Heptanal, 2-(phenylmethylene)-, (2E)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay: Negative (Mutagenic); OECD 471; Fujita& Sasaki 1987, Eder 1993 & Wild 1983

Mouse Lymphoma Assay: Negative; OECD 476; Wild 1983

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published summary of various test material results, omitting some details of specific methods and results that would be present for a standard test guideline compliant study report.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Ames test methods using two tester strains were used - TA97 and TA102. Current guidelines require five tester strains, normally including S. typhimurium and E.Coli

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

The tester strains S.typhimurium TA97 and TA 102 were used.
Tester strain TA 102 has an AT base pair at the primary reversion site and is DNA repair-proficient. It may detect certain oxidizing mutagens, hydrazines and cross-linking agents.
All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrE gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens

Species / strain / cell type:

S. typhimurium, other: TA97 and TA102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction

Test concentrations with justification for top dose:

0, 0.001, 0.005, 0.01, 0.5 and 1.0 mg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

Standard Ames test according to the pre-incubation procedure.

Evaluation criteria:

No information

Statistics:

Kruskal-Wallis analysis

Species / strain:

S. typhimurium, other: TA97 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Non mutagenic in the two tester strains

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Under the conditions of this assay amyl cinnamic aldehyde was not found to be mutagenic in the tester strains.

Conclusions:

Interpretation of results (migrated information):
negative

Amyl cinnamic aldehyde was not mutagenic in the bacterial tester strains used in this study.

Executive summary:

The mutagenicity of 29 food additives including 8 dietary supplements, 13 flavoring agents. 5 bleaching agents and 3 oxidizing agents were examined in an Ames test using the tester strains, Salmonella typhimurium TA 97 and TA 102. The mutation test was carried out by the preincubation procedure described by Ames et al. The test chemicals were preincubated with S9 mix or phosphate buffer (pH 7.4) for 20 min. Potassium bromate showed weak mutagenic activity in TA97 with S9.

The other 28 chemicals, including amyl cinnamic aldehyde, were not mutagenic in the tester strains. Amyl cinnamic aldehyde was not mutagenic in the tester strains.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

no information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The experiment was done according to the standard method in vitro plate incorporation assay according to the methods of Ames. However, in the article there is no mention of GLP status and positive control.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

The tester strains S.typhimurium TA98 , TA100, TA1535, TA1537 and TA1538 were used.

All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrE gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens

Test concentrations with justification for top dose:

up to 3.6 mg/plate

Vehicle / solvent:

DMSO or water

Remarks:

Over 2 years the numbers of revertants/plate in positive controls were in the following ranges: sodium azide at 0.5 µg/plate, 430-760 in TA 1535, 400-700 in TA100; For benzo[a )pyrene at 5 µg/plate, 865-1210 in TA IOO, 235-350 in TA1537, 410-590 in TA1538

Details on test system and experimental conditions:

No information

Evaluation criteria:

no information

Statistics:

no information

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:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

amylcinnamaldehyde was not found to be mutagenic in the Ames test

Executive summary:

Ames tests. The standard plate procedure was followed (Ames, McCann & Yamasaki, 1975). Overnight bacterial cultures had cell. titres of at least 10E9 cells/ml. S-9 liver fractions were prepared from Aroclor-pretreated rats (Aroclor 1254, 500 mg/kg ip) and adjusted to 25 mg protein/ml; 0.5 ml S-9 mix, equivalent to 50111 S·9, was incorporated inlo the plates. Dimethylsulphoxide was used as solvent for test chemicals that were poorly soluble in water.

Five doses of each substance were tested (for non-toxic compounds, up to 3.6 mg/plate) in all five tester strains with and without S·9 mix. Vogel-Bonner medium (Vogel & Bonner, 1956) was used throughout; plates were incubated for 48 hr.

Positive controls were run in each experiment with the reference mutagens sodium azide and benzo[a)pyrene.

Over a period of 2 yr, the numbers of revertants/plate in positive controls were in the following ranges: with sodium azide, at 0.5 µg/plate, 430-760 in TA 1535, 400-700 in TA100; with benzo[a]pyrene. at 5 µg/plate, 865 -1210 in TA100, 235 -350 in TA1537, 410 -590 in TA1538, 660 -1000 in TA98. All chemicals were tested at least twice. Additonally, several flavouring substances were subjected to the test following liquid preincubation (20 min at 37'C) as recommended by Yahagi, Degawa, Seino el al. (1975).

Reference 3

Endpoint:

genetic toxicity in vitro, other

Remarks:

Type of genotoxicity: other: assessment of possible mutagenesis and carcinogenesis effects

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not stated . Publication dated 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published peer-reviewed study results

Qualifier:

no guideline followed

Principles of method if other than guideline:

a, ß-Unsaturated carbonyl compounds are industrially important compounds, ubiquitous in the environment and are formed endogenously. They interact with proteins and enzymes. Genotoxicity was found in eukaryotic cells and some compounds were carcinogenic. Unsaturated carbonyl compounds are considered to play an important role in human cancer. Insufficient and contradictory results were reported on mutagenicity.
The publication demonstrated a clear mutagenic potential for these compounds and have shown interference of their bacterial toxicity with an adequate testing. Structure-mutagenicity relationships were confirmed by the results of the SOS-chromotest. The compounds induce DNA-strand breaks. However, it did not find indications for cross linking. With mutagenic a, ß-unsaturated carbonyl compounds we isolated and characterized I,N 2-cycic deoxyguanosine adducts, 7,8-cyclic and 7-linear guanine adducts as well as I,N2- 7,8-biscyclic adducts and I,N2-cyclic, 7 linear bisadducts. Reactivity of these compounds towards nucleosides runs in parallel with their mutagenic potential. Mutagenic and carcinogenic activities most probably depend on these reactions with DNA, and DNA adducts can be utilized as indicators for the role of these compounds in human carcinogenicity.

GLP compliance:

not specified

Type of assay:

other: various assessments of genotoxicity

Target gene:

The tests included mutagenicity investigations using Salmonella typhimurium strain TA 100, according to Maron and Ames method, with the normal histidine dependency.

SOS chromotest - a second assay used was the Quillardet and Hofnung method using Escherichia coli strain PQ37 including rfa and uvrA mutations. The sfi A gene-linked ß-galactosidase activity is determined in this assay as a measure of the induction of the SOS repair system.

The activity of alkaline phosphatase was used as a measure of protein synthesis and of toxicity.

An assay to detect single strand breaks - alkaline elution assay (Kohn et al) - used 1210 mouse leukeamia cells (L1210 cells) . The F-factor derived in this assay is a measure of the effectivity of the compound to induce strand breaks.

Cytotoxicity was measured using the Trypan Blue test in which damaged cell membranes allow cell to take up Trypan Blue stain.

Binding studies - reaction of test substances with nucleosides and nucleotides and chromatographic isolation of the adducts.

Species / strain / cell type:

other: various strains and species used for a battery of tests

Details on mammalian cell type (if applicable):

L1210 cells used in one of the tests

Additional strain / cell type characteristics:

other: specific strain charcteristics for each test, where available are detailed under "target gene"

Metabolic activation:

with

Metabolic activation system:

S-9 prepared ffrom livers of Aroclor 1254 induced rats for the Ames test

Test concentrations with justification for top dose:

No details typically given for the various test substances in individual tests described in this publication

Vehicle / solvent:

DMSO used in Ames test and SOS chromotest and in alkaline elution test in combination with phosphate buffered saline.

Details on test system and experimental conditions:

The tests included mutagenicity investigations using Salmonella typhimurium strain TA 100, according to Maron and Ames method, with the normal histidine dependency.

SOS chromotest - a second assay used was the Quillardet and Hofnung method using Escherichia coli strain PQ37 including rfa and uvrA mutations. The sfi A gene-linked ß-galactosidase activity is determined in this assay as a measure of the induction of the SOS repair system.

The activity of alkaline phosphatase was used as a measure of protein synthesis and of toxicity.

An assay to detect single strand breaks - alkaline elution assay (Kohn et al) - used 1210 mouse leukeamia cells (L1210 cells) . The F-factor derived in this assay is a measure of the effectivity of the compound to induce strand breaks.

Cytotoxicity was measured using the Trypan Blue test in which damaged cell membranes allow cell to take up Trypan Blue stain.

Binding studies - reaction of test substances with nucleosides and nucleotides and chromatographic isolation of the adducts.

Evaluation criteria:

No details

Statistics:

No details

Species / strain:

other: various tests for mutagenicity

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

Bacterial mutagenicity and genotoxicity was demonstrated in S.typhimurium strain TA100 for the unsaturated carbonyl compound class. The following structure activity relationship were derived:
alkenal mutagenicity decreases with increasing molecule size
2-chloroacrolein and 2-bromoacrolein were found to be extremely strong mutagens
mutagenic activity tends to reduce in presence of S-9
cell toxicity for alkylsubstituted acroleins increases with chain length.

High bacterial toxicity was found to interfere with the SOS chromotest in the E.coli strain PQ37.
Cinnamaldehyde was not positive in the SOS chromotest

THe results from these investigations do not conclusively show the extent of mutagenicity potential for cinnamic aldehyde (results are presented primarily for other unsaturated carbonyl compounds and suggest cinnamicaldehyde is at the lower end of the genotoxic scale for this family of substances). No positive mutagenicity conclusions can be drawn for amyl cinnamic aldehyde
All of the test compounds induced strand breaks in the alkaline elution assay, but at relatively high doses (500 µmol for cinnamaldehyde but not at 250 or 300 µmol) at which cytotoxicity also occurs.

DNA adducts - the interactions of most of the mutagenic a,ß-unsaturated carbonyl compounds with DNA components were investigated. Two isolated regioisomeres of 1,N2-cyclic adducts were identified and most of the test compounds formed one or other of these adducts.
Cinnamicaldehyde is not listed in the publication as forming these adducts.

Remarks on result:

other: other: Ames and SOS-Chromotest and additional investigations

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
other: most of the unsaturated carbonyl compounds tested were demonstrated to be mutagenic

Unsaturated carbonyl compounds are considered to play an important role in human cancer. Insufficient and contradictory results were reported on mutagenicity. A clear mutagenic potential was demonstrated for these compounds and interference of their bacterial toxicity has been demonstrated with adequate testing. Structure-mutagenicity relationships were confirmed by the results of the SOS-chromotest. The compounds induce DNA-strand breaks but there were no indications for cross linking.

Executive summary:

a, ß-Unsaturated carbonyl compounds are industrially important compounds, ubiquitous in the environment and are formed endogenously. They interact with proteins and enzymes. Genotoxicity was found in eukaryotic cells and some compounds were carcinogenic. Unsaturated carbonyl compounds are considered to play an important role in human cancer. Insufficient and contradictory results were reported on mutagenicity. A clear mutagenic potential was demonstrated for these compounds and interference of their bacterial toxicity has been demonstrated with adequate testing. Structure-mutagenicity relationships were confirmed by the results of the SOS-chromotest. The compounds induce DNA-strand breaks but there were no indications for cross linking.

For the mutagenic a, ß-unsaturated carbonyl compounds the authors isolated and characterised 1,N²-cyclic deoxyguanosine adducts; 7,8-cyclic and 7-linear guanine adducts as well as 1,N² -7,8 -biscyclic adducts and 1,N²-cyclic, 7 -linear bis adducts. Reactivity of these compounds towards nucleosides runs in parallel with their mutagenic potential. Mutagenic and carcinogenic activities most probably depend on these reactions with DNA, and DNA adducts can be utilized as indicators for the role of these compounds in human carcinogenicity.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The protocol was approved by Huntingdon Life Sciences Management and by the Study Director on 15 February 2010 and by the Sponsor on 18 February 2010. Experimental start date: 22 February 2010. Experimental completion date: 12 April 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with OECD Guidelines for Testing of Chemicals No. 476: "Genetic toxicology: In vitro mammalian cell gene mutation tests", 1997 under GLP condition.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y mouse lymphoma (3.7.2c) cells (Clive and Spector, 1975), were obtained from American Type Culture Collection (ATCC), Virginia. These cells are heterozygous at the thymidine kinase locus, TK +/-. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock. Cell stocks are periodically checked for freedom from mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

rat S9

Test concentrations with justification for top dose:

Preliminary toxicity test:
1.6, 3.2, 6.3, 12.7, 25.4, 50.7, 101.4, 202.8*, 405.7* and 811.3* μg/mL (*precipitates seen by eye)

Mutation tests:
-S9 mix Test 1 (3 hours) 5, 10, 15, 20, 25, 30, 32.5, 35, 37.5, 40 and 45 μg/mL
+S9 mix Test 1 (3 hours) 5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 μg/mL
-S9 mix Test 2 (24 hours) 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL

Vehicle / solvent:

dimethyl sulphoxide (DMSO)

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide (DMSO)

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: in the presence of S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide (DMSO)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: in the absence of S9 mix

Details on test system and experimental conditions:

Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix.
For 3 hour exposures, cultures contained a total of 6 x 10^6 cells. The final volume of the cultures was 5 mL and the final concentration of the S9 fraction was 2% v/v, if present. For 24 hour exposures, cultures contained a total of 1.5 x 10^6 cells in a total volume of 5 mL.
One culture was prepared for each concentration of the test substance for each test condition. Vehicle controls were tested in duplicate for each test condition. The test substance was formulated and serially diluted in the solvent. Aliquots of 50 μL of test substance dilution (at 100 times the desired final concentration) or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 37°C) or 24 hours (static
humidified incubator, at 37°C, 5% (v/v) CO2). At the end of the 3 hour exposure period, the cells were washed once, resuspended in R10p to nominally 2 x 10^5 cells/mL (assuming no cell loss), incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. At the end of the 24 hour exposure period, the cells were washed once, resuspended in 5 mL R10p and counted, to ascertain treatment growth. The cultures were then diluted to 2 x 10^5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. The RSG was used to determine the concentrations of test substance used in the main test; ideally the maximum concentration should reduce RTG to approximately 10 to 20% of the concurrent vehicle control value. There was evidence of toxicity in the preliminary toxicity test, so the maximum concentrations tested in the 3 hour exposure in the absence and presence of S9 mix were 45 and 30 μg/mL respectively, and in the 24 hour exposure in the absence of S9 mix was 50 μg/mL. The formulations being added at 1% final volume in medium.

The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 10^7 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls Aliquots of 100 μL of test substance dilution (at 100 times the desired final concentration), vehicle or positive control were added, then all cultures were incubated, with continuous shaking, for 3 hours at 37ºC. At least four serial dilutions of the test substance were tested.
Following the 3 hour exposure, the cells were washed once, resuspended in R10p to nominally 2 x 10^5 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 10^5 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 10^3 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air.
After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates, the number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF).
The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The maximum concentrations assessed for mutant frequency in the first main test were 30 and 27.5 μg/mL in the absence and presence of S9 mix respectively.

A second test was carried out, with a 24 hour exposure in the absence of S9 mix. Duplicate 10 mL cultures containing 3 x 10^6 cells were treated for 24 hours with 100 μL of test substance, solvent or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, resuspended in 10 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to
2 x 10^5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p, the intention being to retain at least 1 x 10^7 cells. Following this, the procedure was the same as in the 3 hour treatment. The maximum concentration assessed for mutant frequency in the second main test was 45 μg/mL.

Evaluation criteria:

The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10^-6 (Moore et al., 2006).
The assay was considered valid in accordance with the assay acceptance criteria.

The test agent was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.
If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p < 0.001. Statistics are only reported if the Global Evaluation Factor is exceeded, and this was accompanied by a significant positive linear trend.

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:

Precipitate (observed by eye at the end of treatment) was observed at concentrations of 202.8 μg/mL and greater in the absence and presence of S9 mix, following a 3 hour exposure. Exposure to Hexyl Cinnamic Aldehyde at concentrations from 1.6 to 811.3 μg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values from 105 to 0% and from 97 to 0% respectively.
Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed at any concentration tested. Exposure to concentrations from 1.6 to 811.3 μg/mL resulted in RSG values from 107 to 0%. Concentrations used in the main test were based upon these data.

Cultures were exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 45 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 30 μg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 94 to 22% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. Although this assay did not reach the required 10-20% RTG it is considered valid in accordance with Moore et. al (2002) which states that a chemical can be considered nonmutagenic when there is no culture showing an RTG value between 10-20% if there is no evidence of mutagenicity (e.g., no dose response or mutant frequencies above those seen in the historical background ranges) in a series of data points within 100% to 20% RTG and there is at least one negative data point between 20% and 25% RTG. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Cultures were exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 30 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 27.5 μg/mL were assessed for determination of mutation frequency. RTG values from 93 to 18% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
The results obtained in response to the exposure of cultures to Hexyl Cinnamic Aldehyde in the presence of S9 mix did not demonstrate mutagenic potential. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the historical/acceptable solvent control values and there were no clear increases in the mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05). Therefore it was considered not to be beneficial to perform a direct repeat of the assay.

Cultures were exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 50 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 45 μg/mL were assessed for determination of mutation frequency. RTG values from 92 to 18% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test
concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Main Mutation Test 1 -3 Hour Treatment in the Absence of S9 Mix

Treatment/Concentration (μg/mL)	Replicate	Cell Concentration (x10^5/mL) 24 h	Cell Concentration (x10^5/mL) 48 h	Viability Plate Count^a Day 2	Mutant Plate Count^a Day 2	Mean RTG (%)	Mean MF (x10^4)
Vehicle Control ^b	A	5.22	13.48	42 (192)	165 (192)	100	79
	B	5.63	14.54	36 (192)	160 (192)
	C	5.24	15.12	43 (192)	166 (192)
	D	5.46	16.15	38 (192)	166 (192)
Hexyl Cinnamic Aldehyde 5	A	5.09	15.66	42 (192)	170 (192)	94	73
	B	5.50	13.48	41 (192)	164 (192)
Hexyl Cinnamic Aldehyde 15	A	4.10	13.64	45 (192)	158 (192)	65	98
	B	4.18	14.25	48 (192)	165 (192)
Hexyl Cinnamic Aldehyde 20	A	2.95	10.99	35 (192)	151 (192)	45	101
	B	2.96	10.67	30 (192)	156 (192)
Hexyl Cinnamic Aldehyde 25	A	3.01	10.58	33 (192)	149 (192)	37	108
	B	2.69	8.55	37 (192)	156 (192)
Hexyl Cinnamic Aldehyde 30	A	2.69	8.33	39 (192)	147 (192)	22	156
	B	2.27	5.32	41 (192)	136 (192)
MMS 10	A	3.64	11.41	53 (192)	45 (192)	51	937
	B	4.45	13.05	54 (192)	41 (192)

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Table 2. Main Mutation Test 1 - 3 Hour Treatment in the Absence of S9 Mix, Colony Size Analysis

Treatment/Concentration (μg/mL)	Replicate	Mutant Plate Count ^a Day 2	Total Mutant Colonies	Large Mutant Colonies	Small Mutant Colonies	% Small Mutant Colonies	Mean % Small Mutant Colonies
Vehicle Control ^b	A	165 (192)	27	13	14	52	53
	B	160 (192)	34	16	18	53
	C	166 (192)	30	15	15	50
	D	166 (192)	26	11	15	58
MMS 10	A	45 (192)	209	31	178	85	83
	B	41 (192)	215	41	174	81

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Table 3. Main Mutation Test 1 - 3 Hour Treatment in the Presence of S9 Mix

	Replicate	Cell Concentration (x10^5/mL) 24 h	Cell Concentration (x10^5/mL) 48 h	Viability Plate Count^a Day 2	Mutant Plate Count^a Day 2	Mean RTG (%)	Mean MF (x10^4)
Vehicle Control^b	A	4.30	17.21	46 (192)	166 (192)	100	72
	B	5.30	13.18	36 (192)	169 (192)
	C	3.90	13.06	33 (192)	168 (192)
	D	4.82	13.62	41 (192)	163 (192)
Hexyl Cinnamic Aldehyde 5	A	4.73	12.81	43 (192)	163 (192)	93	75
	B	5.00	11.91	34 (192)	167 (192)
Hexyl Cinnamic Aldehyde 10	A	4.18	12.65	37 (192)	168 (192)	75	64
	B	3.67	12.49	43 (192)	171 (192)
Hexyl Cinnamic Aldehyde 12.5	A	3.00	11.87	43 (192)	166 (192)	59	77
	B	3.27	11.70	32 (192)	162 (192)
Hexyl Cinnamic Aldehyde 17.5	A	1.76 ^c	8.23	52 (192)	155 (192)	21	126
	B	1.75 ^c	8.43	55 (192)	159 (192)
Hexyl Cinnamic Aldehyde 25	A	1.49 ^c	7.24	48 (192)	162 (192)	22	92
	B	1.30 ^c	7.26	35 (192)	160 (192)
Hexyl Cinnamic Aldehyde 27.5	A	1.03 ^c	5.52	38 (192)	156 (192)	18	112
	B	1.02 ^c	4.55	23 (192)	141 (192)
BaP 1	A	4.42	12.10	30 (192)	60 (192)	86	558
	B	4.26	11.78	39 (192)	56 (192)

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)


c. Cell concentration not adjusted due to insufficient growth

BaP - Benzo[a]pyrene

Table 4. Main Mutation Test 1 - 3 Hour Treatment in the Presence of S9 Mix, Colony Size Analysis

Treatment/Concentration (μg/mL)	Replicate	Mutant Plate Count^a Day 2	Total Mutant Colonies	Large Mutant Colonies	Small Mutant Colonies	% Small Mutant Colonies	Mean % Small Mutant Colonies
Vehicle Control^b	A	166  (192)	26	8	18	69	61
	B	169  (192)	23	10	13	57
	C	168  (192)	24	10	14	58
	D	163  (192)	34	13	21	62
BaP 1	A	60 (192)	166	33	133	80	81
	B	56 (192)	159	29	130	82

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

BaP - Benzo[a]pyrene

Table 5. Main Mutation Test 2 - 24 Hour Treatment in the Absence of S9 Mix

Treatment/Concentration (μg/mL)	Replicate	Cell Concentration (x10^5/mL) 04 h	Cell Concentration (x10^5/mL) 24 h	Cell Concentration (x10^5/mL) 48 h	Viability Plate Count^a Day 2	Mutant Plate Count^a Day 2	Mean RTG (%)	Mean MF (x10^4)
Vehicle Control^b	A	16.60	6.85	10.91	44 (192)	170 (192)	100	59
	B	15.03	6.79	11.79	45 (192)	174 (192)
	C	14.54	6.72	11.02	40 (192)	169 (192)
	D	15.69	7.46	12.06	33 (192)	172 (192)
Hexyl Cinnamic Aldehyde 5	A	14.50	6.65	10.96	40 (192)	169 (192)	92	69
	B	14.74	5.78	12.73	34 (192)	164 (192)
Hexyl Cinnamic Aldehyde 15	A	13.16	5.82	11.87	35 (192)	167 (192)	81	70
	B	13.25	6.70	10.06	33 (192)	163 (192)
Hexyl Cinnamic Aldehyde 20	A	8.91	7.53	9.34	27 (192)	168 (192)	63	52
	B	9.17	6.70	10.86	32 (192)	172 (192)
Hexyl Cinnamic Aldehyde 25	A	6.05	4.59	11.07	38 (192)	166 (192)	27	63
	B	5.89	4.50	10.59	30 (192)	169 (192)
Hexyl Cinnamic Aldehyde 30	A	5.11	3.21	10.90	36 (192)	166 (192)	18	81
	B	5.28	3.86	9.36	26 (192)	153 (192)
MMS 10	A	12.38	7.51	7.12	47 (192)	45 (192)	48	938
	B	12.54	6.94	8.50	57 (192)	38 (192)

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Table 6. Main Mutation Test 2 - 24 Hour Treatment in the Absence of S9 Mix, Colony Size Analysis

Treatment/Concentration (μg/mL)	Replicate	Mutant Plate Count^a Day 2	Total Mutant Colonies	Large Mutant Colonies	Small Mutant Colonies	% Small Mutant Colonies	Mean % Small Mutant Colonies
Vehicle Control^b	A	170 (192)	22	8	14	64	52
	B	174 (192)	18	9	9	50
	C	169 (192)	23	12	11	48
	D	172 (192)	20	11	9	45
MMS 5	A	45 (192)	219	29	190	87	86
	B	38 (192)	232	35	197	85

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that Hexyl Cinnamic Aldehyde did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

Hexyl Cinnamic Aldehyde was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). The study consisted of a preliminary toxicity test and two main tests comprising three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. Hexyl Cinnamic Aldehyde was found to be soluble at 216.33 mg/mL in Dimethyl Sulphoxide. A final concentration of 811.3 μg/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the maximum concentration that does not cause a change in osmolality of greater than 50 mOsm/kg. Toxicity was observed in the preliminary toxicity test. Following a 3 hour exposure to Hexyl Cinnamic Aldehyde at concentrations from 1.6 to 811.3 μg/mL, relative suspension growth (RSG) was reduced from 105 to 0% and from 97 to 0% in the absence and presence of S9 mix respectively. Following a 24 hour exposure in the absence of S9 mix RSG was reduced from 107 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG). Following 3 hour treatment in the absence and presence of S9 mix, there were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix were 30 and 27.5 μg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 22 and 18% respectively. In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 45 μg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed at concentrations up to 45 μg/mL, where RTG was reduced to 18%. In all tests the concurrent vehicle and positive controls were within the acceptable historical control ranges. It was concluded that Hexyl Cinnamic Aldehyde did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The protocol was approved by Huntingdon Life Sciences Management and by the Study Director on 15 February 2010 and by the Sponsor on 18 February 2010. Experimental start date: 22 February 2010. Experimental completion date: 12 April 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with OECD Guidelines for Testing of Chemicals No. 476: "Genetic toxicology: In vitro mammalian cell gene mutation tests", 1997 under GLP condition.

Justification for type of information:

Refer to Section 13 for read across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y mouse lymphoma (3.7.2c) cells (Clive and Spector, 1975), were obtained from American Type Culture Collection (ATCC), Virginia. These cells are heterozygous at the thymidine kinase locus, TK +/-. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock. Cell stocks are periodically checked for freedom from mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

rat S9

Test concentrations with justification for top dose:

Preliminary toxicity test:
1.6, 3.2, 6.3, 12.7, 25.4, 50.7, 101.4, 202.8, 405.7 and 811.3 μg/mL

Mutation tests:
-S9 mix Test 1 (3 hours) 5, 10, 15, 20, 25, 30, 32.5, 35, 37.5, 40 and 45 μg/mL
+S9 mix Test 1 (3 hours) 5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 μg/mL
-S9 mix Test 2 (24 hours) 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL

Vehicle / solvent:

dimethyl sulphoxide (DMSO)

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide (DMSO)

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: in the presence of S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide (DMSO)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: in the absence of S9 mix

Details on test system and experimental conditions:

Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix.
For 3 hour exposures, cultures contained a total of 6 x 106 cells. The final volume of the cultures was 5 mL and the final concentration of the S9 fraction was 2% v/v, if present. For 24 hour exposures, cultures contained a total of 1.5 x 106 cells in a total volume of 5 mL.
One culture was prepared for each concentration of the test substance for each test condition. Vehicle controls were tested in duplicate for each test condition. The test substance was formulated and serially diluted in the solvent. Aliquots of 50 μL of test substance dilution (at 100 times the desired final concentration) or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 37°C) or 24 hours (static
humidified incubator, at 37°C, 5% (v/v) CO2). At the end of the 3 hour exposure period, the cells were washed once, resuspended in R10p to nominally 2 x 105 cells/mL (assuming no cell loss), incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 105 cells/mL with R10p where necessary. At the end of the 24 hour exposure period, the cells were washed once, resuspended in 5 mL R10p and counted, to ascertain treatment growth. The cultures were then diluted to 2 x 105 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 105 cells/mL with R10p where necessary. The RSG was used to determine the concentrations of test substance used in the main test; ideally the maximum concentration should reduce RTG to approximately 10 to 20% of the concurrent vehicle control value. There was evidence of toxicity in the preliminary toxicity test, so the maximum concentrations tested in the 3 hour exposure in the absence and presence of S9 mix were 45 and 30 μg/mL respectively, and in the 24 hour exposure in the absence of S9 mix was 50 μg/mL. The formulations being added at 1% final volume in medium.

The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 107 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls Aliquots of 100 μL of test substance dilution (at 100 times the desired final concentration), vehicle or positive control were added, then all cultures were incubated, with continuous shaking, for 3 hours at 37ºC. At least four serial dilutions of the test substance were tested.
Following the 3 hour exposure, the cells were washed once, resuspended in R10p to nominally 2 x 105 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 105 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 105 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 103 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air.
After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates, the number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF).
The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The maximum concentrations assessed for mutant frequency in the first main test were 30 and 27.5 μg/mL in the absence and presence of S9 mix respectively.

A second test was carried out, with a 24 hour exposure in the absence of S9 mix. Duplicate 10 mL cultures containing 3 x 106 cells were treated for 24 hours with 100 μL of test substance, solvent or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, resuspended in 10 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to
2 x 105 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 105 cells/mL with R10p, the intention being to retain at least 1 x 107 cells. Following this, the procedure was the same as in the 3 hour treatment. The maximum concentration assessed for mutant frequency in the second main test was 45 μg/mL.

Evaluation criteria:

The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6 (Moore et al., 2006).
The assay was considered valid in accordance with the assay acceptance criteria.

The test agent was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.
If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p<0.001. Statistics are only reported if the Global Evaluation Factor is exceeded, and this was accompanied by a significant positive linear trend.

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:

Precipitate (observed by eye at the end of treatment) was observed at concentrations of 202.8 μg/mL and greater in the absence and presence of S9 mix, following a 3 hour exposure. Exposure to Hexyl Cinnamic Aldehyde at concentrations from 1.6 to 811.3 μg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values from 105 to 0% and from 97 to 0% respectively.
Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed at any concentration tested. Exposure to concentrations from 1.6 to 811.3 μg/mL resulted in RSG values from 107 to 0%. Concentrations used in the main test were based upon these data.

Cultures were exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 45 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 30 μg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 94 to 22% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. Although this assay did not reach the required 10-20% RTG it is considered valid in accordance with Moore et. al (2002) which states that a chemical can be considered nonmutagenic when there is no culture showing an RTG value between 10-20% if there is no evidence of mutagenicity (e.g., no dose response or mutant frequencies above those seen in the historical background ranges) in a series of data points within 100% to 20% RTG and there is at least one negative data point between 20% and 25% RTG. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Cultures were exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 30 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 27.5 μg/mL were assessed for determination of mutation frequency. RTG values from 93 to 18% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
The results obtained in response to the exposure of cultures to Hexyl Cinnamic Aldehyde in the presence of S9 mix did not demonstrate mutagenic potential. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the historical/acceptable solvent control values and there were no clear increases in the mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05). Therefore it was considered not to be beneficial to perform a direct repeat of the assay.

Cultures were exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 50 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 45 μg/mL were assessed for determination of mutation frequency. RTG values from 92 to 18% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test
concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Main Mutation Test 1 -3 Hour Treatment in the Absence of S9 Mix

Treatment/Concentration (μg/mL)	Replicate	Cell Concentration (x10^5/mL) 24 h	Cell Concentration (x10^5/mL) 48 h	Viability Plate Count^a Day 2	Mutant Plate Count^a Day 2	Mean RTG (%)	Mean MF (x10^4)
Vehicle Control^b	A	5.22	13.48	42 (192)	165 (192)	100	79
	B	5.63	14.54	36 (192)	160 (192)
	C	5.24	15.12	43 (192)	166 (192)
	D	5.46	16.15	38 (192)	166 (192)
Hexyl Cinnamic Aldehyde 5	A	5.09	15.66	42 (192)	170 (192)	94	73
	B	5.50	13.48	41 (192)	164 (192)
Hexyl Cinnamic Aldehyde 15	A	4.10	13.64	45 (192)	158 (192)	65	98
	B	4.18	14.25	48 (192)	165 (192)
Hexyl Cinnamic Aldehyde 20	A	2.95	10.99	35 (192)	151 (192)	45	101
	B	2.96	10.67	30 (192)	156 (192)
Hexyl Cinnamic Aldehyde 25	A	3.01	10.58	33 (192)	149 (192)	37	108
	B	2.69	8.55	37 (192)	156 (192)
Hexyl Cinnamic Aldehyde 30	A	2.69	8.33	39 (192)	147 (192)	22	156
	B	2.27	5.32	41 (192)	136 (192)
MMS 10	A	3.64	11.41	53 (192)	45 (192)	51	937
	B	4.45	13.05	54 (192)	41 (192)

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Table 2. Main Mutation Test 1 - 3 Hour Treatment in the Absence of S9 Mix, Colony Size Analysis

Treatment/Concentration (μg/mL)	Replicate	Mutant Plate Count^a Day 2	Total Mutant Colonies	Large Mutant Colonies	Small Mutant Colonies	% Small Mutant Colonies	Mean % Small Mutant Colonies
Vehicle Control^b	A	165 (192)	27	13	14	52	53
	B	160 (192)	34	16	18	53
	C	166 (192)	30	15	15	50
	D	166 (192)	26	11	15	58
MMS 10	A	45 (192)	209	31	178	85	83
	B	41 (192)	215	41	174	81

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Table 3. Main Mutation Test 1 - 3 Hour Treatment in the Presence of S9 Mix

	Replicate	Cell Concentration (x10^5/mL) 24 h	Cell Concentration (x10^5/mL) 48 h	Viability Plate Count^a Day 2	Mutant Plate Count^a Day 2	Mean RTG (%)	Mean MF (x10^4)
Vehicle Control^b	A	4.30	17.21	46 (192)	166 (192)	100	72
	B	5.30	13.18	36 (192)	169 (192)
	C	3.90	13.06	33 (192)	168 (192)
	D	4.82	13.62	41 (192)	163 (192)
Hexyl Cinnamic Aldehyde 5	A	4.73	12.81	43 (192)	163 (192)	93	75
	B	5.00	11.91	34 (192)	167 (192)
Hexyl Cinnamic Aldehyde 10	A	4.18	12.65	37 (192)	168 (192)	75	64
	B	3.67	12.49	43 (192)	171 (192)
Hexyl Cinnamic Aldehyde 12.5	A	3.00	11.87	43 (192)	166 (192)	59	77
	B	3.27	11.70	32 (192)	162 (192)
Hexyl Cinnamic Aldehyde 17.5	A	1.76^c	8.23	52 (192)	155 (192)	21	126
	B	1.75^c	8.43	55 (192)	159 (192)
Hexyl Cinnamic Aldehyde 25	A	1.49^c	7.24	48 (192)	162 (192)	22	92
	B	1.30^c	7.26	35 (192)	160 (192)
Hexyl Cinnamic Aldehyde 27.5	A	1.03^c	5.52	38 (192)	156 (192)	18	112
	B	1.02^c	4.55	23 (192)	141 (192)
BaP 1	A	4.42	12.10	30 (192)	60 (192)	86	558
	B	4.26	11.78	39 (192)	56 (192)

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)


c. Cell concentration not adjusted due to insufficient growth

BaP - Benzo[a]pyrene

Table 4. Main Mutation Test 1 - 3 Hour Treatment in the Presence of S9 Mix, Colony Size Analysis

Treatment/Concentration (μg/mL)	Replicate	Mutant Plate Count^a Day 2	Total Mutant Colonies	Large Mutant Colonies	Small Mutant Colonies	% Small Mutant Colonies	Mean % Small Mutant Colonies
Vehicle Control^b	A	166  (192)	26	8	18	69	61
	B	169  (192)	23	10	13	57
	C	168  (192)	24	10	14	58
	D	163  (192)	34	13	21	62
BaP 1	A	60 (192)	166	33	133	80	81
	B	56 (192)	159	29	130	82

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

BaP - Benzo[a]pyrene

Table 5. Main Mutation Test 2 - 24 Hour Treatment in the Absence of S9 Mix

Treatment/Concentration (μg/mL)	Replicate	Cell Concentration (x10^5/mL) 04 h	Cell Concentration (x10^5/mL) 24 h	Cell Concentration (x10^5/mL) 48 h	Viability Plate Count^a Day 2	Mutant Plate Count^a Day 2	Mean RTG (%)	Mean MF (x10^4)
Vehicle Control^b	A	16.60	6.85	10.91	44 (192)	170 (192)	100	59
	B	15.03	6.79	11.79	45 (192)	174 (192)
	C	14.54	6.72	11.02	40 (192)	169 (192)
	D	15.69	7.46	12.06	33 (192)	172 (192)
Hexyl Cinnamic Aldehyde 5	A	14.50	6.65	10.96	40 (192)	169 (192)	92	69
	B	14.74	5.78	12.73	34 (192)	164 (192)
Hexyl Cinnamic Aldehyde 15	A	13.16	5.82	11.87	35 (192)	167 (192)	81	70
	B	13.25	6.70	10.06	33 (192)	163 (192)
Hexyl Cinnamic Aldehyde 20	A	8.91	7.53	9.34	27 (192)	168 (192)	63	52
	B	9.17	6.70	10.86	32 (192)	172 (192)
Hexyl Cinnamic Aldehyde 25	A	6.05	4.59	11.07	38 (192)	166 (192)	27	63
	B	5.89	4.50	10.59	30 (192)	169 (192)
Hexyl Cinnamic Aldehyde 30	A	5.11	3.21	10.90	36 (192)	166 (192)	18	81
	B	5.28	3.86	9.36	26 (192)	153 (192)
MMS 10	A	12.38	7.51	7.12	47 (192)	45 (192)	48	938
	B	12.54	6.94	8.50	57 (192)	38 (192)

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Table 6. Main Mutation Test 2 - 24 Hour Treatment in the Absence of S9 Mix, Colony Size Analysis

Treatment/Concentration (μg/mL)	Replicate	Mutant Plate Count^a Day 2	Total Mutant Colonies	Large Mutant Colonies	Small Mutant Colonies	% Small Mutant Colonies	Mean % Small Mutant Colonies
Vehicle Control^b	A	170 (192)	22	8	14	64	52
	B	174 (192)	18	9	9	50
	C	169 (192)	23	12	11	48
	D	172 (192)	20	11	9	45
MMS 5	A	45 (192)	219	29	190	87	86
	B	38 (192)	232	35	197	85

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that Hexyl Cinnamic Aldehyde did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

Hexyl Cinnamic Aldehyde was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). The study consisted of a preliminary toxicity test and two main tests comprising three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. Hexyl Cinnamic Aldehyde was found to be soluble at 216.33 mg/mL in Dimethyl Sulphoxide. A final concentration of 811.3 μg/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the maximum concentration that does not cause a change in osmolality of greater than 50 mOsm/kg. Toxicity was observed in the preliminary toxicity test. Following a 3 hour exposure to Hexyl Cinnamic Aldehyde at concentrations from 1.6 to 811.3 μg/mL, relative suspension growth (RSG) was reduced from 105 to 0% and from 97 to 0% in the absence and presence of S9 mix respectively. Following a 24 hour exposure in the absence of S9 mix RSG was reduced from 107 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG). Following 3 hour treatment in the absence and presence of S9 mix, there were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix were 30 and 27.5 μg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 22 and 18% respectively. In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 45 μg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed at concentrations up to 45 μg/mL, where RTG was reduced to 18%. In all tests the concurrent vehicle and positive controls were within the acceptable historical control ranges. It was concluded that Hexyl Cinnamic Aldehyde did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No evidence of bacterial mutation was seen in an Ames test performed in S. typhimurium TA97 and TA102 using amyl cinnamic aldehyde.  No evidence of clastogenicity is seen in a mouse bone marrow micronucleus assay performed with the read-across substance hexyl cinnamic aldehyde.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The experiment was done according to the standard method of in vivo micronucleus. However, in the article there is no mention of GLP status and positive control.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

. No positive control, only one sampling time (30hr)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: purchased from Ivanovas GmbH, Kisslegg
- Age at study initiation: 10-14 week old
- Weight at study initiation: Not reported
- Assigned to test groups randomly: [no/yes, under following basis: ] Not reported
- Fasting period before study: Not applicable
- Housing: Not reported
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not reported

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Olive oil

Details on exposure:

The dose was given by i.p. injection once, using olive oil as a vehicle. Eight animals were used for each dose group.

Duration of treatment / exposure:

The mice were killed and bone-marrow smears were prepared 30 hr after treatment.

Frequency of treatment:

Single injection

Post exposure period:

The mice were killed and bone-marrow smears were prepared 30 hr after treatment.

Remarks:

Doses / Concentrations:
0 (olive oil), 324, 540, 756 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Eight

Control animals:

yes, concurrent vehicle

Positive control(s):

Not reported

Tissues and cell types examined:

The mice were killed and bone-marrow smears were prepared 30 hr after treatment.

Details of tissue and slide preparation:

No further details available

Evaluation criteria:

If significantly different from concurrent vehicle control, the result was judged as positive.

Statistics:

Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970).

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not specified

  

Dose (mg/kg)	Surviving/treated mice	Mean no. of micronucleated PE/1000 PE
1 x 756	8/8	2.4
1 x 540	8/8	1.8
1 x 324	8/8	2.1
0	8/8	1.0

Conclusions:

Interpretation of results (migrated information): negative
Exposure to HCA did not result in an increase in the proportion of micronucleated PCEs.

Executive summary:

In an in vivo micronucleus assay performed similarly to the OECD guideline No. 474 with minor deviations, male/female NMRI mice were exposed to alpha-Hexylcinnamaldehyde (HCA) diluted in olive oil by a single intraperitoneal dose. Different dose levels (324, 540, 756 mg/kg bw) were tested (8 animals per dose). 30 hours after the injection, the bone marrow cells were collected and one thousand polychromatic erythrocytes were observed in order to quantify the number of micronucleated polychromatic erythrocytes. Concurrent vehicle animals were used as negative control, no data was available on positive control. Under the test conditions, there was no significant increase of micronucleated polychromatic erythrocytes, if compared with the concurrent control. No mortality was observed. Therefore, under the study conditions it is concluded that HCA is not clastogenic.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The experiment was done according to the standard method of in vivo micronucleus. However, in the article there is no mention of GLP status and positive control.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Read-across is proposed for this endpoint, using data available for the analogue substance hexyl cinnamic aldehyde. A detailed justification for the proposed read-across approach is attached.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

. No positive control, only one sampling time (30hr)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: purchased from Ivanovas GmbH, Kisslegg
- Age at study initiation: 10-14 week old
- Weight at study initiation: Not reported
- Assigned to test groups randomly: [no/yes, under following basis: ] Not reported
- Fasting period before study: Not applicable
- Housing: Not reported
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not reported

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Olive oil

Details on exposure:

The dose was given by i.p. injection once, using olive oil as a vehicle. Eight animals were used for each dose group.

Duration of treatment / exposure:

The mice were killed and bone-marrow smears were prepared 30 hr after treatment.

Frequency of treatment:

Single injection

Post exposure period:

The mice were killed and bone-marrow smears were prepared 30 hr after treatment.

Remarks:

Doses / Concentrations:
0 (olive oil), 324, 540, 756 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Eight

Control animals:

yes, concurrent vehicle

Positive control(s):

Not reported

Tissues and cell types examined:

The mice were killed and bone-marrow smears were prepared 30 hr after treatment.

Details of tissue and slide preparation:

No further details available

Evaluation criteria:

If significantly different from concurrent vehicle control, the result was judged as positive.

Statistics:

Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970).

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not specified

  

Dose (mg/kg)	Surviving/treated mice	Mean no. of micronucleated PE/1000 PE
1 x 756	8/8	2.4
1 x 540	8/8	1.8
1 x 324	8/8	2.1
0	8/8	1.0

Conclusions:

Interpretation of results (migrated information): negative
Exposure to HCA did not result in an increase in the proportion of micronucleated PCEs.

Executive summary:

In an in vivo micronucleus assay performed similarly to the OECD guideline No. 474 with minor deviations, male/female NMRI mice were exposed to alpha-Hexylcinnamaldehyde (HCA) diluted in olive oil by a single intraperitoneal dose. Different dose levels (324, 540, 756 mg/kg bw) were tested (8 animals per dose). 30 hours after the injection, the bone marrow cells were collected and one thousand polychromatic erythrocytes were observed in order to quantify the number of micronucleated polychromatic erythrocytes. Concurrent vehicle animals were used as negative control, no data was available on positive control. Under the test conditions, there was no significant increase of micronucleated polychromatic erythrocytes, if compared with the concurrent control. No mortality was observed. Therefore, under the study conditions it is concluded that HCA is not clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not genotoxic and as such no mode of action is required.

Additional information

Additional information from genetic toxicity in vivo:

No evidence of bacterial mutation was seen in an Ames test performed in S. typhimurium TA97 and TA102 using amyl cinnamic aldehyde. No evidence of clastogenicity is seen in a mouse bone marrow micronucleus assay performed with the read-across substance hexyl cinnamic aldehyde.

Justification for selection of genetic toxicity endpoint

Higher tier (in vivo) study.

Justification for classification or non-classification

No classification for genetic toxicity is proposed in the absence of any indication of mutagenic or clastogenic activity in standard studies.