Benzyl propionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2015 and March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl propionate

EC Number:

204-559-3

EC Name:

Benzyl propionate

Cas Number:

122-63-4

Molecular formula:

C10H12O2

IUPAC Name:

benzyl propanoate

Method

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Doubling time: 12 – 16 h
- Modal number of chromosomes: 22


MEDIA USED
- Type and identity of media including CO2 concentration: Minimum Essential Medium (MEM) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1%)
- 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:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Concentrations applied: 12.5, 25.0, 50.0, 100.0, 200.0 and 300.0 µg/mL
The maximum test item concentration of the pre-experiment (1642 μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiment was limited by phase separation of the test item.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

- Cell density at seeding: 0.7 – 1.2 x 10E+7 cells

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time: 8 days

SELECTION AGENT: 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: two independent experiments with each 5 replicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The colonies were stained with 10% methylene blue in 0.01% KOH solution.
NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted.

DETERMINATION OF CYTOTOXICITY
- Method: adjusted cloning efficiency

- OTHER: In a pre-experiment the medium was checked for precipitation as well as pH and osmolarity changes.

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated 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 was considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Other confounding effects: Phase separation occurred at 300 µg/mL at the beginning and at the end of treatment with and without S9 mix.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 12.8 μg/mL and 1642.0 μg/mL (equal to a molar concentration of approximately 10 mM) were used. In the pre-experiment a relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 410.5 μg/mL and above in the absence of metabolic activation. In the presence of metabolic activation no relevant cytotoxic effect was determined up to the highest concentration.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Phase separation occurred at 205.3 μg/mL and above after 4 hours treatment without and 102.6 μg/mL and above with metabolic activation. The dose range of the main experiment was set based on the main experiment. The individual concentrations were spaced by a factor of 2.0. Narrower spacing was used at the two highest concentrations to cover possible toxic effects more closely.

HISTORICAL CONTROL DATA
- Positive historical control data (in mutant colonies per 10E+6 cells):
EMS: range = 53.9 – 889.0, mean = 153.0, SD = 88.5
DMBA: range = 59.6 – 2042.6, mean = 424.6, SD = 291.4

- Negative (solvent/vehicle) historical control data (in mutant colonies per 10E+6 cells):
+ S9: Solvent control (water, DMSO, medium): range = 1.6 – 42.8, mean = 15.0, SD = 7.4, 95% CL = 0.2 – 29.7
- S9: Solvent control (water, DMSO, medium): range = 2.4 – 44.2, mean = 14.6, SD = 7.0, 95% CL = 0.6 – 28.7

Any other information on results incl. tables

Summary of results

	Conc. μg/mL	PS	S9 mix	Relative cloning efficiency I	Relative cell density	Relative adjusted CE I	Mutant colonies / 10^6cells	95% confidence interval	Relative cloning efficiency I	Relative cell density	Relative adjusted CE I	Mutant colonies / 10^6cells		95 % confidence interval
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12		13
Experiment I /4h treatment				Culture I					Culture II
Solvent control with DMSO			-	100.0	100.0	100.0	17.0	0.2 – 29.7	100.0	100.0	100.0		6.4	0.2 – 29.7
Positive control (EMS)	300.0		-	123.9	67.9	84.1	300.3	0.2 – 29.7	81.9	95.3	78.1		211.7	0.2 – 29.7
Test item	12.5		-	124.3	71.2	88.5	#		94.7	95.0	89.9		#
Test item	25.0		-	113.4	111.8	126.8	19.1	0.2 – 29.7	116.8	80.5	94.1		9.2	0.2 – 29.7
Test item	50.0		-	118.6	88.0	104.4	19.2	0.2 – 29.7	99.9	72.3	72.3		15.2	0.2 – 29.7
Test item	100.0		-	115.7	74.9	86.7	24.5	0.2 – 29.7	98.3	88.4	86.9		8.6	0.2 – 29.7
Test item	200.0		-	101.0	96.7	97.6	12.9	0.2 – 29.7	49.8	83.2	41.5		16.3	0.2 – 29.7
Test item	300.0	PS	-	86.4	80.7	69.7	24.2	0.2 – 29.7	54.6	65.9	36.0		17.9	0.2 – 29.7
Solvent control with DMSO			+	100.0	100.0	100.0	6.4	0.6 – 28-7	100.0	100.0	100.0		16.6	0.6 – 28-7
Positive control (DMBA)	2.2		+	75.6	102.1	77.3	95.6	0.6 – 28-7	94.0	128.3	120.6		133.6	0.6 – 28-7
Test item	12.5		+	76.6	118.9	91.1	#		98.4	112.9	111.1		#
Test item	25.0		+	69.5	101.1	70.3	8.7	0.6 – 28-7	102.5	121.2	124.3		17.9	0.6 – 28-7
Test item	50.0		+	86.8	101.6	88.2	12.3	0.6 – 28-7	102.8	86.9	89.3		15.0	0.6 – 28-7
Test item	100.0		+	79.8	95.0	75.8	12.3	0.6 – 28-7	102.3	103.5	105.9		24.1	0.6 – 28-7
Test item	200.0		+	66.1	106.8	70.6	11.5	0.6 – 28-7	93.5	100.4	93.8		10.5	0.6 – 28-7
Test item	300.0	PS	+	65.9	107.5	70.8	18.6	0.6 – 28-7	89.1	97.5	86.9		6.7	0.6 – 28-7

PS = Phase separation at the beginning and at the end of treatment

# = culture was not continued as a minimum of only four analysable concentrations is required

Applicant's summary and conclusion

Conclusions:

The test item did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Thus, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

A GLP conform study according to OECD TG 476 was performed to assess the potential of the test item to induce gene mutations at the HPRT locus in V79 cells. An appropriate cell number was treated with test item concentrations of 12.5, 25.0, 50.0, 100.0, 200.0 and 300.0 µg/mL for 4 hours in the absence and presence of S9 mix.

Phase separation was observed at the highest concentration of 300 μg/mL in the presence and absence of metabolic activation.

No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation. Moderate cytotoxic effects limited to just one of the parallel cultures were noted at 200 and 300 μg/mL without metabolic activation.

The 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.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Thus, the test item did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Thus, the test item is considered to be non-mutagenic in this HPRT assay.