2,2'-ethylenedioxydiethyl bis(2-ethylhexanoate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant OECD guideline study, available as unpublished report, no restrictions, adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-ethylenedioxydiethyl bis(2-ethylhexanoate)

EC Number:

202-319-2

EC Name:

2,2'-ethylenedioxydiethyl bis(2-ethylhexanoate)

Cas Number:

94-28-0

Molecular formula:

C22H42O6

IUPAC Name:

2-(2-{2-[(2-ethylhexanoyl)oxy]ethoxy}ethoxy)ethyl 2-ethylhexanoate

Details on test material:

- Name of test material (as cited in study report): Eastman TEG-EH
- Description: Eastman(TM) TEG-EH (Triethylene Glycol Bis (2-EthylHexanoate)) Plasticizer
- Physical state: clear colourless liquid
- Specific gravity: 0,968 (20/20C)
- Analytical purity: 99,1% (ester by GC)
- Impurities (identity and concentrations): see confidential details on test material
- Composition of test material, percentage of components: see confidential details on test material
- Purity test date: 2007-07-11
- Lot/batch No.: TD-7022702
- Expiration date of the lot/batch : not provided (production date = 2007-07-11)
- Storage condition of test material: room temperature in the dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB- Salmonella strains

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: WP2uvrA-

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was immiscible in water according to the information provided by the Sponsor. However, the test material was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Controls 2

Negative solvent / vehicle controls:

yes

Controls 3

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9-mix

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix

Controls 5

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9-mix

Controls 7

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: approximately 10 hours at 37°C
- Exposure duration: approximately 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test material will be considered mutagenic (positive) in the test system if a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation are observed. Biological relevance of the results will be considered first, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, -tested up to the maximum recommended dose level of 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 and Table 2 for Experiment 1 and Table 3 and Table 4 for Experiment 2.

Table 1: Test Results: Experiment 1 – Without metabolic activation

Test period		From:16 August 2007					To:19 August 2007
With or without S9-mix	Test substance cc (µg/plate)	Mean number (M) of revertant colonies and the standard deviations (SD)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
		M	SD	M	SD	M	SD	M	SD	M	SD
-	0	101	9.6	18	3.5	20	3.8	20	2.1	16	1.2
-	50	112	58.6	18	3.2	17	1.7	18	4.4	15	5.0
-	150	105	10.0	25	8.4	16	5.3	21	5.5	14	2.9
-	500	98	1.5	22	4.2	18	3.8	19	7.2	15	1.7
-	1500	96	11.5	14	6.0	17	1.5	14	3.0	17	3.8
-	5000	87	18.6	17	4.2	21	2.0	13	3.1	13	4.0
Positive controls -	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Cc (µg/plate)	3		5		2		0.2		80
	N° colonies per plate	480	88.0	79	16.5	291	47.6	435	43.5	566	72.3

 

Table 2: Test Results: Experiment 1 – With metabolic activation

Test period		From:16 August 2007					To:19 August 2007
With or without S9-mix	Test substance cc (µg/plate)	Mean number (M) of revertant colonies and the standard deviations (SD)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
		M	SD	M	SD	M	SD	M	SD	M	SD
+	0	98	13.6	14	0.0	26	6.8	30	6.1	14	2.6
+	50	83	13.6	11	1.5	19	2.5	28	3.1	17	3.2
+	150	82	12.9	11	2.3	17	6.9	27	3.5	16	2.3
+	500	91	16.3	9	1.2	20	8.1	29	1.5	18	2.1
+	1500	92	7.4	11	2.5	25	5.5	22	4.6	13	1.7
+	5000	86	16.1	14	0.6	26	9.0	27	3.5	13	7.8
Positive controls +	Name	2AA		2AA		2AA		BP		2AA
	Cc (µg/plate)	1		2		10		5		2
	N° colonies per plate	559	42.9	206	25.2	332	20.2	252	28.4	331	48.0

 

Table 3: Test Results: Experiment 2 – Without metabolic activation

Test period		From:23 August 2007					To:26 August 2007
With or without S9-mix	Test substance cc (µg/plate)	Mean number (M) of revertant colonies and the standard deviations (SD)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
		M	SD	M	SD	M	SD	M	SD	M	SD
-	0	100	3.2	19	4.0	21	4.9	22	2.5	15	3.5
-	50	91	2.3	16	2.5	18	4.4	22	2.3	14	1.2
-	150	88	2.6	16	1.5	21	4.2	22	0.6	11	2.6
-	500	97	4.0	17	1.2	19	3.5	23	2.5	13	1.5
-	1500	95	9.1	17	4.2	24	4.9	18	2.9	14	0.6
-	5000	74	3.1	17	2.6	21	0.6	23	3.1	14	1.5
Positive controls -	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Cc (µg/plate)	3		5		2		0.2		80
	N° colonies per plate	662	25.7	303	6.7	614	25.7	367	61.5	653	20.1

 

Table 4: Test Results: Experiment 2 – With metabolic activation

Test period		From:23 August 2007					To:26 August 2007
With or without S9-mix	Test substance cc (µg/plate)	Mean number (M) of revertant colonies and the standard deviations (SD)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
		M	SD	M	SD	M	SD	M	SD	M	SD
+	0	99	2.5	15	0.6	27	2.5	22	4.5	12	5.6
+	50	91	14.6	12	1.5	28	3.8	20	2.6	13	9.2
+	150	87	4.2	15	0.6	24	4.0	22	3.0	7	3.8
+	500	92	12.5	12	3.0	23	6.8	22	4.4	7	3.1
+	1500	88	10.1	13	2.5	22	5.5	21	3.5	10	3.5
+	5000	94	11.7	12	0.6	24	2.1	21	2.6	8	1.5
Positive controls +	Name	2AA		2AA		2AA		BP		2AA
	Cc (µg/plate)	1		2		10		5		2
	N° colonies per plate	684	18.0	200	35.0	432	26.6	230	26.1	175	9.5

 

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-l-oxide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella trphimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with solutions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat

liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment.The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) and positive controls were valid. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.