Allyl heptanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro (Ames test): negative (±S9 mix)

in vitro (Micronucleus assay): negative (±S9 mix), read across from allyl hexanoate

in vitro (Mammalian cell gene mutation assay): negative (±S9 mix), read across from allyl hexanoate

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-28 till 2009-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study in accordance with OECD TG

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals, December 13, 2007, Draft Proposal for a new Guideline No. 487 “In vitro Mammalian Cell Micronucleus Test” (3rd version).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed by Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz (2009-03-30)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: from human donors

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a 27 year-old male donor for the first experiment and from a 29 year-old female donor for Experiment II. Blood samples were drawn by venous puncture and collected in heparinised tubes.
- Type and identity of media: The culture medium was DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1) already supplemented with 15 mM HEPES. The antibiotic solution contained 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. Additionally, the medium was supplemented with the mitogen Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), 10 % FBS (fetal bovine serum), the anticoagulant heparin (25,000 U.S.P.-U/mL) and L-glutamin 200 mM. All incubations were done at 37 °C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).
- Properly maintained: yes
Treatments were commence at around 44 - 48 hours after culture initiation when the cells are actively proliferating.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment:
- without metabolic activation, 4 hours treatment: 10.2, 17.8, 31.2, 54.7, 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
- with metabolic activation, 4 hours treatment: 10.2, 17.8, 31.2, 54.7, 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Dose selection of Experiment II was based on the results obtained in Experiment I.
Experiment II:
- without metabolic activation, 20 hours treatment: 31.2, 54.7, 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
- with metabolic activation, 4 hours treatment: 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
For more detail see table under "any other information on material and methods incl. tables".

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO (E. MERCK, 64293 Darmstadt, Germany; purity 99.9 %). The final concentration of DMSO in the culture medium was 0.5 % (v/v).
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Demecolcin; 4.0 µg/mL (Exp.I) and 0.05 µg/mL (Exp.II), dissolved in deionised water

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation Migrated to IUCLID6: 1.0 µg/mL (Exp.I) and 0.3 µg/mL (Exp.II), dissolved in deionised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation Migrated to IUCLID6: 12.5 µg/mL (Exp. I) and 10.0 µg/mL (Exp. II), dissolved in 0.9% saline

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (+16 hours recovery period) or 20 hours; Cells were either treated for 4 hours in the absence of S9 mix (Exp.I) and in the presence of S9 mix (Exp.I and II) or for 20 hours without S9 mix (Exp.II).
The culture medium was replaced with serum-free medium (Experiment I without S9 mix; Experiment I and II in with S9 mix) or complete medium with 10 % FBS (v/v) (Experiment II without S9 mix), containing the test item.
- Recovery period: After treatment for 4 hours, the cells were re-suspended in "saline G" and incubatet for further 16 hours.
- Cytokinesis blocker: After washing, the cells were re-suspended in complete culture medium containing Cytochalasin B (4 µg/mL) and cultured another approximately 20 hours until preparation.
- Fixation time (start of exposure up to fixation or harvest of cells): The cultures were harvested by centrifugation 40 hours after beginning of treatment. Thereafter the cells were fixed and slides were prepared.

STAIN (for cytogenetic assays): The cells were stained with Giemsa.

NUMBER OF REPLICATIONS: In each experimental group two parallel cultures were analysed.

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

DETERMINATION OF CYTOTOXICITY
- Method: other: cytokinesis block proliferation index
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the cytokinesis block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described for the mutagenicity assay.
The pre-experiment was performed with 10 concentrations of the test item and the negative, solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hours (with and without S9 mix) and the cells were prepared 40 hours after start of the exposure.

OTHER EXAMINATIONS:
The frequency of micronucleated cells was reported as % micronucleated cells.

OTHER: Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area.

Evaluation criteria:

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory’s historical control data and
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.

Statistics:

Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together.

Species / strain:

lymphocytes: from humans

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

In Experiment I no cytotoxicity was observed up to the highest applied concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: from humans

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

In Experiment II in the presence of S9 mix no cytotoxicity was observed up to the highest applied concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: from humans

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at the two highest evaluated concentrations. However, in Experiment II in the presence of S9 mix, the highest applied concentration was not evaluable due to a reduced cell number.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the pre-test on toxicity, precipitation of the test item was observed at the end of treatment at 512.7 µg/mL and above in the presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA: In Experiment I, in the presence of S9 mix and in Experiment II in the absence of S9 mix, statistically significant increases in cells carrying micronuclei were observed. These values were in the range of the laboratory’s historical solvent control data and therefore considered as being biologically irrelevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no further data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Experiment I

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, Allyl capronate (Sym09 /611045) is considered to be non-aneugenic/clastogenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 25, 1999 - July 02, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study in accordance with OECD TG

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (ICR Harmonised Tripartite Guideline of July 19, 1995)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

This test uses several specially constructed histidine-requiring (his-) mutants of Salmonella typhimurium and measures his- to his+ reversion induced by chemicals which cause base changes or frameshift mutations in the genome of this organism.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate (S9) from Aroclor 1254 pretreated male rats

Test concentrations with justification for top dose:

According to an initial toxicity test, Allyl heptanoate was tested in concentrations of 15 to 5000 µg per plate in the absence and of 0.5 to 500 µg per plate in the presence of S9.

Vehicle / solvent:

dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

2-Aminoanthracene (A3880-0, Aldrich, Steinheim); 1.0 - 4.0 µg/plate for TA1535, TA1537, TA102 and 0.5 - 1.0 µg/plate for others strains

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

H2O

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: 0.25 µg/plate for TA102

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: 2.5 µg/plate for TA98

Negative solvent / vehicle controls:

yes

Remarks:

H2O

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: 0.5 µg/plate TA1535 and TA100

Negative solvent / vehicle controls:

yes

Remarks:

H2O

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 50 µg /plate for TA1537

Details on test system and experimental conditions:

Principle
Special mutant strains of Salmonella typhimurium are used. Each tester strain contains a different type of mutation in the histidine operon (his), thereby imposing a requirement for histidine in the growth medium. In addition to the histidine mutation, the tester strains contain other mutations that greatly increase their ability to detect mutagens (see Table 1). On a minimal medium containing traces of histidine the bacteria grow and divide until all histidine is consumed. Spontaneous reverse mutations to the histidine prototrophy of the wild type (his) occurring during these few cell divisions lead to revertants. These grow in the histidine free growth medium and form visible colonies. The exposure of bacteria to various mutagens increase the frequency of reverse mutations to the manyfold of the spontaneous value. The metabolic conversion of the test substance by mammalian liver enzymes is examined by the comparison of the tests with and without the addition of the S9 fraction of liver homogenate.

Tester strains
Five strains of Salmonella typhimurium will be used in the study. They are originally obtained from Dr. Bruce N. Ames in 1975. Their designation and genotypic characterisation are summarized in Table 1.

TABLE 1
Strain Designation Histidine Mutation Additional Repair Mutation Affecting Mutation Type Detected
LPS-coat R-Factor
TA1537 hisC3076 uvrB- rfa Frameshift
TA98 hisD3052 uvrB- rfa pKM101 Frameshift
TA1535 hisG46 uvrB- rfa Base Pair
TA100 hisG46 uvrB- rfa pKM101 Substitution
TA1O2 hisUG8476 rfa pKM101, pAQ1 Base Pair
hisG428 on plasmid pAQ1 Substitution


hisC3076: This mutation in the C-gene of the his-operon contains a (+ 1) frameshift mutation leading to a strech of 4-GC- base pairs. This mutation is reverted by mutagens like ICR191, and aminoacridine.

hisD3052: This is a (-1) frameshift mutation in the hisD-gene coding for the histidinol dehydrogenase. Near the site of the mutation is a sequence of eight repetitive -GC- residues in which deletions of two base pairs reverte the auxotrophy by the generation of the proper reading frame. Aromatic amines and derivatives can revert the mutation.

hisG46: This is a mis-sense mutation in the hisG-gene which codes for the first enzyme of the histidine biosynthesis. The mutation substitutes proline for (leucine) in the wild type organism. Mutagens which cause base pair substitutions (transitions and transversions) primarily at one of the -GC- pairs can revert the mutation to prototrophy.

Strain TA1O2 carries the unrevertable deletion hisUG8476 in the histidine operon of the bacterial chromosome, but offers the revertable mutation hisG428 on the plasmid pAQ1 which normally exists in the cells in multiple copies (Lewin et. al., 1982). TA102 was developped to detect mutations at -AT- base pairs in contrast to mutations at -GC- base pairs in the strains TA1535 and TA100.

uvrB-: This a deletion of the uvrB-gene of the DNA excision repair system which renders the bacteria more susceptible to mutagens. The deletion extends through the bio-gene and as a consequence the strains require biotin for growth. Deletions cannot be reverted.

rfa: The mutation rfa is the deep rough mutation, which causes loss of the polysaccharide side chain of the lipopolysaccharide molecules in the bacterial cell wall. This increases cell permeability to large molecules that do not penetrate the normal cell wall and greatly decreases the patho-genicity of these organisms.

pKM101: This a plasmid containing an R-factor for resistance to ampicillin. pKM101 increases the chemical and spontaneous mutagenesis probability by enhancing an error-prone DNA-repair system, which is normally present in Salmonella.

Bacteria
Frozen permanents of the tester strains are stored in liquid nitrogen. They are prepared from fresh overnight cultures to which 8% DMSO is added as a cryoprotective agent.
From the permanents master plates are prepared on Vogel-Bormer (VB) minimal-medium plates enriched with histidine and biotin. Ampicillin is added to the plates used for strains with the R-factor. Master plates are used as a source of bacteria for inoculating the overnight cultures. Master plates are always kept at 4°C and discarded after one month or sooner if the number of spontaneous revertants per plate falls out of the normal range specific for a strain.
Tester strain cultures are grown in Oxoid nutrient broth No. 2 or Difco bacto nutrient broth on a shaker (12-13 h, 37°C) to a density of 1-3 x 10(9) cells per ml. Only fresh cultures are used for mutagenicity assays

Metabolic activation system :
The rat liver homogenate fractions used were prepared by King & Harnasch GmbH (lot KH4997), 79199 Kirchzarten. The preparation of the 9000g supernatant of liver homogenates (S9) from Sprague-Dawley male rats (8-10 weeks old) induced with Aroclor 1254 (500 mg/kg body weight) is in accordance with the method recommended by Ames et al. (1975).

Each S9 lot was tested for
* protein concentration (Lowry Method)
* ability of enzymatic stimulation of benzo(a)pyrene and 2-aminoanthracene
induced mutagenesis with tester strains TA98 and TA100.
* sterility

The S9 preparations (in 0.15 M KCl) are stored in liquid nitrogen.
S9-mix containing 10% S9 is freshly prepared for each mutagenicity assay. The concentration of the cofactors in the S9-mix (per ml) are: distilled water, 0.335 ml; phosphate buffer (0.2 M, pH 7.4), 0.5 ml; NADP (0.1 M), 0.04 ml; glucose-6-phosphate (1 M), 0.005 ml; MgCl2/KCl (0.4M/1.65M), 0.02ml; S9 fraction, 0.1 ml.

Top agar
Autoclaved top agar containing 0.6% Difco agar and 0.5% NaCl is melted and 10% of a sterile solution of 0.5 mM L-histidine-HCI / 0.5 mM biotin are added and thoroughly mixed prior to the main test procedure. The molten agar is kept at 45°C in portions of 2 ml in small sterile tubes.

Test procedure
The tests will be performed using the plate incorporation method.
To each tube containing 2 ml molten top agar 0.1 ml of bacteria will be added followed by the test solution (dissolved in H20 or DMSO) and 0.5 ml of S9-mix or phosphate buffer in the assays without metabolic activation. The test components are mixed with a vortex and immediatly poured onto coded minimal agar plates and carefully spread to achieve an uniform distribution of the top agar on the surface of the plate. The mixing, pouring, and distribution is done as quick as possible. The minimal agar plates contain 20 ml of 1.5% Difco-Bacto agar in VB medium E with 2% glucose. Plates are kept for 72 h at 37°C in the dark and then examined. Besides the counting of the number of revertant colonies (his revertants), the plates are examined for the existence of a normal background lawn and/or precipitates and microscopically for microcolony growth.

Dose Level
An initial test is performed to find a suitable dose range. This will be done with the strain TA100 with and without S9-mix. The following 7 dosages are tested in half-log dilutions, starting with the maximum dose of 5.000 µg/plate using the method of the plate incorporation test. After the incubation of the plates for 72 h (37°C) the number of revertant colonies will be noted and the quality of the background lawn of auxotrophs carefully examined. The maximum concentration not inhibiting the growth of prototrophs and auxotrophs will be the maximum dose in the main test. If toxicity is not observed 5.000 µg per plate will be the maximum dose.

Controls
The solvent used for the test substance will be used as the negative control and plated with each strain in triplicate with and without S9-mix. Control without solvent will also be done in the same manner.
The following chemical mutagens will serve as positive control substances

Confirming genotypes of tester strains
Tests for the genotypes of the strains are included in each mutagenicity assay and comprise:

a) his- mutation
Each of the tester strains will be streaked on two selective plates:
1. VB-minimal plates with 0.1 ml of 0.1 M L-histidine and 0.1 ml of 0.5 mM biotin per plate.
2. VB-minimal plates with 0.1 ml of 0.5 mM biotin but without histidine.
All strains should grow on plate 1 none on plate 2

b) rfa mutation
All strains will be tested for the deep rough (rfa) character by their sensitivity for crystal violet. Each strain is plated on a nutrient agar plate. A sterile filter paper disc with 10 µl of a 1 mg/ml solution of crystal violet is transferred to the seeded plate. After incubation, a clear zone of inhibition appears around the disc indicating the presence of the rfa mutation. The rfa mutation permits large molecules such as crystal violet to enter and kill the bacteria. Crystal violet cannot penetrate the cell wall of wild type strains

c) uvrB mutation
The uvrB mutation will be confirmed by demonstrating the UV-sensitivity of the strains. The strains will be streaked across a nutrient agar plate in parallel stripes. A piece of card-board is placed over the uncovered plate so that half of each bacterial streak is covered. With a germicidal lamp at a distance of 33 cm the TA1535 and TA1537 plates are irradiated for 6 seconds, and the TA98 and TA100 plates for 8 seconds. Growth should only be seen on the unirradiated side of the plates.

d) pKM101 plasmid
The pKM101 plasmid renders its host cell resistance to ampicillin. They will be tested by streaking the strains across the surface of a VB minimal plate with additional histidine, biotin and 25 pg/ml ampicillin in the medium. Alternatively, a 10 pg ampicillin paper disc is placed in the middle of agar plates. TA1535 and TA1537 should be sensitive judged on the zones of inhibition.

Sterility controls
The c) uvrB mutation
The uvrB mutation will be confirmed by demonstrating the UV-sensitivity of the strains. The strains will be streaked across a nutrient agar plate in parallel stripes. A piece of card-board is placed over the uncovered plate so that half of each bacterial streak is covered. With a germicidal lamp at a distance of 33 cm the TA1535 and TA1537 plates are irradiated for 6 seconds, and the TA98 and TA100 plates for 8 seconds. Growth should only be seen on the unirradiated side of the plates.

d) pKM101 plasmid
The pKM101 plasmid renders its host cell resistance to ampicillin. They will be tested by streaking the strains across the surface of a VB minimal plate with additional histidine, biotin and 25 pg/ml ampicillin in the medium. Alternatively, a 10 pg ampicillin paper disc is placed in the middle of agar plates. TA1535 and TA1537 should be sensitive judged on the zones of inhibition.

Sterility controls
The VB-minimal plates, the test substance, the stock solutions and the 59-mix will be checked for sterility.

Evaluation criteria:

A mutagenic activity of a substance will be suspected if the number of revertants on the test plates in comparison to that of spontaneous revertants on the concurrent vehicle control plates is significantly increased. Positive results have to be reproduced and a dose related increase of the mutagenic response has to be ascertained. This might implicate the necessity of repeating the test with a narrow dosage

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>500 µg/plate w/o metabolic activation, >50 µg/plate w metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>500 µg/plate w/o metabolic activation, >150 µg/plate w metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>500 µg/plate w/o metabolic activation, >50 µg/plate w metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>500 µg/plate w/o metabolic activation, >150 µg/plate w metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>500 µg/plate w/o metabolic activation, >150 µg/plate w metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results of the mutagenicity tests of the substance Allyiheptoate show individual plate counts and the mean number of revertant colonies per plate from two independent experiments.

The number of spontaneous revertants observed using each of the five strains was very close to those previously established in our laboratory and was within the range obtained by Ames et al. (1975) as well as reported by Dc Serres and Shelby (1979).
Similarly, the results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.

The experiments were done with and without metabolic activation by rat liver homogenate (59). Allylheptoate was dissolved in DMSO and tested in concentrations of 15 to 5000 µg per plate in the absence of 59 and of 0.5 to 500 µg per plate in the presence of S9. In the absence of S9 Allylheptoate was bacteriotoxic at 500 µg/plate towards all strains. In the presence of S9 Allylheptoate was bacteriotoxic towards the strains TA98 and TA1535 at 50 µg/plate and towards the strains TAI00, TA102, and TA1537 at 150 µg/plate. The bacteriotoxic effect of the test substance was rather acute and reduced more the density of the background lawn at lower concentrations than the number of emerging revertants.

The test compound Allylheptoate failed to induce a significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. Similarly, the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a X2-test (Mohn and Ellenberger, 1977), did not reveal a significant effect at any of the test points in the absence and presence of a metabolizing system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, these results indicate that Allyl heptoate under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the absence and presence of a metabolizing system.

Executive summary:

The mutagenicity of the substance Allylheptoate was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried out using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor1254 pretreated male rats as metabolic activation system.

Allylheptoate was dissolved in DM50 and tested in concentrationsof 15 to 5000 µg per plate in the absence of S9 and of 0.5 to 500 µg per plate in the presence of S9. In the absence of S9 Allylheptoate was bacteriotoxic at 500 µg/plate towards all strains. In the presence of S9 Allylheptoate was bacteriotoxic towards the strains TA98 and TA1535 at 50 µg/plate and towards the strains TA100, TA102, and TA1537 at 150 µg/plate.

Sodium azide, 2-nitrofluorene, 9-aminoacridine, mytomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.
In the concentration range investigated, Allylheptoate did not induce a significant increase in the mutation frequency in the tester strains in the absence and presence of a metabolic activation system.

Reference 3

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform GLP study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - 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:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: for seeding and treatment of the cell cultures medium was MEM (minimal essential medium) containing Hank's salts, 10 % FBS (except during 4 hour treatment), neomycin (5 µg/mL) and amphotericin B (1 %). For selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I:
4 h (-S9 mix): 12.5, 25.0, 50.0, 100.0, 200.0, 400.0, 800.0, 1600.0 µg/mL
4 h (+S9 mix): 3.1, 6.3, 12.5, 25.0, 50.0, 75.0 µg/mL

Experiment II:
24 h (-S9 mix): 12.5, 25.0, 50.0, 100.0, 200.0, 400.0, 800.0, 1600.0 µg/mL
4 h (+S9 mix): 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen according to its solubility properties and its non-toxicity to the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration:
experiment I: 4 h (±S9 mix)
experiment II: 24 h (-S9 mix), 4 h (+S9 mix)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): after 7 days of expression time

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 1.5x10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10^6 cells found in the solvent controls fall within the laboratory historical control data range.
- the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
- the cloning efficiency II (absolute value) of the solvent controls must exceed 50%.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together (see Overal remarks, attachments).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 200 µg/mL (-S9mix) and at 75 µg/mL (+S9mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:

other: strain/cell type: V79

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

The study was performed to investigate the potential of Allylcaproate to induce gene mu­tations at the HPRT locus in V79 cells of the Chinese hamster. The study was performed in two independent complete experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum concentration of the pre-experiment (1600 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and phase separation. DMSO was used as solvent. The tested concentrations are:

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	12.5	25.0		50.0		100.0	200.0^PS	400.0^PS		800.0^PS		1600.0^PS
4 hours	+	3.1		6.3		12.5		25.0		50.0		75.0
		Experiment II
24 hours	-	12.5	25.0		50.0		100.0	200.0	400.0^PS		800.0^PS		1600.0^PS
4 hours	+	6.3		12.5		25.0		50.0		100.0		200.0^PS

PS  phase separation visible at the end of treatment

The evaluated experimental points and the results are summarised in Table1 (see section "Attached background material").No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. It can be stated that under the ex­perimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Allylcaproate is considered to be non-mutagenic in this HPRT assay.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: from human donors

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a 27 year-old male donor for the first experiment and from a 29 year-old female donor for Experiment II. Blood samples were drawn by venous puncture and collected in heparinised tubes.
- Type and identity of media: The culture medium was DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1) already supplemented with 15 mM HEPES. The antibiotic solution contained 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. Additionally, the medium was supplemented with the mitogen Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), 10 % FBS (fetal bovine serum), the anticoagulant heparin (25,000 U.S.P.-U/mL) and L-glutamin 200 mM. All incubations were done at 37 °C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).
- Properly maintained: yes
Treatments were commence at around 44 - 48 hours after culture initiation when the cells are actively proliferating.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment:
- without metabolic activation, 4 hours treatment: 10.2, 17.8, 31.2, 54.7, 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
- with metabolic activation, 4 hours treatment: 10.2, 17.8, 31.2, 54.7, 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Dose selection of Experiment II was based on the results obtained in Experiment I.
Experiment II:
- without metabolic activation, 20 hours treatment: 31.2, 54.7, 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
- with metabolic activation, 4 hours treatment: 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
For more detail see table under "any other information on material and methods incl. tables".

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO (E. MERCK, 64293 Darmstadt, Germany; purity 99.9 %). The final concentration of DMSO in the culture medium was 0.5 % (v/v).
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Demecolcin; 4.0 µg/mL (Exp.I) and 0.05 µg/mL (Exp.II), dissolved in deionised water

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation Migrated to IUCLID6: 1.0 µg/mL (Exp.I) and 0.3 µg/mL (Exp.II), dissolved in deionised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation Migrated to IUCLID6: 12.5 µg/mL (Exp. I) and 10.0 µg/mL (Exp. II), dissolved in 0.9% saline

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (+16 hours recovery period) or 20 hours; Cells were either treated for 4 hours in the absence of S9 mix (Exp.I) and in the presence of S9 mix (Exp.I and II) or for 20 hours without S9 mix (Exp.II).
The culture medium was replaced with serum-free medium (Experiment I without S9 mix; Experiment I and II in with S9 mix) or complete medium with 10 % FBS (v/v) (Experiment II without S9 mix), containing the test item.
- Recovery period: After treatment for 4 hours, the cells were re-suspended in "saline G" and incubatet for further 16 hours.
- Cytokinesis blocker: After washing, the cells were re-suspended in complete culture medium containing Cytochalasin B (4 µg/mL) and cultured another approximately 20 hours until preparation.
- Fixation time (start of exposure up to fixation or harvest of cells): The cultures were harvested by centrifugation 40 hours after beginning of treatment. Thereafter the cells were fixed and slides were prepared.

STAIN (for cytogenetic assays): The cells were stained with Giemsa.

NUMBER OF REPLICATIONS: In each experimental group two parallel cultures were analysed.

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

DETERMINATION OF CYTOTOXICITY
- Method: other: cytokinesis block proliferation index
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the cytokinesis block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described for the mutagenicity assay.
The pre-experiment was performed with 10 concentrations of the test item and the negative, solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hours (with and without S9 mix) and the cells were prepared 40 hours after start of the exposure.

OTHER EXAMINATIONS:
The frequency of micronucleated cells was reported as % micronucleated cells.

OTHER: Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area.

Evaluation criteria:

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory’s historical control data and
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.

Statistics:

Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together.

Species / strain:

lymphocytes: from humans

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

In Experiment I no cytotoxicity was observed up to the highest applied concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: from humans

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

In Experiment II in the presence of S9 mix no cytotoxicity was observed up to the highest applied concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: from humans

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at the two highest evaluated concentrations. However, in Experiment II in the presence of S9 mix, the highest applied concentration was not evaluable due to a reduced cell number.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the pre-test on toxicity, precipitation of the test item was observed at the end of treatment at 512.7 µg/mL and above in the presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA: In Experiment I, in the presence of S9 mix and in Experiment II in the absence of S9 mix, statistically significant increases in cells carrying micronuclei were observed. These values were in the range of the laboratory’s historical solvent control data and therefore considered as being biologically irrelevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no further data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Experiment I

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, Allyl capronate (Sym09 /611045) is considered to be non-aneugenic/clastogenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

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

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: for seeding and treatment of the cell cultures medium was MEM (minimal essential medium) containing Hank's salts, 10 % FBS (except during 4 hour treatment), neomycin (5 µg/mL) and amphotericin B (1 %). For selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I:
4 h (-S9 mix): 12.5, 25.0, 50.0, 100.0, 200.0, 400.0, 800.0, 1600.0 µg/mL
4 h (+S9 mix): 3.1, 6.3, 12.5, 25.0, 50.0, 75.0 µg/mL

Experiment II:
24 h (-S9 mix): 12.5, 25.0, 50.0, 100.0, 200.0, 400.0, 800.0, 1600.0 µg/mL
4 h (+S9 mix): 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen according to its solubility properties and its non-toxicity to the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration:
experiment I: 4 h (±S9 mix)
experiment II: 24 h (-S9 mix), 4 h (+S9 mix)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): after 7 days of expression time

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 1.5x10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10^6 cells found in the solvent controls fall within the laboratory historical control data range.
- the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
- the cloning efficiency II (absolute value) of the solvent controls must exceed 50%.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together (see Overal remarks, attachments).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 200 µg/mL (-S9mix) and at 75 µg/mL (+S9mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:

other: strain/cell type: V79

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

The study was performed to investigate the potential of Allylcaproate to induce gene mu­tations at the HPRT locus in V79 cells of the Chinese hamster. The study was performed in two independent complete experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum concentration of the pre-experiment (1600 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and phase separation. DMSO was used as solvent. The tested concentrations are:

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	12.5	25.0		50.0		100.0	200.0^PS	400.0^PS		800.0^PS		1600.0^PS
4 hours	+	3.1		6.3		12.5		25.0		50.0		75.0
		Experiment II
24 hours	-	12.5	25.0		50.0		100.0	200.0	400.0^PS		800.0^PS		1600.0^PS
4 hours	+	6.3		12.5		25.0		50.0		100.0		200.0^PS

PS  phase separation visible at the end of treatment

The evaluated experimental points and the results are summarised in Table1 (see section "Attached background material").No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. It can be stated that under the ex­perimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Allylcaproate is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

- genetic toxicity in vitro:

The mutagenicity of the substance Allyl heptanoate was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102) with the conditions of validity of current Guidelines (OECD 471). In the concentration range investigated, Allyl heptanoate did not induce a significant increase in the mutation frequency in the tester strains in the absence and presence of a metabolic activation system (King 1999).

From the read across substance allyl hexanoate (see section 13) it was shown that the test item is not mutagenic or clastogenic for in vitro micronucleus assay (Bohnenberger 2010). From the read across substance allyl hexanoate (see section 13) it was shown that the test item is negative for mammalian cell gene mutation assay (Wollny 2013).

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential of allyl heptanoate is deemed non-genotoxic and does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.