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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Cyclamen Aldehyde Extra:

-Ames - Negative

-Micronucleus - Negative

-Gene mutation in mammalian cells - Negative

Link to relevant study records

Referenceopen allclose all

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 102
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not applicable
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
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
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.

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
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
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
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
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
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.

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

 

          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                                         

          3

          2

12 ±       1

121 ±     9

187 ±     27

33                                         

12 ±       6

          1             

13 ±       3             

108 ±     19

172 ±     12

100                                                       

11±        3

          2             

11 ±       2

100 ±     2

154 ±     13

200                                                      

          1

2 ±         2

8 ±         1

 

128 ±     9

300                                                       

          o'

M:: 2

M:: 2                      

 

104 ±     8

333                                       

 

 

 

M::: 2

 

1000

 

 

 

          o'3

 

3330 SP

 

 

 

          o'3

 

5000 SP

 

 

 

          o'3

 

With S9-mix

positive cx:ntml

 

solvent cx:ntml    

110 ±     15

 

13 ±       3

546 ±     51

 

          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

          2

17 ±       3

99 ±       9

246 ±     15

100        

12 ±       9

          5

15 ±       6

74 ±       3

189 ±     17

200                                                      

8 ±         2

          2

16 ±       1

 

186 ±     9

300        

11 ±       2

7 ±         1

15 ±       1

 

213 ±     14

333                       

 

 

 

M::: 2

 

1000

 

 

 

          o'3

 

3330 SP

 

 

 

          o'3

 

5000 SP

 

 

 

          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±        02

0±        02

M:: 1

122 ±   7

 Solvent control: 0.1 ml dimethyl sulfoxide

1           Bacterial background lawn extremelyreduced

2       Bacterial background lawnabsent

MC    Microcolonies

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:
experimental study
Adequacy of study:
key study
Study period:
06 Oct. 2020 - 16 Feb.2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
under GLP conditions
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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with β-Naphthoflavone/Phenobarbital. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Treatments were carried out both in the absence and presence of S-9 by addition of either 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test system was 1% (v/v).
Test concentrations with justification for top dose:
Test concentrations:
- without S9: 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 85 μg/mL
- with S9: 20, 40, 60, 80, 90, 100, 110, 120, 130, 140, 150, 175 μg/mL
The highest concentrations analysed were 65 μg/mL in the absence of S-9 and 90 μg/mL in the presence of S-9 due to the high cytotoxicity of the other highest doses.

Justification for top dose:
In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 2.344 to 300 μg/mL (limited by solubility of the formulated test article in culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested in the absence and presence of S-9 (75 to 300 μg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest two concentrations in the absence and presence of S-9 (150 and 300 μg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and the higher concentration was discarded. The highest concentrations to give >10% RS were 37.5 μg/mL in the absence of S-9 and 75 μg/mL in the presence of S-9, which gave 75% and 45% RS, respectively.
Vehicle / solvent:
The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).
Negative solvent / vehicle controls:
yes
Remarks:
Negative (vehicle) controls comprised treatments with the vehicle DMSO diluted 100-fold in the treatment medium
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Remarks:
100-fold dilution
Details on test system and experimental conditions:
Cytotoxicity Range-Finder Experiment
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment culture volume was 20 mL. Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10. Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air for 7 days. Wells containing viable clones were identified by eye using background illumination and counted.

Mutation Experiment
Treatment of Cell Cultures
At least 107 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL. After 3 hours in an incubator set to 37°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and, where sufficient cells survived, the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described. Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies (Brusick, 1986; Scott et al., 1991). Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.

Plating for Survival
Following adjustment of the cultures to 2 x 105 cells/mL after treatment, samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.

Expression Period
Cultures were maintained in flasks for a period of 7 days during which the hprt mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6TG resistance: without S9: 10-65 µg/mL; with S9: 20-90 µg/mL.

Plating for Viability
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for 6TG Resistance
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (14 days). Wells containing viable clones were identified by eye using background illumination and counted.
Rationale for test conditions:
The test conditions follow the requirements of OECD Guideline 476.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The Mutation Frequency (MF) at one or more concentrations was significantly greater than that of the vehicle control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical vehicle control database (mean
MF +/- 2 standard deviations).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
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
Positive controls validity:
valid
Additional information on results:
In the Mutation Experiment twelve concentrations, ranging from 10 to 85 μg/mL in the absence of S-9 and from 20 to 175 μg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 65 μg/mL in the absence of S-9 and 90 μg/mL in the presence of S-9, which gave 16% and 13% RS, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by one or both concentrations of the positive control chemicals 4-nitroquinoline 1-oxide (NQO) without S-9 and benzo(a)pyrene (B[a]P) with S-9. Therefore, the study was accepted as valid.

When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with Cyclamen Aldehyde Extra at any
concentration tested in the absence and presence of S-9 and there were no statistically significant linear trends.

A mean MF value which marginally exceeded the historical vehicle control range of 1.10 to 7.72 mutants/106 viable cells was observed in the absence of S-9 at a Cyclamen Aldehyde Extra concentration of 45 μg/mL (8.14 mutants/106 viable cells). This was acceptable as the MF value was not statistically significant by comparison with the mean vehicle control MF value (4.58 mutants/106 viable cells) and there was no statistically significant linear trend over the concentration range analysed. The data were considered indicative of a negative result under both treatment conditions.

Tables could be found in the attached study report

Conclusions:
It is concluded that tested substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.
Executive summary:

Tested substance was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol.

The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by a β-Naphthoflavone/Phenobarbital-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 2.344 to 300 μg/mL (limited by solubility of the formulated test article in culture medium). The highest concentrations to give >10% relative survival (RS) were 37.5 μg/mL in the absence of S-9 and 75 μg/mL in the presence of S-9, which gave 75% and 45% RS, respectively.

In the Mutation Experiment twelve concentrations, ranging from 10 to 85 μg/mL in the absence of S-9 and from 20 to 175 μg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 65 μg/mL in the absence of S-9 and 90 μg/mL in the presence of S-9, which gave 16% and 13% RS, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by one or both concentrations of the positive control chemicals 4-nitroquinoline 1-oxide (NQO) without S-9 and benzo(a)pyrene (B[a]P) with S-9. Therefore, the study was accepted as valid.

When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with Cyclamen Aldehyde Extra at any concentration tested in the absence and presence of S-9 and there were no statistically significant linear trends.

A mean MF value which marginally exceeded the historical vehicle control range of 1.10 to 7.72 mutants/106 viable cells was observed in the absence of S-9 at a Cyclamen Aldehyde Extra concentration of 45 μg/mL (8.14 mutants/106 viable cells).

This was acceptable as the MF value was not statistically significant by comparison with the mean vehicle control MF value (4.58 mutants/106 viable cells) and there was no statistically significant linear trend over the concentration range analysed. The data were considered indicative of a negative result under both treatment conditions.

It is concluded that tested substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2020 - August 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.
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 (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: Human peripheral blood lymphocytes obtained from a healthy non-smoking individual
Cytokinesis block (if used):
Cytochalasin B (cytoB) was dissolved in DMSO to a stock concentration of 2 mg/mL. It was used at 6 µg/mL concentration to block cytokinesis.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice.
Upon arrival at the testing laboratory, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the doses tested ranged from 0.19 to 1900 µg/mL (10 mM), which was the limit dose for this assay.
At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 570 µg/mL in all three exposure groups (4-hour with and without S9, 24-hour without S9). Based upon these results, the doses chosen for the micronucleus assay ranged from 26.7 to 160 µg/mL for the non-activated 4-hour exposure group, from 52.2 to 250 µg/mL for the S9-activated 4-hour exposure group, and from 13.1 to 65.3 µg/mL for the non-activated 24-hour exposure group.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO) was used as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
vinblastine
Details on test system and experimental conditions:
Test system: human peripheral blood lymphocytes (HPBL) were obtained from a healthy non-smoking individual. The donor had no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals.

No. of Replicate Cultures: 2

Frequency and route of administration: target cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9, by incorporation of the test substance vehicle mixture into the treatment medium.

Preliminary Toxicity Test for Selection of Dose Levels: HPBL were exposed to vehicle alone and to nine concentrations of test substance with halflog dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose, lowest precipitating dose, and the highest soluble dose was measured. Dose levels for the micronucleus assay were based upon post-treatment toxicity (CBPI relative to the vehicle control).

Micronucleus Assay: based on the results of the preliminary toxicity test, the doses selected for testing in the micronucleus assay were as follows:
-26.7 to 160 µg/mL for the non-activated 4-hour exposure group
-52.2 to 250 µg/mL for the S9-activated 4-hour exposure group,
-13.1 to 65.3 µg/mL for the non-activated 24-hour exposure group.
Precipitation of the test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose evaluated for the micronuclei was selected based on the following: 55 ± 5% cytotoxicity (CBPI relative to the vehicle control). Two additional doses were included in the evaluation of micronuclei.
Rationale for test conditions:
Test conditions follow the OECD guideline 487.
Evaluation criteria:
The test substance was considered to have induced a positive response if
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity (≥ 55 ± 5% CBPI relative to the vehicle control) at doses ≥ 102 µg/mL in the non-activated 4-h exposure groups, at doses ≥ 128 µg/mL in the S9-activated 4-h exposure group; and at doses ≥ 41.8 µg/mL in the non-activated 24-h exposure group.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within de the 95% control limit of the historical negative control data.

Tables could be found in the attachement report

Conclusions:
Under the conditions of the assay described in this report, Cyclamen Aldehyde was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.
Executive summary:

The test substance, Cyclamen Aldehyde, was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.19 to 1900 µg/mL (10 mM), which was the limit dose for this assay. Cytotoxicity [≥ 55 ± 5% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 190 µg/mL in the non-activated and S9-activated 4-hour exposure groups, and at doses ≥ 57 µg/mL in the non-activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 570 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 26.7 to 160 µg/mL for the non-activated 4-hour exposure group, from 52.2 to 250 µg/mL for the S9-activated 4-hour exposure group, and from 13.1 to 65.3 µg/mL for the non-activated 24-hour exposure group.

In the micronucleus assay, cytotoxicity (≥ 55 ± 5% CBPI relative to the vehicle control) was observed at doses ≥ 102 µg/mL in the non-activated 4-hour exposure groups, at doses ≥ 128 µg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 41.8 µg/mL in the non-activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was not observed at any dose in any of the three exposure groups.

The doses selected for evaluation of micronuclei were 52.2, 81.6, and 102 µg/mL for the non-activated 4-hour exposure group; 52.2, 102, and 128 µg/mL for the S9-activated 4-hour exposure group; and 13.1, 33.4, and 41.8 µg/mL for the non-activated 24-hour exposure group.

Neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within de the 95% control limit of the historical negative control data.

These results indicate Cyclamen Aldehyde was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

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).