Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not Mutganeic to S. typhirmurium in In-Vitro Bacterial Reverse Mutation Assay (Equivalent to OECD TG 471)

Not Mutagenic to Chinese hamster lung fibroblasts (V79) in In-Vitro Gene Mutation Study in Mammalian Cells (OECD TG 476)

Cytoxicity-induced increase in break type aberrations in Human peripheral lymphocytes in In-Vitro Chromosome Aberration Study in Mammalian Cells (OECD TG 473)

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- Principle of test: The mutagenicity of n-hexyl acrylate was tested in the Salmonella-microsome assay according to the method of Ames et al. (1975).
- Short description of test conditions: N-hexyl acrylate was tested in triplicate in S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 with and without microbial activation (phenobarbital and aroclor 1254 induced rat liver S9 fractions). N-hexyl acrylate was tested up to cytotoxic concentrations.
- Parameters analysed / observed: Revertant colonies were counted manually after incubation in the dark at 37C for 48-72 hours.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polyscience Inc.
- Expiration date of the lot/batch: not specified
- Purity test date:not specified
Target gene:
Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98, and TA100 also contain the pKM101 plasmid (carrying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 are reverted only by mutagens that cause base substitutions.
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and Aroclor 1254 induced S9 mix
Test concentrations with justification for top dose:
Main assay: 25, 100, 400 and 1600 ug/plate
The top dose was chosen based on the presence of cytotoxicity as indicated by a cleared background lawn
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not specified
Untreated negative controls:
yes
Remarks:
historical average of spontaneous revertants for TA1535
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-Aminoanthracene
Untreated negative controls:
yes
Remarks:
historical average of spontaneous revertants for TA 1537
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
other: 2-aminoanthracene Strain TA1537 Phenobarbital induced S9
Untreated negative controls:
yes
Remarks:
historical average of spontaneous revertants for TA 1538
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 4-Nitro-o-phenylenediamine Strain TA1538 without S9; 2 Aminoathracene Strain TA1538 Phenobarbital induced S9
Untreated negative controls:
yes
Remarks:
historical average of spontaneous revertants for TA98
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 4-Nitro-o-phenylenediamine Strain TA98 without S9; 2 Aminoanthracene Strain TA98 Phenobarbital induced S9
Untreated negative controls:
yes
Remarks:
historical average of spontaneous revertants for TA100
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene Strain TA100 Phenobarbital induced S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 0.5 - 1.0 x 10^9 bacteria/mL

DURATION
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS: 3 replicate plates per dose

DETERMINATION OF CYTOTOXICITY
- Method: cleared background lawn
Rationale for test conditions:
The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al (1975). This methodology has been shown to detect a wide range of classes of chemical mutagens.
Evaluation criteria:
For tester strains TA98, TA1535, and TA1537, and TA100, an individual dose was considered positive if the mean revertant colony count on the test plates was equal to or greater than two times the mean number of spontaneous revertants on the vehicle control plates. A positive result for the assay was defined as a dose-related increase in the mean number of revertant colonies over at least three concentrations of test substance, including at least one positive dose.
Statistics:
The mean revertant colony count and standard deviation were determined for each dose point.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
N-hexyl acrylate was not mutagenic in five strains of Salmonella typhimurium bacteria in the presence or absence of metabolic activation.
Executive summary:

The substance was tested for its mutagenic potential based on the ability to induce back mutations in selected loci in Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98, and TA1538 at concentrations ranging from 25 to 1600 µg/plate according to the methods described in Ames et al. (1975) (equivalent or similar to OECD 471). The assay was performed using the Standard plate test with and without metabolic activation (phenobarbital/aroclor 1254 induced rat liver microsomes), respectively. The test material caused visible reduction in the growth of the bacterial background lawn at at a concentration of 1600 µg/plate and was used as the top dose level in the main assay. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29th July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In-Vitro Mammalian Chromosome Aberration Test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sartomer Batch KAL1908
- Expiration date of the lot/batch: Jan 2019
- Purity test date: Feb 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark, in flammable cabinet, used under safety lighting.
- Stability under test conditions: stable under conditions of test
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Primary lymphocytes were obtained from fresh blood samples drawn from the peripheral circulation of a volunteer between the ages of 18 to 35 years old, non-smoker and not knowingly having been exposed to high levels of radiation hazardous chemicals or recent viral infection.
- Suitability of cells: Human peripheral blood lymphocytes are recognized in the OECD TG 473 as a suitable cell line for the Mammalian Chromosome Aberration test
- Cell cycle length, doubling time or proliferation index: ~16 h in stock cultures
- Methods for maintenance in cell culture if applicable: Bulk human lumphocyte cultures were prepared and batched out into test flasks or exposure tubes after 48 ±2 hours of incubation at ~37°C, 5% CO2 in humidifed air.
- Normal (negative control) cell cycle time: Average Generation time is considered to be ~ 16 hours based on over 20 years of in house experience with human lymphocytes using BrdU incorporation to assess the number of first, second and third division metaphase cells.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Lymphocyte cultures were established in Modified Eagle's Medium (MEM) supplemented with: sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer (10% fetal bovine serum), heparinised whole blood, phytohaemagglutinin, and lithium heparin.
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 (20% S9 fraction in S9 mix; dosed at 10% in culture media for final concentration of 2%) from liver homogenates of Male CD Sprague Dawley Rats treated with Phenobarbital (80mg/kg)/B-Naphtha flavone (100mg/kg) in Arachis Oil
Test concentrations with justification for top dose:
Preliminary toxicity test were performed to determine the top dose used in each exposure condition.

Three exposure groups were used:
1. 4-hours exposure without S9; 20-hour recovery
2. 4-hours with S9; 20-hour recovery
3. 24-hour continous exposure without S9

The dose ranges of test item used was 6.09 to 1560 ug/mL (maximum recommended dose level 10mM). A precipitate was observed at concentrations above 390 ug/mL in the 4-hour exposure groups and above 195 ug/mL in the continous exposure group. Haemolysis was observed at and above 24. 39 ug/mL in all exposure groups. Microscopic assessment showed the presence of metaphase cells up to 50 ug/mL in the 4-hour exposure without S9 and in the 24-hour continous exposure group. Metaphase cells were observed up to 97.5 ug/mL in the 4-hour exposure with S9. See attached for table of mitotic indices.

The selection for the maximum dose level for the main experiment was base don toxicity and was 100 ug/mL for the 4-hour exposure group without S9 and the 24 hour continous exposure group and was 175 ug/mL for the 4-hour exposure with S9.

The dose levels of the controls and test item are given below:
4-hour without S9: 0*, 3.13, 6.25, 12.5*, 25*, 50*, 75*, 100 ug/mL
4-hour with S9: 0*, 12.5, 25, 50*, 100*, 125*, 150*, 175 ug/mL
24-hour withou S9: 0*, 3.13, 6.25*, 12.5*, 25*, 50*, 75, 100 ug/mL

* = dose levels selected for metaphase analysis
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO (final concentration 1%)
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance in a vehicle compatible with this test system was assessed. The test item was immiscible at 15.6 mg/mL in aqueous media but was fully miscible in DMSO at 156.0 mg/mL
Untreated negative controls:
yes
Remarks:
Frequency range of spontaenous chromosoem aberration in historical untreated control lymphocytes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):

DURATION
- Exposure duration: 4-hour (+20 hour recovery) and 24-hour continous without S9; 4-hour (+20 hour re3covery) with S9
-Fixation time: 24 hours after start of treatment

SPINDLE INHIBITOR (cytogenetic assays): Mitosis was arrested by addition of demecolcine ~2-3 hours prior to harvest.

STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: Duplicate cultures per dose level

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 consecutive well spread metaphases per dose concentration (150 per duplicate); If the analysis results in a large frequency of aberrant cells, the analysis will be terminated after a total of 15 metaphases with aberrations (excluding gaps) have been recorded.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: In cells with 69 chromosmes or more (including endoreduplicated cells) will be scored as polyploid cells.
- Determination of endoreplication: see above
- OTHER: If the metaphase cell has 44 to 48 chromosomes, any breaks, fragments, deletions, exchanges, and chromosomal disintegrations are recorded as structural chromosome aberrations according to the simplified system of Savage (1976).
Rationale for test conditions:
Human peripheral lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration test.
Evaluation criteria:
The following criteria were used to determine a valid assay:
1. The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures will be within the laboratory historical control data range.
2. All positive control chemicals must induced positive responses (P≤0.01).
3. All three exposure conditions use a top dose that meets the requirements of the testing guideline
The required number of cells and concentration are analyzed where possible.

Providing all acceptability criteria are met, a test item will be considered negative :
1. The number of cells with structural chromosoem aberrations in all evaluated dose groups should be within the range of laboratory historical control data
2. No toxicologically or statistically significant increase in the number of cells with structural chromosome aberrations is observed.
3. There is no concentration-related increase at any dose level.

Providing all acceptability criteria are met, a test item will be considered positive:
1. The number of cells with structural chromosoem aberrations in all evaluated dose groups is outside the range of laboratory historical control data
2. At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent solvent control.
3. The observed increase in frequency of cells with structural aberrations is considered to be dose-related.
Statistics:
When there is no indication of any increased aberrations at any dose level then statistical analysis may not be necessary. In all other circumstances comparisons will be made between appropriate vehicle control value and each individual dose level using FIsher's Exact Test on observed numbers of cells with aberrations excluding gaps. A toxicologically significant response is recorded when the p-value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control.
Key result
Species / strain:
lymphocytes:
Remarks:
Human peripheral blood
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes:
Remarks:
Human peripheral blood
Metabolic activation:
without
Genotoxicity:
ambiguous
Remarks:
Statistically significant increase only at highest dose in presence of cytotoxicity beyond recommended range
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: Human Peripheral Blood
Metabolic activation:
without
Genotoxicity:
ambiguous
Remarks:
Statistically significant increase only at highest dose tested in presence of cytotoxicity beyond recommended range
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when test item was dosed into media
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm when the test item was dosed into media
- Evaporation from medium: No evaporation from medium was expected
- Water solubility: see field "Vehicle" above
- Precipitation: No precipitate was observed at any dose used.

RANGE-FINDING/SCREENING STUDIES: See field "Concentration and Justification for top dose" above

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached figures
Remarks on result:
other: 4-Hour Exposure
Conclusions:
N-hexyl acrylate produced a significant increase in the frequency of cells with chromosome aberrations following 24 hour continous exposure without S9 only at the highest dose tested which was associated with significant cytotoxicity (43% itotic index; a 57% reduction in mitotic index). Per OECD TG 473, care should excercised when interpreting positive results only to be found in the higher end of the recommended 55 +/- 5% cytotoxicity range.
Executive summary:

N-hexyl acrylate was examined for its potential to induce structural alterations in human peripheral lymphocytes, in both the presence and absence of an S9 metabolic activation system. The test substance did not induce a statistically significant increases in the frequency of structural aberrations in the 4-hour exposure condition with metabolic activation at any of the doses chosen. A statistically significant increase in the frequency of chromosome aberrations (predominantly break type) was observed at the highest dose tested in 4-hour and 24 hour exposure without metabolic activation. This increase occurred in the presence of significant cytotoxicity with mitotic indices of 40% and 43% for the 4-hour and 24-hour exposure respectively. This measure cytotoxicity is on the high end of the recommended range for the high dose (reduction in mitotic index of 55 +/- 5%). Additionally, both results were driven by one of the two replicates where cytotoxicity was greater. Positive results of predominantly break type observed only at the highest dose in the presence of pronounced cytotoxicity may be more indicative of a cytotoxic-induced response as opposed to a true genotoxic mechanism and are considered to have reduced biological relevance.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Feb 2018 - 14 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In-Vitro Gene Mutation study in Mammalian Cells
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Harlan CCR and originated from Labor für Mutagenitätsprüfungen (LMP) Technical University 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in-vitro experiments for many years. The high proliferation rate (doubling time 12 - 16h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22.
- Cell cycle length, doubling time or proliferation index: 12 - 16 h in stock cultures
- Methods for maintenance in cell culture if applicable: Laboratory stock cell cultures are periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen.
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 C with 5% CO2 in humidified air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 (20% S9 fraction in S9 mix; dosed at 10% in culture media for final concentration of 2%) from liver homogenates of Male CD Sprague Dawley Rats treated with Phenobarbital (80mg/kg)/B-Naphtha flavone (100mg/kg) in Arachis Oil
Test concentrations with justification for top dose:
Preliminary Cytotoxicity Screen:
4-hour –S9 exposure group: 0.63, 1.25, 2.5, 5, 10, 20, 30, 40, and 50 µg/mL, the dose levels were selected following excessive precipitate observed in the solubility test.
4-hours + S9 exposure group: 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL, the dose levels were selected to avoid excessive precipitation and toxicity as indicated in the solubility test.

Mutagenicity test- Main Experiment
The maximum concentration selected for the main mutagenicity experiment was limited by a combination of the onset of test item induced toxicity observe din the preliminary cytotoxicity screen and precipitate formation.

The concentrations of the test item are given below:
4-hour without S9: 0*, 2.5, 5*, 10*, 20*, 25*, 30*, 35*, 40 ug/mL
4-hour with S9 (2%): 0*,0.78, 1.56, 3.13, 6.25*, 12.5*, 25*, 50*, 100* ug/mL
* Concentrations plated out for cloning efficiency and mutant frequency

Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO (final concentration 1%)
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance in a vehicle compatible with this test system was assessed. The test item was immiscible at 15.6 mg/mL in aqueous media but was fully miscible in DMSO at 156.0 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1E07 cells/ 225 cm2 flask

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-Thioguanine (6TG) at 11 ug/mL

NUMBER OF REPLICATIONS: Five concentrations of test item in duplicate are used during the exposure period. Mutant frequencies are normally derived from sets of ten dishes/flasks per dose for the mutant colony count and three dishes per dose for cloning colonoy counts.

NUMBER OF CELLS EVALUATED: 2E06 cells from each culture were seeded to allow expression of the mutant phenotype.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (200 cells/plate)
Evaluation criteria:
The following criteria will be used to determine a valid assay:
i) The average absolute cloning efficiency of the Day 7 negative controls should exceed 50%. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls is generally within the historical range. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays.
iii) The concurrent positive controls should induce responses that are comparable with those generated in the historical positive control range and produce a statistically significant increase compared with the concurrent negative control.
iv) The criteria for selection of the maximum concentration have been met. Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended concentration (10 mM, 2 mg/mL or 2 µL/mL whichever is lower), or include the lowest precipitating concentration.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
Statistics:
When there is no indication of any marked increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium of more than 1.0 unit compared with the vehicle control were observed up to 1560 µg/mL.
- Effects of osmolality: The osmolality of the test substance in medium was tested up to concentration of 1560 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Evaporation from medium: Not applicable
- Precipitation: At the end of the exposure period, precipitate of the test item was observed at 35 and 40 µg/mL in the absence of metabolic activation and at 100 µg/mL in the presence of metabolic activation.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity test, a four-hour exposure to concentrations from 0.63 to 50 ug/mL in the absence of S9 and from 1.56 to 400 ug/mL in the presence of S9 mix, resulted in Day 1 relative survivals of 95 to 0% and 95 to 0% respectively. Precipitate was seen at 40 μg/mL and above post-dosing in the absence of S9 and at 50 μg/mL and above in the presence of S9. Concentrations for the main test were based upon these data.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table below
- Negative (solvent/vehicle) historical control data: see table below

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Main Test (-S9 mix): Exposure to the test item resulted in Day 0 Viability of between 97% and 14%. The Day 7 cloning efficiencies were 105% to 96% relative to solvent controls.
-Main Test (+S9 mix): Exposure to the test item resulted in Day 0 Viability of between 99% and 90%. The Day 7 cloning efficiencies were 108% to 114% relative to solvent controls.

Table 1 Historical Control Rnages for Vehicle Control Cultures

       Vehicle Control Mutant Frequencies per survivor x 10^-6
   4 -Hour without S9  4 -Hour with S9
 Range  5 -23

5 -24 

 Mean  12.15  12.20
 Standard Deviation  5.26  4.30
 N  20  20

Table 2 Historical Control Rnages for Positive Control Substances

             Positive Cotnrol Mutant Frequencies per survivor x 10^-6
 

 4 -Hour without S9

(EMS)

500 ug/mL

4 -Hour without S9

(EMS)

750 ug/mL

4 -Hour With S9

(DMBA)

1 ug/mL 

4 -Hour with S9

(DMBA)

2 ug/mL 

 Range  171 - 346  291 - 574  209 - 409  202 - 786
 Mean  260.50  419.05  295.95  124.91
 Standard Deviation  40.63  71.81  56.44  124.91
 N  20  19  20  20
Conclusions:
The test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in V79 cells at any dose level, either in the presence or absence of metabolic activation.
Executive summary:

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, Chinese hamster lung fibroblasts (V79) were exposed to the test material diluted in DMSO, in duplicate in the presence and absence of metabolic activation (S9-mix). Four-hour exposures were used both with and without activation (S9) in both tests. The highest final concentration used in the preliminary toxicity test was 100 μg/mL. Cytotoxicity was measured as Day 1 relative survival; values were from 97 to 14% in the absence of S9 mix respectively. In the presence of S9 mix, no cytotoxicity was observed, however the test item was tested up to precipitating concentrations.

In the absence of S9 mix, cells were exposed to concentrations from 2.5 to 40 μg/mL. Day 1 survival values ranged from 97 to 1% relative to the solvent control. The test material did not cause a statistically significant increase in mutant frequency.

In the presence of S9 mix cells were exposed to concentrations from 0.78 to 100 μg/mL. Day 1 survival values ranged from 92 to 90% relative to the solvent control. The test material did not cause a statistically significant increase in mutant frequency.

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

Additional information

Galloway S.M. (2000) Cytotoxicity and Chromosome Aberrations In Vitro: Experience in INdustry and the Case for an Upper Limit on Toxicity in the Aberration Assay. Env. Mol. Mut. 35:191 -201

Justification for classification or non-classification

N-hexyl acrylate was not mutagenic in five strains of Salmonella typhimurium bacteria at any dose tested in the presence or absence of metabolic activation in a study equivalent or similar to OECD TG 471. In an in-vitro gene mutation study in mammalian cells (OECD TG 476), N-hexyl acrylate did not induce any statistically significant increases in mutant frequency at the HPRT locus in Chinese hamster lung fibroblasts (V79) tested in duplicate in the presence or absence of metabolic activation. In an in-vitro chromosome aberration study in mammalian cells (OECD TG 473), N-hexyl acrylate did not induce a statistically significant increase in the frequency of structural aberrations in the 4-hour exposure condition with metabolic activation at any of the doses chosen. A statistically significant increase in the frequency of chromosome aberrations (predominantly break type) was observed at the highest dose tested in 4-hour and 24 hour exposure without metabolic activation. This increase occurred in the presence of significant cytotoxicity with mitotic indices of 40% and 43% for the 4-hour and 24-hour exposure respectively. This measured cytotoxicity is on the high end of the recommended range for the high dose per OECD TG 473 (reduction in mitotic index of 55 +/- 5%). Additionally, both results were driven by one of the two replicates where cytotoxicity was greater. Positive results of predominantly break type observed only at the highest dose in the presence of pronounced cytotoxicity may be more indicative of a cytotoxic-induced response as opposed to a true genotoxic mechanism and are considered to have reduced biological relevance (Galloway 2000). Finally, no neoplastic lesions were observed in a 2 -year chronic inhalation bioassay with a structurally similar compound N-butyl acrylate which is used as the read-across source for repeat dose toxicity. Taking all of the available data together, these findings do not warrant the classification of N-hexyl acrylate as a genotoxin under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).


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