15 ±       1		213 ±     14
333				M::: 2
1000				o±          o'3
3330 SP				o±          o'3
5000 SP				o±          o'3

  

Solvent control: 0.1 ml dimethyl sulfoxide

1            Bacterial background lawn slightlyreduced

2       Bacterial background lawn extremelyreduced

3       Bacterial background lawnabsent

SP     SlightPrecipitate

MC   Microcolonies

MUTAGENIC RESPONSE OFCYCLAMEN ALDEHYDE EXTRAINTHE

SALMONELLA TYPHIMUR/UMREVERSE MUTATION

  

Direct plate assay 2Day of performance:

TA1535, TA1537, TA98 and TA102: 24 September 2002

 

Dose(µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) withdifferent strains ofSalmonellatyphimurium
	TA1535	TA15 37	TA98	TA102
positiveoc:ntrol	176 ±   16	541 ±   25	1094 ± 49	918 ±   38
solventa:ntrcl	12 ±     1	8±        2	24 ±     6	323 ±   24
	With S9-mix
100	10 ±     1	5± 2	19 ± 3	239 ±   28
300	8±        3	5±        4	21 ±     3	239 ±   54
600	M:: 1	M:: 1	M:: 1	210 ±   13
1000	0±        0^2	0±        0^2	M:: 1	122 ±   7

 Solvent control: 0.1 ml dimethyl sulfoxide

1           Bacterial background lawn extremelyreduced

2       Bacterial background lawnabsent

MC    Microcolonies

Applicant's summary and conclusion

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.