Ethylene di(acetate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): S. typhimurium TA 1535, TA 1537, TA 98, and TA 100 and E. coli WP2uvrA: negative with and without metabolic activation

Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): Chinese hamster ovaries (CHO) cells: negative with and without metabolic activation

Gene mutation in mammalian cells (OECD 476, HGPRT Test): Mouse lymphoma L5178Y cells: negative 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:

22 Nov - 21 Dec 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no test substance purity specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

yes

Remarks:

analytical purity of test substance not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I:
Salmonella strains: 10, 33.3, 100, 333, 1000, 5000 µg/plate (with and without S9 mix)
E.coli strain: 33.3, 100, 333, 1000, 3330, 5000 µg/plate (with and without S9 mix)
Experiment II:
all strains: 33.3, 100, 333, 1000, 3330, 5000 µg/plate (with and without S9 mix)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (2-AA, all, 2.5 + 5.0 µg/plate, +S9); ICR-191 (TA1537, 2.0 µg/plate, -S9); sodium azide (SA, TA100 + 1535, 2.0 µg/plate, -S9); 2-nitrofluorene (2NF, TA98, 1.0 µg/plate, -S9); 4-nitroquinoline-N-oxide (4NQO, WP2, 2.0 µg/plate, -S9)

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 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: density of bacterial background lawn

Evaluation criteria:

1) Tester strains TA98, TA100 and WP2 uvrA
For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
2) Tester strain TA1535 and TA1537
For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least on of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose-response to increasing concentrations of the test article.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed

RANGE-FINDING/SCREENING STUDIES:
The cytotoxicity of the test item was determined in TA100 and WP2 uvrA in order to allow selection of appropriate doses to be tested in the mutagenicity assay. The test article was tested up to a maximum concentration of 5 mg/plate (dose range 6.67 - 5000 µg/plate). Cytotoxicity was observed with tester strain TA100 at 667 µg/plate and above in the presence of S9 mix and 333 µg/plate and above in the absence of S9 mix as evidenced by thinning of the bacterial background lawn. No cytotoxicity was observed for tester strain WP2 uvrA. In general no test article precipitate was observed. The highest dose chosen for the main study gave a reduction of revertants per plate and/or thinning or disappearance of the bacterial background lawn.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study no changes of the background lawn were observed.

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	vehicle	97 ± 6	11 ± 4	184 ± 28	23 ± 4	9 ± 3
–	10	101 ± 1	13 ± 3	--	18 ± 3	5 ± 1
–	33.3	100 ± 8	14 ± 5	175 ± 6	16 ± 3	5 ± 3
–	100	117 ± 12	13 ± 2	191 ± 8	20 ± 4	3 ± 1
–	333	94 ± 5	12 ± 2	183 ± 10	19 ± 6	6 ± 3
–	1000	101 ± 10	9 ± 1	197 ± 16	17 ± 6	5 ± 2
–	3330	--	--	161 ± 17	--	--
–	5000	100 ± 21	14 ± 4	188 ± 7	22 ± 3	6 ± 3
Positive controls, –S9	Name	SA	SA	4NQO	2NF	ICR-191
	Concentrations (μg/plate)	2	2	2	1	2
	Mean No. of colonies/plate (average of 3 ± SD)	751 ± 35	590 ± 44	1319 ± 59	298 ± 19	427 ± 36
+	vehicle	98 ± 3	13 3	197 ± 39	30 ± 5	4 ± 2
+	10	130 ± 10	16 3	--	33 ± 4	7 ± 2
+	33.3	133 ± 6	12 5	186 ± 12	34 ± 5	8 ± 0
+	100	124 ± 5	13 4	198 ± 20	34 ± 4	7 ± 0
+	333	143 ± 8	16 2	203 ± 9	31 ± 2	6 ± 1
+	1000	138 ± 8	15 5	226 ± 64	30 ± 2	9 ± 1
+	3330	--	--	178 ± 12	--	--
+	5000	130 ± 8	14 3	204 ± 15	25 ± 3	4 ± 2
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	5	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	774 ± 19	134 ± 6	840 ± 191	804 ± 36	128 ± 9

 

4NQO = 4-nitroquinoline-N-oxide

2AA = 2-Aminoanthracene

SA = Sodium Azide

2NF = 2-Nitrofluorene

 

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

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	vehicle	96 ± 17	17 ± 3	159 ± 28	12 ± 1	6 ± 3
–	33.3	99 ± 7	15 ± 5	135 ± 10	15 ± 4	6 ± 2
–	100	90 ± 4	20 ± 5	142 ± 12	12 ± 3	4 ± 1
–	333	103 ± 9	15 ± 6	120 ± 19	14 ± 4	4 ± 1
–	1000	109 ± 11	13 ± 5	138 ± 27	19 ± 3	8 ± 1
–	3330	108 ± 10	11 ± 5	150 ± 19	17 ± 3	5 ± 1
–	5000	95 ± 11	13 ± 4	150 23	15 ± 2	5 ± 2
Positive controls, –S9	Name	SA	SA	4NQO	2NF	ICR-191
	Concentrations (μg/plate)	2	2	2	1	2
	Mean No. of colonies/plate (average of 3 ± SD)	581 ± 33	523 ± 13	1355 ± 87	268 ± 42	411 ± 64
+	vehicle	85 ± 9	16 ± 5	178 ± 18	18 ± 4	6 ± 1
+	33.3	93 ± 25	10 ± 1	131 ± 14	15 ± 4	7 ± 2
+	100	97 ± 18	11 ± 2	141 ± 19	17 ± 3	7 ± 2
+	333	88 ± 10	14 ± 5	139 ± 6	24 ± 6	7 ± 1
+	1000	104 ± 15	12 ± 1	156 ± 14	21 ± 3	9 ± 4
+	3330	91 ± 15	8 ± 3	138 ± 6	27 ± 3	9 ± 2
+	5000	89 ± 8	8 ± 3	127 ± 6	24 ± 10	8 ± 2
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	5	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	834 ± 16	105 ± 17	1019 ± 50	842 ± 65	127 ± 11

 

4NQO = 4-nitroquinoline-N-oxide

2AA = 2-Aminoanthracene

SA = Sodium Azide

2NF = 2-Nitrofluorene

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:

22 Nov 1999 - 21 Jan 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no analytical purity of test substance specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 21 July, 1997

Deviations:

yes

Remarks:

no analytical purity of test substance specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5a culture medium supplemented with 10% fetal bovine serum (FBS), L-glutamine (2mM), penicillin G (100 units/mL and streptomycin (100 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I:
3 hours with and without metabolic activation: 515, 735, 1050, 1500 µg/mL
Experiment II:
18 hours without and 3 hours with metabolic activation: 500, 750 1000, 1500 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: water

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

cell culture grade water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: 2 concentrations used for both, one was evaluated; Cyclophosphamide (CP, 5 + 10 µg/mL, +S9); Mitomycin C (MMC, I: 0.75 + 1.5 µg/mL, II: 0.2 + 0.4 µg/mL, -S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): 0.1 µg/mL Colcemid
STAIN (for cytogenetic assays): 5% Giemsa solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy
- Determination of endoreplication

Evaluation criteria:

Evaluation of a positive response:
A test substance was considered positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when p<0.01) in the number of cells with chromosomal aberrations is observed at one or more concentrations. Statistical evaluation of the percentage of cells with more than one aberration provided an indication of the severity of the positive response observed. The linear trend test evaluated the dose responsiveness. If a significant increase was seen at one or more concentrations, a dose-response should be observed.
Evaluation of a negative response:
A test substance as considered negative for inducing chromosomal aberrations if no significant increase was observed in the number of cell with chromosomal aberrations at any of the concentrations.
Equivocal evaluation:
Although most assays give clear positive or negative results, in rare cases the data set would preclude making a definitive judgement about the activity of the test substance. Results might remain equivocal or questionable regardless of the number of times the assay is repeated.

Statistics:

Statistical Analysis employed a Cochran-Armitage test for linear trend and Fisher's Exact Test to compare the percentage of cells with aberrations (and, if applicable, the percentage of cells with more than one aberration) in treated cells to the results obtained for the vehicle control. Statistical analysis was also performed for cells exhibiting polyploidy and/or endoreduplication in order to indicate significant (p<0.01) increases in these events as indicators of possible induction of nummerical aberrations; however, the test substance was evaluated only for structural aberrations not for numerical aberrations by this protocol.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: an initial chromosomal aberration test without metabolic activation was done (Experiment I without S9 mix), based on these results the other 3 experiments were set up

COMPARISON WITH HISTORICAL CONTROL DATA:
the values of the controls are in range with historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no visual signs of toxicity could be observed for all cultures but reductions of mitotic index were seen for all experiments but for varying concentrations (see any other information on results including tables)

Table 1. Test results of experiment I.

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	without gaps
Exposure period 3h, fixation time 20h, without S9 mix
Vehicle control	10 µL/mL	100	0
MMC	1.5 µg/mL	--	74*
Negative control	0	76	0
Test substance	515	106	0.5
	735	133	0
	1050	126	0
	1500	104	0.5
Exposure period 3h, fixation time 24h, with S9 mix
Vehicle control	10 µL/mL	100	2.0
CP	5 µg/mL	--	36
Negative control	0	66	0
Test substance	515	134	1.5
	735	137	2.5
	1050	95	2.5
	1500	85	2.0

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

 

Table 2. Test results of experiment II.

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	without gaps
Exposure period 18h, fixation time 20h, without S9 mix
Vehicle control	10 µL/mL	100	0
MMC	1.5 µg/mL	--	50*
Negative control	0	50	1.5
Test substance	500	78	0.5
	750	74	1.0
	1000	83	0
	1500	74	0
Exposure period 3h, fixation time 24h, with S9 mix
Vehicle control	10 µL/mL	100	1.0
CP	5 µg/mL	--	32
Negative control	0	112	0
Test substance	500	74	1.5
	750	96	0.5
	1000	95	0.5
	1500	101	0

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

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:

13 Apr - 31 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Government of India, Department of Science and Technology, New Delhi, India

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the batch: 20 Oct 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 to 25 °C
- Solubility and stability of the test substance in the vehicle were confirmed by analytical methods

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The Dow Chemical Company, Michigan, USA
- Cell cycle length, doubling time or proliferation index: 10 - 12 h
- Modal number of chromosomes: 40


MEDIA USED
- Type and identity of media: RPMI 1640 medium containing penicillin/streptomycin
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I
With S9 mix: 54, 162, 487 amd 1462 µg/mL (3 h)

Experiment II
Without S9 mix: 54, 162, 487 amd 1462 µg/mL (3 h)

Experiment III
Without S9 mix: 54, 162, 487 amd 1462 µg/mL (24 h)

The selection of the concentrations used in the main experiments was based on data from the range-finding experiment. No cytotoxocicity was observed up to 1462 µg/mL (10 mM), therefore this concentration was chosen as top dose for the main study.

Vehicle / solvent:

sterile water (Milli-Q)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 6 x 10E6 cells/tube

DURATION
1st experiment: 3 h exposure with and without S9 mix
2nd experiment: 3 h exposure with S9 mix
3rd experiment: 24 h exposure without S9 mix
- Expression time (cells in growth medium): 2 days after the end of treatment, cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtiter plates containing trifluorothymidine (TFT) selective medium. The microtiter plates were incubated for 12 days.
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days

SELECTION AGENT (mutation assays): 1-4 µg/mL TFT

NUMBER OF REPLICATIONS: single samples each in two independent experiments in 96-well microtiter plates

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency and relative total growth

- OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.

Evaluation criteria:

Increase of mutation frequency in a dose-dependent manner im comparison with the concurrent vehicle controls.

Statistics:

X2 test, one-sided, p ≤ 0.05

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
The test item did not precipitate in medium and did not cause any appreciable change in the pH and osmolarity of the test medium. It was found stable in water for 24 h at room temperature at concentrations of 2500 to 150000 µg/mL. Therefore a maximum concentration of 1462 µg/mL (10 mM) was tested.

RANGE-FINDING/SCREENING STUDIES:
The test item did not show evidence of significant growth inhibition (80 - 90% inhibition over the control) as relative total growth in any of the tested concentrations (25 - 1462 µg/mL) either in the presence or absence of S9 mix.

HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minumum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Table 1: Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Presence of Metabolic Activation (Experiment 1)

Treatment (µg/mL)	Non-selective medium		Selective medium No. of Empty Wells			Cem	MF (x 10^-6)	Induced MF	% RTG
	P(0)	Cev	R1*	R2*	Total
sterile water	0.1875	104.62	88.5	87	175.5	0.008622	0.0000824		100
54	0.1979	101.25	88	88.5	176.5	0.008078	0.0000798	-0.0000026	76
162	0.2031	99.63	88.5	88.5	177	0.007807	0.0000784	-0.000004	67
487	0.2135	96.51	88	87	175	0.008896	0.0000922	0.0000098	54
1462	0.2396	89.3	88	86	174	0.009445	0.0001058	0.0000232	46
CPA 12µg/mL	0.3281	69.75	22	23	45	0.13927	0.0019996+	0.0019172	31

MF: Mutant Frequency

CE: Cloning Efficiency

CPA: Cyclophosphamide

+: Significantly higher than control (p ≤0.05)

*: Mean of two replicates

RTG: Relative Total Growth = RCE x RSG x 100

Table 2: Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Absence of Metabolic Activation (Experiment 2)

Treatment (µg/mL)	Non-selective medium		Selective medium No. of Empty Wells			Cem	MF (x 10^-6)	Induced MF	% RTG
	P(0)	Cev	R1*	R2*	Total
sterile water	0.1771	108.19	86.5	87.5	174	0.009445	0.0000873		100
54	0.1875	104.62	88	86.5	174.5	0.00917	0.0000877	0.0000004	78
162	0.1979	101.25	87	88	175	0.008896	0.0000879	0.0000006	71
487	0.2031	99.63	86.5	87.5	174	0.009445	0.0000948	0.00000948	60
1462	0.2135	96.51	88.5	86.5	175	0.008896	0.0000922	0.0000922	57

MF: Mutant Frequency

CE: Cloning Efficiency

+: Significantly higher than control (p≤0.05)

*: Mean of two replicates

RTG: Relative Total Growth = RCE x RSG x 100

Table 3: Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Absence of Metabolic Activation (Experiment 3)

Treatment (µg/mL)	Non-selective medium		Selective medium No. of Empty Wells			Cem	MF (x 10^-6)	Induced MF	% RTG
	P(0)	Cev	R1*	R2*	Total
sterile water	0.2031	99.63	88.5	88	176.5	0.008078	0.0000811		100
54	0.2135	96.51	86.5	88.5	175	0.008896	0.0000922	0.0000111	79
162	0.2188	94.97	86.5	87	173.5	0.009731	0.0001025	0.0000214	75
487	0.2448	87.96	86	87	173	0.010008	0.0001138	0.0000327	56
1462	0.2708	81.65	87.5	87.5	175	0.008896	0.000109	0.0000279	43
MMS 10µg/mL	0.401	57.11	25.5	24.5	50	0.129171	0.0022618+	0.0021807	23

MF: Mutant Frequency

CE: Cloning Efficiency

MMS: Methylmethanesulfonate

+: Significantly higher than control (p≤0.05)

*: Mean of two replicates

RTG: Relative Total Growth = RCE x RSG x 100

Conclusions:

Under the tested conditions, the test compound was not mutagenic in mouse lymphoma L517Y TK+/- cells with and without metabolic activation up to 1462 µg/mL (10 mM).

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 bacteria

One study investigating the induction of gene mutation in bacteria by ethylene diacetate (CAS 111 -55 -7) is available (Covance, 2000a). The study was conducted according to OECD guideline 471 under GLP conditions. The tester strains, S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and the E. coli strain WP2 uvr A pKM 101 were used.

The experiments were performed according to the plate incorporation procedure at concentrations from 10-5000 µg/plate in the first experiment and from 33.3-5000 µg/plate in the second experiment with and without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). The included positive and negative controls in the experiments showed the expected results and were therefore considered as valid. No increase in the number of revertant colonies was observed in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed in the main study up to the limit concentration of 5000 µg/plate. Under the conditions of this study, ethylene diacetate (CAS 111 -55 -7) did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested.

 

In vitro cytogenicity in mammalian cells

A study investigating the cytogenicity in mammalian cells in vitro with ethylene diacetate (CAS 111 -55 -7) was performed in accordance with OECD guideline 473 under GLP conditions (Covance, 2000b). The induction of structural chromosome aberrations was evaluated in vitro in Chinese hamster ovary (CHO) cells, incubated for 3 h with and without and for 18 h without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). Concentrations of 515-1500 µg/mL (3 h incubation, experiment I) and 500-1500 µg/mL (18 h and 3 h incubation, experiment II) of the test substance in the vehicle (water) were applied. The negative as well as the positive controls showed the expected results and were within the range of historical control data. The frequency of polyploid cells with and without metabolic activation was within the expected range. In the experiments without metabolic activation and 18 h incubation, an influence of the test substance was observed which led to a reduction in the mitotic index. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity in the chromosomal aberration test with and without metabolic activation performed in CHO cells in vitro.

 

In vitro gene mutation in mammalian cells

Ethylene diacetate (CAS 111 -55 -7) was tested for its mutagenic potential in mammalian cells according to OECD guideline 490 and in compliance with GLP (Eurofins, 2018b). Two independent experimental runs were performed with L5178Y TK+/- c-3.7.2C mouse lymphoma cells (heterozygous thymidine kinase locus). The cells were treated with the test item at four concentrations (range 54 – 1462 µg/mL) for three hours with and without metabolic activation (1^st and 2^nd experiment), and for 24 hours without metabolic activation (3^rd experiment). In all experiments, the reduction in cell growth as relative suspension growth, as well as the reduction in cell growth as relative total growth were analysed and compared to those of vehicle controls.

A preliminary cytotoxicity test revealed that a concentration of 1462 µg/mL (10 mM) with and without metabolic activation was not cytotoxic, therefore this concentration was selected as maximum dose in the main study. Concurrent vehicle controls (sterile water) and positive control substances were included. Mutant frequencies of solvent controls were within the range of historical control data. The positive control substances cyclophosphamide (+ S9 mix) and methylmethanesulfonate (–S9 mix) markedly increased the number of mutants for L5178Y cells, thus demonstrating the sensitivity and validity of the test system.

Under the conditions of the test, the test item did not induce a significant, dose-related increase in mutant frequencies in any of the tested concentrations, either in the presence or absence of metabolic activation. Thus, the test item was not considered to be mutagenic in mammalian cells.

 

Conclusion for genetic toxicity

The studies with ethylene diacetate (CAS 111 -55 -7) investigating genetic mutations in bacteria in vitro, cytogenicity in mammalian cells in vitro and gene mutation in mammalian cells in vitro provided negative results. Therefore, the available data do not provide any indications for a potential regarding genetic toxicity of ethylene diacetate.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.