Octanoic acid, 2-phenoxyethyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

Chromosome aberration test (OECD 473): negative in cultured peripheral human lymphocytes cells with and without metabolic activation

HPRT test (OECD 476): negative in Chinese Hamster V79 cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Nov - 8 Dec 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted in 2008

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His operon (S. typhimurium strains)
Trp operon (E. coli strains)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and beta-naphtoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate with and without metabolic activation for TA100 and WP2uvrA

Main study (experiment 1 and 2):
10, 33, 100, 333 and 1000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance is soluble and stable in the vehicle throughout the test

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-nitrofluorene (NF; 10 μg/plate in DMSO, -S9, TA98); 2-Amino-anthracene (2-AA; 1 to 10 μg/plate in DMSO, +S9, all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test substance is considered positive if the total number of revertants is greater than 2 (TA 100) or three (TA 98, TA 1535, TA 1537, WP2uvrA) times the concurrent control. Positive responses should be reproducible in at least one additional independent experiment. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 1000 μg/plate and above, the test material precipitated in the medium.

RANGE-FINDING/SCREENING STUDIES: In the range finding test, the test substance was tested up to 5000 µg/plate in the absence and presence of S9 mix in TA100 and WP2uvrA. No reduction of the bacterial lawn and no biologically relevant decrease in the number of revertants were observed. Due to the precipitation of the test substance in medium at a concentration of 1000 µg/plate and above, this concentration was for the main experiment (TA1535, TA1537 and TA98 in experiment 1 and all strains for experiment 2).

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain specific positive control values were within laboratory historical control data ranges (table 3, any other information on results)

Table 1. Test results of experiment 1 (plate incorporation) and range finding study (TA100 and WP2 uvrA)           

With or without S9-Mix (5%)	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		cross-linking type	Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	0	137 ± 1	12 ± 3	25 ± 6	29 ± 3	9 ± 3
–	3	139 ± 14	-	28 ± 6	-	-
–	10	133 ± 16	10 ± 3	24 ± 4	31 ± 2	6 ± 2
–	33	137 ± 9	8 ± 2	23 ± 4	34 ± 5	4 ± 2
–	100	131 ± 9	11 ± 3	24 ± 5	26 ± 8	6 ± 2
–	333	132 ± 7	7 ± 4	26 ± 7	27 ± 10	5 ± 2
–	1000	137 ± 19P	7 ± 1P	21 ± 3P	28 ± 4P	6 ± 3P
–	3330	140 ± 9P	-	17 ± 1P	-	-
–	5000	129 ± 13P	-	23 ± 4P	-	-
Positive controls, -S9	Name	MMS	SA	4NQO	NF	9AA
	Concentrations (μg/plate)	650	5	10	10	60
	Mean No. of colonies/plate (average of 3 ± SD)	1132 ± 23	933 ± 31	1271 ± 22	1122 ± 62	357 ± 38
+	0	140 ± 11	9 ± 2	26 ± 9	35 ± 6	6 ± 3
+	3	127 ± 8	-	24 ± 3	-	-
+	10	125 ± 10	9 ± 3	27 ± 5	37 ± 6	6 ± 2
+	33	130 ± 8	9 ± 2	25 ± 2	38 ± 7	4 ± 1
+	100	136 ± 6	9 ± 4	32 ± 7	34 ± 4	6 ± 1
+	333	117 ± 4	7 ± 2	26 ± 4	34 ± 5	6 ± 2
+	1000	125 ± 10P	5 ± 1P	26 ± 1P	34 ± 11P	6 ± 1P
+	3330	106 ± 7P	-	24 ± 6P	-	-
+	5000	102 ± 9P	-	21 ± 2P	-	-
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	2.5	10	1	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	1442 ± 40	406 ± 27	586 ± 62	1075 ± 21	539 ± 15

 

Table 2. Test results of experiment 2 (plate incorporation)

With or without S9-Mix (10%)	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		cross-linking type	Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	0	106 ± 11	12 ± 1	23 ± 5	29 ± 6	6 ± 1
–	10	123 ± 24	9 ± 2	23 ± 2	20 ± 5	6 ± 1
–	33	130 ± 20	8 ± 1	23 ± 2	21 ± 4	5 ± 2
–	100	103 ± 4	9 ± 1	26 ± 4	20 ± 5	3 ± 1
–	333	115 ± 6	10 ± 3	26 ± 4	18 ± 7	7 ± 4
–	1000	112 ± 9P	10 ± 4P	21 ± 5P	21 ± 5P	6 ± 2P
Positive controls, -S9	Name	MMS	SA	4NQO	NF	9AA
	Concentrations (μg/plate)	650	5	10	10	60
	Mean No. of colonies/plate (average of 3 ± SD)	921 ± 74	1022 ± 31	937 ± 20	1094 ± 8	322 ± 28
+	0	107 ± 4	8 ± 3	31 ± 5	36 ± 8	6 ± 2
+	10	94 ± 10	11 ± 1	29 ± 4	33 ± 9	4 ± 1
+	33	100 ± 5	10 ± 2	27 ± 4	27 ± 9	6 ± 1
+	100	105 ± 8	4 ± 1	34 ± 3	28 ± 2	7 ± 1
+	333	116 ± 9	8 ± 4	30 ± 4	23 ± 5	4 ± 1
+	1000	82 ± 5P	9 ± 1P	36 ± 3P	13 ± 6P	5 ± 2P
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	10	1	5
	Mean No. of colonies/plate (average of 3 ± SD)	1261 ± 141	260 ± 43	317 ± 67	541 ± 123	526 ± 12

                                  

MMS = methylmethanesulfonate

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

SA = sodium azide

NF = 7-nitrofluorene

P = Precipitate

Table 3. Laboratory historical control data

	TA1535		TA1537		TA98		TA100		WP2uvrA
Negative control	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Min	3	3	3	3	12	12	63	60	4	4
Max	28	29	19	21	45	51	194	195	44	44
Mean	12	11	7	6	21	26	119	99	19	20
3xSD	23	23	92	83	22	19	10	9	14	14
	TA1535		TA1537		TA98		TA100		WP2uvrA
Positive control	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Min	375	58	79	57	286	186	458	228	67	56
Max	1923	538	927	833	1790	1703	1593	2061	1403	1146
Mean	1061	202	352	370	1059	736	1057	1056	849	315
3xSD	596	229	389	383	545	935	530	991	892	377

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Oct - 21 Dec 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

(200 metaphases scored)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in Jul 2016

Deviations:

yes

Remarks:

(200 metaphases scored)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in 2008

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Collected from healthy adult, non-smoking, male volunteers
- Sex, age and number of blood donors: Dose range stude (male, 34 years, average generation time = 14.6 h), first cytogenetic study (male, 27 years, average generation time = 14.4 h), second cytogenetic study (male, 25 years, AGT = 14.5 h)
- Whole blood was used

MEDIA USED
- Type and identity of media including CO2 concentration:
RPMI 1640 medium supplemented with
- 20% (v/v) heat-inactivted foetal calf serum
- L-glutamine (2 mM)
- penicillin/streptomycin (50 U/mL and 50 µg/mL)
- 30 U/mL heparin
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of adult male wistar rats treated with phenobarbital and ß-naphthoflavone in corn oil

Test concentrations with justification for top dose:

Experiment I
3 h treatment with and without metabolic activation: 10, 33 and 100 µg/mL
Experiment II
24 and 48 h treatment without metabolic activation: 10, 100 and 150 µg/mL
3 h treatment with metabolic activation: 10, 33 and 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h, 24h and 48h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment: 48 h fixation time, 24 h treatment: 24 h fixation time, 48 h treatment: 48 h fixation time

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 µg/mL culture medium
STAIN (for cytogenetic assays): Giemsa Solution 5% (v/v) in tap water

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 (Parallel cultures were treated at each conenctration. 100 metaphases per culture were scored.)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p< 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0,05) increase in the number of cells with chromosome aberrations.

Statistics:

Chi-square test, one sided, p < 0.05

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was already seen at a concentration of 100 µg/mL at the 3-, 24- and 48-h exposure time without metabolic activation and at the 3-h exposure time with metabolic activation.
- Other confounding effects:
1) To make sure a positive response was observed the positive control cultures were exposed to 2 concentrations of MMC-C. The concentration stated and a double amount was used. Since the concentration stated in the protocol gave a positive response, the higher concentration was not used for scoring chromosomal aberrations.
2) In the first cytogenetic assay the spindle inhibitor colchicine was added 7 min late, 2 h and 23 min before end of experiment. However, the number of cells in metaphase was within the normal range and the deviation was considered to have no effect on the outcome of the study.
3) In the dose range finding study the lymphocytes were not cultured in duplicate and no positive control cultures were included. In the solubility test the test substance precipitated in the culture medium at the concentration of 333 µg/mL and therefore single lymphocytes cultures were cultured. This deviation in the study design has no influence on the results of the study.
4) In the first and second cytogenetic assay the culture temperature was below the range. However, these deviations occured at the beginning of the test only for a short time and the number of cells with aberrations found in the negative control were within the laboratory historical range.

RANGE-FINDING/SCREENING STUDIES:
The highest concentration analysed was selected based on the solubility of the test substance in culture medium or on toxicity, inhibition of the mitotic index of about 50% or greater.

COMPARISON WITH HISTORICAL CONTROL DATA:
Test results are in range with historical laboratory control data (for historical data see table 1 and 2 under any other informations on materials and methods incl. tables).

Table 1: Results of Range Finding Study

Without metabolic activation (-S9)	Number of metaphases per 1000 cells
3 h exposure time, 24 h fixation time (µg/mL)	Absolute	Percentage of control
DMSO	77	100
3	75	97
10	74	96
33	70	91
100 P	68	88
333 P	52	68
24 h exposure time, 24 h fixation time (µg/mL)
DMSO	72	100
3	71	99
10	68	94
33	35	90
100 P	63	88
333 P	22	31
1000 P	18	25
2644 P	15	21
48 h exposure time, 48 h fixation time (µg/mL)
DMSO	70	100
3	65	93
10	63	90
33	60	86
100 P	63	90
333 P	14	20
1000 P	9	13
2644 P	7	10
With metabolic activation (-S9)
3 h exposure time, 24 h fixation time (µg/mL)
DMSO	71	100
3	68	96
10	67	94
33	66	93
100 P	57	80
333 P	53	75

P: Precipitation in the culture medium

Table 2: Results of experiment 1

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 3h, fixation time 24h, without S9 mix
DMSO	1.0% (v/v)	100	0	0
MMC-C	0.5	87	33***	30***
Test substance	10	99	0	0
	33	97	1	1
	100 P	90	1	1
Exposure period 3h, fixation time 24h, with S9 mix
DMSO	1.0% (v/v)	100	0	0
CP	10	79	34***	31***
Test substance	10	92	0	0
	33	85	0	0
	100 P	79	1	1

Significantly different from control group (Chi-square test), *** P < 0.001

P: Precipitation

Table 3: Results of experiment 2

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 24h, fixation time 24h, without S9 mix
DMSO	1.0% (v/v)	100	1	1
MMC-C	0.2	39	57	53
Test substance	10	96	1	0
	100 P	66	1	1
	150 P	48	0	0
Exposure period 48h, fixation time 48h, without S9 mix
DMSO	1.0% (v/v)	100	3	1
MMC-C	0.1	64	76***	73***
Test substance	10	95	0	0
	100 P	66	0	0
	150 P	45	1	0
Exposure period 3h, fixation time 48h, with S9 mix
DMSO	1.0% (v/v)	100	1	1
CP	10	- a)	42***	41***
Test substance	10	96	0	0
	33	90	3	3
	100 P	89	1	0

a) CP was fixed after 24 hours, MI not calculated compared to 48 h control.

Significantly different from control group (Chi-square test), *** P < 0.001

P: Precipitation

Table 4: Historical control data for lymphocyte chromosome aberration studies

	aberrant cells per 100 metaphases
	gaps included		gaps excluded
	with S9	without S9	with S9	without S9
Range	0 - 6	0 - 6	0 - 5	0 - 5
Mean	0.7	0.8	0.6	0.6
SD	1.0	1.0	0.9	0.9
n	404	599	405	601

n = number of observations

SD = Standard Deviation

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Mar - 13 Jun 2017

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)

Version / remarks:

adopted in Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: In the cell bank at Eurofins BioPharma, Planegg, Germany
- Suitability of cells: Yes
- Proliferation rate: 12 - 14 h
- Methods for maintenance in cell culture: Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C incubation temperature.

MEDIA USED
- Type and identity of media: MEM medium supplemented with 10% fetal bovine serum, 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES and 2.5 µg/mL amphotericin B
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

Experiment I
Without S9 mix: 25, 50, 100, 150, 200, 250, 300, 400 and 500 µg/mL (4 h)
With S9 mix: 25, 50, 100, 150, 200, 250, 300, 400 and 500 µg/mL (4 h)

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: Based on the results of a solubility test DMSO was used as solvent.

Untreated negative controls:

yes

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: 3.0 x 10E6

DURATION
- Exposure duration: 4 h
- Expression time: 7- 9 days
- Selection time: 7 - 12 days

SELECTION AGENT: 11 µg/mL thioguanine

STAIN: Giemsa

NUMBER OF REPLICATIONS: Duplicates in 1 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative survival

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration-related increase of the mutations within their range has to be discussed.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was observed at 500 µg/mL without metabolic activation and at 400 and 500 µg/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4)
- Effects of osmolality: Osmolality of the highest test item concentration was 445 mOsm/kg (solvent control: 425 mOsm/kg).
- Precipitation: Precipitation of the test item was noted at concentrations of 500 μg/mL (without metabolic activation) and at concentrations of 400 and 500 μg/mL (with metabolic activation).

RANGE-FINDING/SCREENING STUDIES:
The selection of the concentrations used in the main experiments was based on data from the pre-experiments. No biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item with and without metabolic activation (please refer to Table 1 under "any other information on results incl. tables").

HISTORICAL CONTROL DATA (please refer to Table 4 under "any other information on results incl. tables")
- Positive historical control data: The positive control values were within the range of the historical control data.
- Negative (solvent/vehicle) historical control data: The negative and solvent control values were within the range of the historical control data.

Table 1: Summary of results of the range-finding test

Dose Group	Concentration [μg/mL]	Number of cells at the		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative Survival (RS) [%]
		beginning of treatment	end of treatment	I	II	mean
Without metabolic activation
NC1	0	4000000	3808000	165	150	158	79	75	113
NC2		4000000	3349000	169	147	158	79	66	100
S1	0	4000000	3383000	171	170	171	85	72	100
S2		4000000	3026000	178	143	161	80	61
1	25	4000000	3111000	137	191	164	82	64	96
2	50	4000000	3485000	168	163	166	83	72	109
3	100	4000000	2924000	180	172	176	88	64	97
4	250	4000000	2023000	173	173	173	87	44	66
5 P	500	4000000	2244000	164	147	156	78	44	66
6 P	1000	4000000	2380000	163	156	160	80	47	71
7 P	1500	4000000	2159000	188	160	174	87	47	71
8 P	2000	4000000	2278000	188	170	179	90	51	77
With metabolic activation
NC1	0	4000000	5287000	141	149	145	73	96	90
NC2		4000000	5695000	139	159	149	75	106	100
S1	0	4000000	5168000	154	164	159	80	103	100
S2		4000000	5542000	176	140	158	79	109
1	25	4000000	4658000	151	159	155	78	90	85
2	50	4000000	5066000	137	132	135	67	85	80
3	100	4000000	4437000	136	163	150	75	83	78
4	250	4000000	4029000	190	170	180	90	91	85
5 P	500	4000000	4437000	159	164	162	81	90	84
6 P	1000	4000000	3859000	171	163	167	84	81	76
7 P	1500	4000000	3655000	209	179	194	97	89	84
8 P	2000	4000000	3383000	170	165	168	84	71	67

NC: negative control

S: solvent control

P: precipitation at the end of treatment

CE: cloning efficiency

RS: relative survival

Table 2: Summary of results (experiment I)

		CE in non-selective medium				CE in selective medium
Dose Group	Concentration [μg/mL]	Number of colonies per flask			CE [%]	Number of colonies per flask							Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
Without metabolic activation
NC1	0	143	175	159	80	7	8	8	3	10	7.2	2.3	22.6
NC2		160	153	157	78	3	11	8	5	5	6.4	2.8	20.4
S1	0	155	154	155	77	7	8	8	9	5	7.4	1.4	23.9
S2		148	147	148	74	2	7	10	8	5	6.4	2.7	21.7
1	25.00	166	152	159	80	14	6	16	12	10	11.6	3.4	36.5
2	50.00	144	154	149	75	19	14	9	10	11	12.6	3.6	42.3
3	100.00	174	157	166	83	10	7	7	3	8	7.0	2.3	21.1
4	150.00	169	160	165	82	7	9	4	3	9	6.4	2.5	19.5
5	200.00	144	157	151	75	8	11	7	12	11	9.8	1.9	32.6
6	250.00	161	144	153	76	3	6	3	9	3	4.8	2.4	15.7
7	300.00	187	176	182	91	1	2	3	4	6	3.2	1.7	8.8
8	400.00	183	170	177	88	10	11	11	15	6	10.6	2.9	30.0
9 P	500.00	163	156	160	80	2	3	1	7	5	3.6	2.2	11.3
EMS	300 μg/ml	177	134	156	78	62	83	75	70	82	74.4	7.8	239.2
With metabolic activation
NC1	0	179	141	160	80	2	6	11	4	12	7.0	3.9	21.9
NC2		175	177	176	88	7	5	11	8	4	7.0	2.4	19.9
S1	0	153	156	155	77	8	8	7	2	10	7.0	2.7	22.7
S2		152	158	155	78	6	11	10	7	11	9.0	2.1	29.0
1	25.00	162	169	166	83	2	6	5	6	7	5.2	1.7	15.7
2	50.00	159	179	169	85	4	15	8	6	8	8.2	3.7	24.3
3	100.00	164	158	161	81	13	13	6	7	9	9.6	2.9	29.8
4	150.00	171	157	164	82	3	10	6	4	7	6.0	2.4	18.3
5	200.00	172	162	167	84	10	2	9	2	9	6.4	3.6	19.2
6	250.00	158	133	146	73	10	8	8	9	7	8.4	1.0	28.9
7	300.00	147	169	158	79	4	1	2	9	4	4.0	2.8	12.7
8	400.00	177	154	166	83	4	4	9	7	4	5.6	2.1	16.9
9 P	500.00	154	171	163	81	12	13	6	7	6	8.8	3.1	27.1
DMBA	1 μg/ml	158	158	158	79	93	101	100	97	129	104.0	12.8	329.1

NC: negative control

S: solvent control (DMSO)

P: precipitation at the end of treatment

CE: cloning efficiency

EMS: Ethylmethanesulfonate [300 μg/mL]

DMBA: 7,12-dimethylbenz(a)anthracene [1 μg/mL]

Table 3: Results of the statistical analysis

Dose Group	Concentration [μg/mL]	Mutant Frequency	p-value	Statistical Significance
Without metabolic activation
NC1	0	22.6	/	/
NC2		20.4	/
S1	0	23.9	/	/
S2		21.7	/	/
1	25.00	36.5	0.0373	+
2	50.00	42.3	0.0023	+
3	100.00	21.1	0.6540	-
4	150.00	19.5	0.6563	-
5	200.00	32.6	0.0500	-
6	250.00	15.7	0.2414	-
7	300.00	8.8	0.0077	+*
8	400.00	30.0	0.0926	-
9	500.00	11.3	0.0120	+*
EMS	300 μg/ml	239.2	0.0007	+
With metabolic activation
NC1	0	21.9	/	/
NC2		19.9	/	/
S1	0	22.7	/	/
S2		29.0	/	/
1	25.00	15.7	0.0110	+*
2	50.00	24.3	0.5738	-
3	100.00	29.8	0.6590	-
4	150.00	18.3	0.0926	-
5	200.00	19.2	0.4169	-
6	250.00	28.9	0.5661	-
7	300.00	12.7	0.0356	+*
8	400.00	16.9	0.0899	-
9	500.00	27.1	0.9291	-
DMBA	1 μg/ml	329.1	0.0003	+

NC: Negative control

S: Solvent Control

DMBA: 7,12-dimethylbenz(a)anthracene [1.0 μg/mL]

EMS: Ethylmethanesulfonate [300 μg/mL]

+: significant

-: not significant

+*: significantly decreased compared with solvent control, therefore not relevant for interpretation of results

Table 4: Historical control data

	NC		PC
	Experiment I		Experiment I
	-S9	+S9	EMS	DMBA
Mean	24	24	301	402
Min	9	8	193	132
Max	36	37	631	709
SD	7.98	7.40	79.86	172.21
RSD [%]	33.61	30.81	26.52	42.88
n =	40	46	44	44
LCL	7.8	9.2	141.1	57.2
UCL	39.7	38.8	460.9	746.1

NC: negative control

PC: positive controls (-S9 EMS; +S9 DMBA)

S9: metabolic activation

Mean: mean of mutants/10⁶ cells

Min.: minimum of mutants/10⁶ cells

Max.: maximum of mutants/10⁶ cells

SD: standard deviation

RSD: relative standard deviation

n: number of control values

LCL: lower control limit

UCL: upper control limit

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacterial cells

The Mutagenicity of 2-phenoxyethyl octanoate was tested in a GLP-conform bacterial reverse mutation assay performed according to OECD guideline 471. The test was performed with a standard battery of Salmonella typhimurium tester strains TA 1535, TA 1537, TA 98 and TA 100, and WP2uvrA bacterial cells (Notox, 2009). In the preliminary cytotoxicity test, concentrations of up to 5000 µg/plate were tested in TA 100 and WP2uvrA. 2-phenoxyethyl octanoate precipitated on the plates at dose levels of 1000 µg/plate and upwards. Cytotoxicity, based on a reduced background lawn and a decreased number of revertants, could not be observed at any of the concentrations. 2-phenoxyethyl octanoate did not have mutagenic properties in the absence or presence of metabolic activation. Positive control substances induced a distinct increase in the number of revertants in all strains with and without metabolic activation thereby proving validity of the assay. Based on the results of the conducted study, 2-phenoxyethyl octanoate was not considered to have mutagenic properties in bacterial cells.

 

In vitro cytogenicity in mammalian cells

An in vitro mammalian chromosome aberration test was performed with the test substance in cultured peripheral human lymphocytes similar to OECD guideline 473 and under GLP conditions (Notox, 2012). Duplicate cultures were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). Based on the results of a range-finding study, concentrations of 10, 33 and 100 µg/mL were applied for 3 h in the first experiment with and without metabolic activation, respectively, with a 24-h fixation time. In the second experiment cells were exposed to 10, 100 and 150 μg/mL without S9 mix for 24 h with a 24-h fixation time and for 48 h with a 48-h fixation time, and to 10, 33 and 100 µg/mL with S9 mix for 3 h with a 48-h fixation time. Precipitation was observed at 100 µg/mL and above. Mitomycin C and Cyclophosphamide were used as positive control substances, which validated the test conditions and/or the activity of the metabolizing system. No cytotoxicity was observed at any concentrations tested. Precipitation was observed at concentrations of 100 µg/mL in the range-finding study. The evaluation of 100 well-spread metaphases per concentration revealed no biologically relevant increase in the frequency of chromosome aberrations at non-cytotoxic concentrations with and without metabolic activation in comparison to the negative controls. Thus, based on the results of this study, 2-phenoxyethyl octanoate is considered to be non-clastogenic to cultured peripheral human lymphocytes in vitro with and without metabolic activation.

 

In vitro gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay was performed according to OECD guideline 476 and under GLP conditions with the test substance, in Chinese hamster lung fibroblasts (V79) (Notox, 2017). The cells were treated for 4 h with and without metabolic activation (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with phenobarbital and β-naphthoflavone). The test substance was tested at 25, 50,100, 150, 200, 250, 300, 400 and 500 μg/mL with and without metabolic activation. Precipitation of the test substance was noted at concentrations of 500 μg/mL (with metabolic activation) and at concentrations of 400 and 500 μg/mL (without metabolic activation). 7,12-dimethylbenz(a)anthracene and ethylmethanesulfonate were used as positive controls with and without S9 mix, respectively, showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems. A vehicle control (DMSO) and a negative control (treatment medium) were included, and the results fell within the historical control data range. No growth inhibition was observed in the experiments with and without metabolic activation. In the experiment without metabolic activation, statistical analysis showed that the mutant frequencies (25 and 50 μg/mL) were significantly increased over those of the negative controls (solvent and treatment medium), but there was no evidence for a dose-response relationship, since the mutant frequencies at higher concentrations (300 and 500 μg/mL) were significantly decreased when compared with the solvent control. Therefore, this effect was considered to be not biologically relevant. In the experiment with metabolic activation, the mutant frequencies at 25 and 300 μg/mL were significantly decreased compared with the solvent control. There was no dose-response relationship, and therefore this effect was also considered to be not biologically relevant. Therefore, it was concluded that the test substance was not mutagenic in Chinese hamster lung fibroblasts (V79) under the experimental conditions described.

Justification for classification or non-classification

Based on the available data on genetic toxicity, there is no indication that 2-phenoxyethyl octanoate induces mutagenic properties in bacterial cells or mutagenic and clastogenic properties in mammalian cells, respectively. In conclusion, 2-phenoxyethyl octanoate does not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.