Allyl 3-cyclohexylpropionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative (±S9 mix) HPRT test: negative (±S9 mix) MNT test: negative (± S9 mix)

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:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Non-GLP study similar to OECD TG 471, only four relevant strains are tested

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only four relevant strains tested

Principles of method if other than guideline:

A standard plate assay was followed according to Ames et al. (1975): Ames BN, McCann J and Yamasaki E, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Research 31, 347-363, 1975

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor-pretreated rats

Test concentrations with justification for top dose:

5 concentrations tested per strain, no details on concentrations provided, concentration up to 3.6 mg/plate tested

Vehicle / solvent:

Dimethylsulphoxide may have been used as solvent

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: soidum azide, benzo[a]pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation), Vogel-Bonner medium was used

DURATION
- Preincubation period: if applicable, then liquid pre-incubation for 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Positive result if 1) statistical significance, 2) a reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency; agents producing reproducible, dose-related and significant (p equal to or less than 0.01) but less than two-fold elevations were classified as marginally mutagenic

Statistics:

Statistical significance in difference between test groups and negative controls was determined according to the methods of Kastenbaum and Bowman (Tables for determining the statistical significance of mutation frequencies, Mutation Research 9, 527-549, 1970)

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No further details on results reported

Number of revertants in positive controls with sodium azide: 400 to 700 in TA100, 430 to 760 in TA1535

Number of revertants in positive controls with benzo[a]pyrene: 660 to 1000 in TA98, 865 to 1210 in TA100, 235 to 350 in TA1537, 410 to 590 in TA1538

Conclusions:

The substance was non-mutagenic in an Ames test with Salmonella typhimurium in the absence and presence of metabolic activation (S9-mix).

Executive summary:

The potential of the substance allyl 3-cyclohexylproprionate to cause reverse mutations in bacteria (Salmonella typhimurium) was studied in a non-GLP Ames test with the strains TA98, TA100, TA1535, TA1537 and TA1538. The standard plate testing procedure was followed (Ames et al. 1975) that was widely similar to OECD TG 471, with the exception that only four relevant bacterial strains were tested. Overnight bacterial cultures had cell titres of at lest 10E09 cells/mL. S9-mix was prepared from Aroclor-pretreated rats (intraperitoneal injection of 500 mg/kg bw) and adjusted to 25 mg protein/mL. An aliquot of 0.5 mL S9-mix equivalent to 50 µL S9 -mix was incorporated into the plates. Dimethylsulphoxide was used as solvent for test substances that were poorly soluble in water. Five doses of the substance were tested (up to 3.6 mg/plate) in all five tester strains with and without S9-mix. Vogel-Bonner medium was used and plates were incubated for 48 hours. Positive controls were run in parallel with the reference substances sodium azide (0.5 µg/plate tested with TA100 and TA1535) and benzo[a]pyrene (5 µg/plate tested with TA98, TA100, TA1537 and TA1538). The substance was tested at least twice. The substance allyl 3-cyclohexylproprionate was not mutagenic in any of the tested bacterial strains in the absence and presence of metabolic activation system under the conditions of the test.

Reference 2

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:

2012-06-26 to 2013-04-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimal essential medium (MEM) containing Hank's salts, 10 % FBS (exept during 4 hour treatment), neomycin (5 µg/mL) and amphotericin B (1 %).
- 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:

S9 mix

Test concentrations with justification for top dose:

Dose selection
Experiment I:
4-hour exposure group (-S9 mix): 1.9, 3.8, 7.5, 15.0, 30.0, 60.0, 90.0, 120.0
4-hour exposure group (+S9 mix): 3.8, 7.5, 15.0, 30.0, 60.0, 90.0

Experiment II:
24-hour exposure group (-S9 mix): 1.9, 3.8, 7.5, 15.0, 30.0, 60.0, 120.0, 180.0
4-hour exposure group (+S9 mix): 7.5, 15.0, 30.0, 60.0, 90.0, 120.0


The evaluated cell cultures
Experiment I:
4-hour exposure group (-S9 mix): 1.9, 3.8, 7.5, 15.0, 30.0
4-hour exposure group (+S9 mix): 7.5, 15.0, 30.0, 60.0, 90.0

Experiment II:
24-hour exposure group (-S9 mix): 1.9, 3.8, 7.5, 15.0, 30.0
4-hour exposure group (+S9 mix): 15.0, 30.0, 60.0, 90.0, 120.0

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. The final concentration of DMSO in the culture medium was 0.5 % v/v.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 h (±S9 mix), 24 h (-S9 mix)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days

SELECTION AGENT (mutation assays): 6-Thioguanine
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1.5X10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concenentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

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 was 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.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 30 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 60 µg/mL and above

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
- Effects of osmolality: no
- Evaporation from medium: no data
- Water solubility: turbdity indicating beginning phase separation was observed at 90.0 µg/mL (4-hour exposure, +S9 mix in Experiment I) and at and above 90.0 µg/mL (4-hour exposure, +S9 mix in Experiment II).
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Allyl Cyclo Hexyl Propionate is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The purpose of this study is to assess the potential mutagenicity of a test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. The test methods described are designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No 440/2008 of 30 May 2008 and US EPA OPPTS 870.5300 Guideline.

The study was performed to investigate the potential of Allyl Cyclo Hexyl Propionate 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 (2000 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 insolubility. DMSO was used as solvent.

The tested concentrations and evaluated experimental points are described in the part "Test concentrations" and the results are summarised in Table 1 (see Attached document).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, Allyl Cyclo Hexyl Propionate is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2012-06-20 to 2013-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/Ham's F12 medium, mixture 1:1) supplemented with 200 mM GlutaMax
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Mammalian microsomal fraction S9 Mix

Test concentrations with justification for top dose:

pre-test on cytotoxicity: 4.2 to 1963.0 µg/mL

Further concentrations please see Table 1

The highest treatment concentration in this study, 1963.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; final concentration of DMSO in the culture medium was 0.5 % (v/v)

- Justification for choice of solvent: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

mitomycin C

other: Demecolcin

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48h
- Exposure duration: 4 or 20h (see Table 1)
- Expression time (cells in growth medium): cells exposed for 4h have 16h recovery period before fixation, no recovery period for 20h exposure cells
- Fixation time (start of exposure up to fixation or harvest of cells): 20h with Cytochalasin B (4 µg/mL)
- Cells were prepared 40 hrs after start of the exposure

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
cytotoxic effect the CBPI: ca 500 cells per culture and cytotoxicity is expressed as % cytostasis
micronuclei effects: 1000 binucleate cells per culture. The frequency of micronucleated cells was reported as % micronucleated cells

DETERMINATION OF CYTOTOXICITY
- percentages of reduction in the CBPI (cytokinesis-block proliferating index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate
- Exposure time 4 hrs (with and without S9 mix), cells were prepared 40 hrs after start of the exposure

Evaluation criteria:

cytotoxic effect: percentages of reduction in the CBPI (cytokinesis-block proliferating index) in comparison with the controls (% cytostasis)

micronuclei effects: 1000 binucleate cells per culture. The frequency of micronucleated cells was reported as % micronucleated cells

criteria for the evaluation of micronuclei:
The micronuclei were counted in cells showing a clearly visible cytoplasm area. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.


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 historical control data and/or
- 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:

For statistical significance: Chi square test

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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
- Effects of osmolarity: no
- Evaporation from medium: no
- Water solubility: no data
- Precipitation: no
- Other confounding effects: no

COMPARISON WITH HISTORICAL CONTROL DATA: no

ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Table 2: Summary of results of the in vitro micronucleus test in human lymphocytes with Allyl Cyclo Hexyl Propionate

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
Exposure period 4 hrs without S9 mix
I	40 hrs	Solvent control^1	1.94		0.35
		Positive control^2	1.76	19.7	7.75^S
		39.1	1.96	n.c.	0.30
		641.0^PS	2.00	n.c.	0.05
		1121.7^PS	2.00	n.c.	0.30
		1963.0^PS	2.01	n.c.	0.15
Exposure period 20 hrs without S9 mix
IIA	40 hrs	Solvent control^1	1.88		0.35
		Positive control^3	1.55	38.1	3.05^S
		39.1	1.85	3.2	0.30
		68.3	1.80	8.8	0.60
		119.6	1.61	30.6	0.40
IIB	40 hrs	Solvent control^1	1.89		0.40
		Positive control^4	1.41	54.5	6.55^S
		300.0	1.58	34.5	0.55
		350.0	1.53	40.6	0.20
		400.0	1.52	41.4	0.65

*     For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^PS    Phase separation occurred microscopically at the end of treatment

^S     The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

¹     DMSO             0.5 % (v/v)

²           MMC                2.0 µg/mL

³           Demecolcin  150.0 ng/mL

⁴           Demecolcin    75.0 ng/mL

 

Table 2 (cont.): Summary of results of the in vitro micronucleus test in human lymphocytes withAllyl Cyclo Hexyl Propionate

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
Exposure period 4 hrs with S9 mix
I	40 hrs	Solvent control^1	2.07		0.45
		Positive control^2	1.48	55.1	3.30^S
		39.1	2.00	6.8	0.50
		641.0^PS	1.96	10.1	0.40
		1121.7^PS	1.98	8.4	0.40
		1963.0^PS	1.89	16.5	0.20
IIA	40 hrs	Solvent control^1	2.10		0.65
		Positive control^2	1.33	69.9	5.75^S
		68.3	2.10	0.1	0.50
		641.0^PS	2.02	7.4	0.65
		1121.7^PS	2.06	3.8	0.45
		1963.0^PS	1.97	11.7	0.50

*     For the positive control groups and for the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^PS    Phase separation occurred at the end of treatment

^S     The number of micronucleated cells is statistically significantly higher than corresponding control values

¹     DMSO      0.5 % (v/v)
²           CPA        15.0 µg/mL

 

Conclusions:

Under the experimental conditions reported, the test item Allyl Cyclo Hexyl Propionate did not induce micronuclei in human lymphocytes in vitro when tested up to the highest required or evaluable concentrations

Executive summary:

The test item Allyl Cyclo Hexyl Propionate, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytesin vitroin the absence and presence of metabolic activation by S9 mix.

Three independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment IIA, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. In Experiment IIB, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis.

The highest treatment concentration in this study, 1963.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

No precipitation of the test item in the culture medium was observed at the end of treatment. No relevant influence on osmolarity or pH value was observed. Phase separation was observed in Experiment I at 68.3 µg/mL and above in the absence and presence of S9 mix and in Experiment IIA at 119.6 µg/mL in the presence of S9 mix.

In Experiment I in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA and IIB in the absence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. However, in Experiment IIB with narrow concentration spacing a cytostasis of 41.4 % was reached at the highest evaluated concentration and the total cell number was already markedly reduced (not determined quantitatively).

In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.05 – 0.65 % micronucleated cells) did not exceed the range of the solvent control values (0.35 – 0.65 % micronucleated cells) and were within the range of the laboratory historical control data.

Either Demecolcin (75.0 or 150.0 ng/mL), MMC (2.0 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item Allyl Cyclo Hexyl Propionate did not induce micronuclei in human lymphocytes in vitro when tested up to the highest required or evaluable concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance was considered to be not mutagenic in an in-vivo mouse bone marrow micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

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

Remarks:

The study was performed under non-GLP conditions but in accordance with principles similar to those of OECD TG 475

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

GLP compliance:

no

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: Ivanovas GmbH, Kisslegg
- Age at study initiation: 10 to 14 weeks
- Weight at study initiation: not reported
- Assigned to test groups randomly: not reported
- Fasting period before study: not reported
- Housing: not reported
- Diet (e.g. ad libitum): Altromin standard chow ad libitum
- Water (e.g. ad libitum): not reported
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

IN-LIFE DATES: not reported

Route of administration:

intraperitoneal

Vehicle:

Olive oil

Details on exposure:

Test substance was mixed with olive oil and administered intraperitoneal.

Duration of treatment / exposure:

Test animals received an intraperitoneal injection of test substance at 0 and 24 hours.

Frequency of treatment:

Twice

Post exposure period:

Animals were killed 30 hours after the last dose.

Dose / conc.:

156 mg/kg bw/day (nominal)

Remarks:

in olive oil as injected

Dose / conc.:

78 mg/kg bw/day (nominal)

Remarks:

in olive oil as injected

Dose / conc.:

49 mg/kg bw/day (nominal)

Remarks:

in olive oil as injected

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

in olive oil as injected

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

No

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

Bone marrow smears were prepared 30 hours after the last treatment. The smears were stained with May-Grünwald and Giemsa according to the method of Schmid (1976, The micronucleus test for cytogenetic analysis. In Chemical Mutagens. Edited by A. Hollaender. Vol. 4, page 31. Plenum Press, New York).

Evaluation criteria:

Slides were scored as described by Wild and co-workers (1980, Cytogenetic effect of orthophenylenediamine in the mouse, Chinese hamster, and guinea pig and of derivatives, evaluated by micronucleus test. Archives of Toxicology 43, 249-255). 1000 polychromatic erythrocytes from each animal were analysed for micronuclei. An increased frequency of micronucleus-containing polychromatic erythrocytes indicates cytogenetic damage. The proportion of polychromatic among total (poly- and normochromatic) erythrocytes is based on the differentiation of a total number of 1000 erythrocytes.

Statistics:

Statistical significance in difference between test groups and negative controls was determined according to the methods of Kastenbaum and Bowman (Tables for determining the statistical significance of mutation frequencies, Mutation Research 9, 527-549, 1970)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

no signs of toxicity were reported

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): there was no induction of formation of micronuclei
- Ratio of PCE/NCE (for Micronucleus assay): not reported
- Appropriateness of dose levels and route: yes
- Statistical evaluation: not reported

Conclusions:

The substance was considered to be not mutagenic in an in-vivo mouse bone marrow micronucleus assay.

Executive summary:

An in-vivo mouse bone marrow micronucleus assay was conducted under non-GLP conditions according to principle similar to those of OECD TG 475. Male and female NMRI mice aged 10 to 14 weeks were used in the test. The test substance allyl 3-cyclohexylproprionate was mixed with olive oil and administered to four animals per dose group twice by intraperitoneal injection at 0 and 24 hours. The substance was tested at 49, 78 and 156 mg/kg bw. A group receiving olive oil only was tested in parallel. Animals were sacrificed 30 hours after the last injection, bone marrow was isolated and smears were prepared and stained with May-Grünwald and Giemsa following the method of Schmid (1976). A total of 1000 polychromatic erythrocytes from each animal was analysed for micronuclei. Results were expressed as mean number of micronucleated polychromatic erythrocytes (MNPE) per 1000 polychromatic erythrocytes (PE) and the numbers found in the experiment were 2.2 MNPE/1000 PE in the controls, 1.8 MNPE/1000 PE in the group exposed to 49 mg/kg bw, 1.7 MNPE/1000 PE in the group exposed to 78 mg/kg bw and 4.0 MNPE/1000 PE in the group exposed to 156 mg/kg bw. The authors of the study found that the findings in the exposed animals were not statistically different from the results observed in the controls. It was concluded that the test substance allyl 3-cyclohexylproprionate was not mutagenic in the in-vivo mouse bone marrow micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:
Three genetic toxicity in vitro assays were conducted with the substance covering different modes of action of genetic toxicity. The substance did not produce significant genetic toxicity in a reliable bacterial reverse mutation assay with S. typhimurium and E. coli, in a in vitro micronucleus assay with human lymphoctyes and in an in vitro Gene Mutation Assay in Chinese Hamster V79 Cells (V79/HPRT). It is therefore concluded that the substance is not genotoxic. Further testing on the genetic toxicity of allylcyclohexylpropionate is not required.

Justification for selection of genetic toxicity endpoint: in vivo
A GLP and guideline study was performed in mammalian cells to detect the activity of clastogenic and aneugenic chemicals. A total of 1000 polychromatic erythrocytes from each animal was analysed for micronuclei.  The authors of the study concluded that the findings in the exposed animals were not statistically different from the results observed in the controls. It was therefore stated that the test substance allyl 3-cyclohexylproprionate was not mutagenic in the in-vivo mouse bone marrow micronucleus assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available studies are considered reliable for this assessment.

 

The substance was neither mutagenic in a battery of three reliable in vitro assays nor clastogenic in a reliable bone marrow micronucleus assay. The substance is considered to be not genotoxic. The substance does not need to be classified for this endpoint according to to Regulation (EC) No 1272/2008 (CLP), as amended for the 17th time in Regulation (EU) 2021/849.