1,3-butylene diacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

22 Nov - 21 Dec 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (no test substance purity specified).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

analytical purity of test substance not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene di(acetate)

EC Number:

203-881-1

EC Name:

Ethylene di(acetate)

Cas Number:

111-55-7

Molecular formula:

C6H10O4

IUPAC Name:

ethane-1,2-diyl diacetate

Method

Target gene:

his operon

Species / strain

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

Controls

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.

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

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

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The results of the assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of metabolic activation.