Bis(2-(2-butoxyethoxy)ethyl) adipate

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:

22 July to 17 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2011

Reference 2

Reference Type:

secondary source

Title:

EPA High Production Volume Chemical Challenge Program CAS 141-17-3 adipic acid, bis[2-(2-butoxyethoxy)ethyl] ester

Author:

US EPA

Year:

2011

Bibliographic source:

High Production Volume Chemical Challenge Program

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-(2-butoxyethoxy)ethyl) adipate

EC Number:

205-465-5

EC Name:

Bis(2-(2-butoxyethoxy)ethyl) adipate

Cas Number:

141-17-3

Molecular formula:

C22H42O8

IUPAC Name:

bis(2-(2-butoxyethoxy)ethyl) adipate

Method

Target gene:

his operon (Salmonella typhimurium strains); trp operon (E.coli strains)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

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:

Preliminary Toxicity Test
0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 1 and 2
50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide

The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthtracene (2AA)

Remarks:

+S9: 2AA (all strains); -S9: N-ethyl-N-nitro-N-nitrosoguanidine (ENNG), benzo(a)pyrene (BP), 9-aminoacridine (9AA), 4-nitroquinoline-N-oxide (4NQO)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Standard plate incorporation method (experiment 1) and preincubation method (experiment 2)

DURATION
- Preincubation period: 20 min (only exp 2)
- Exposure duration: 48 h at 37 °C (exp 1 and 2)

NUMBER OF REPLICATIONS:
3 replicates/strain

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.

Evaluation criteria:

Acceptance Criteria:

The following criteria must be met for acceptance:
- All tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate in vehicle and untreated controls.
- The appropriate characteristics of each tester strain must be confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor.
- All tester strain cultures should be in the range of 0.9 to 9.9 x 10^9 bacteria per mL.
- Each mean positive control value should be at least 2x the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains and the integrity of the S9-mix
- The test should include a minimum of four non-toxic dose levels.

Evaluation Criteria:

There are several criteria for determining a positive result. Any, one, or all of the following may be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested.
- A reproducible increase at one or more concentrations.
- The biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by the UKEMS (Mahon et al., 1989).
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Mean values with standard deviations were calculated.

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

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

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

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:

RANGE-FINDING STUDIES
In order to select appropriate dose levels for use in the main test, a preliminary test were conducted in the presence or absence of metabolic activation. Ten dose levels and controls were tested up to and including 5000 µg/plate at approximately half-log intervals. The assay was conducted by mixing 0.1 mL the bacterial culture (TA 100 or WP2uvrA-), and 0.1 mL of the vehicle or test chemical mixture, 0.5 mL of S9-mix or phosphate buffer and 2.0 mL of molten agar supplemented with trace histidine or tryptophan and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 °C, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Numbers of revertant colonies in the preliminary toxicity test

With (+) or without (-) S9-mix	Strain	Dose (µ/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA 100	90	88	99	108	113	114	107	86	114	118	113*
+	TA 100	120	112	108	121	100	98	92	96	90	98	105*
-	WP2 uvrA-	43	40	40	43	48	42	38	38	45	41	42*
+	WP2 uvrA-	47	43	48	43	38	37	42	41	37	38	42*

* Precipitate present

Table 2: Range-finding test without metabolic activation

	Revertant colony counts (mean of 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	105	19	28	24	13
50	111	16	24	25	10
150	120	14	28	29	12
500	105	15	31	25	11
1500	109P	17P	26P	28P	12P
5000	115P	14P	30P	24P	13P
ENNG (3)	497
ENNG (5)		162
ENNG (2)			283
4NQO (0.2)				116
9AA (80)					2530

P - precipitate

Abbreviations: ENNG, N-ethyl-N’-nitro-N-nitrosoguanidine; 9AA, 2-aminoacridine; BP, benzo(a)pyrene; 2AA, 2-aminoanthracene; 4NQO, 4- nitroquinoline-1-oxide

Table 3: Range-finding test with metabolic activation

	Revertant colony counts (mean 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	115	14	33	29	12
50	98	13	32	27	12
150	109	16	32	30	11
500	99	10	29	29	12
1500	115P	14P	31P	29P	12P
5000	116P	14P	32P	29P	14P
2AA (1)	801
2AA (2)		292			95
2AA (10)			130
BP (5)				189

P - preicipitate

Table 4: Main Test without metabolic activation

	Revertant colony counts (mean 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	117	21	26	34	9
50	115	20	35	27	11
150	117	20	30	27	9
500	115	21	31	30	10
1500	117P	20P	31P	31P	11P
5000	124P	18P	30P	32P	7P
ENNG (3)	439
ENNG (5)		174
ENNG (2)			182
4NQO (0.2)				122
9AA (80)					730

P - precipitate.

Table 5: Main Test with metabolic activation

	Revertant colony counts (mean 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	124	24	41	29	13
50	117	18	38	24	10
150	127	18	36	30	9
500	119	11	34	33	12
1500	114P	13P	29P	26P	10P
5000	130P	18P	35P	22P	9P
2AA (1)	952
2AA (2)		261			217
2AA (10)			202
BP (5)				309

P - precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative