Ethyl 3,5-dichloro-4-hexadecyloxycarbonyloxybenzoate

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:

Feb 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions. No TA 102 or E. coli strain was tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

current version

Deviations:

yes

Remarks:

no TA 102 or E.coil strain was tested

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in May 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

404-740-9

EC Name:

-

Cas Number:

115895-09-5

Molecular formula:

C26H40Cl2O5

IUPAC Name:

ethyl 3,5-dichloro-4-{[(hexadecyloxy)carbonyl]oxy}benzoate

Details on test material:

- Name of test material (as cited in study report): AF-366
- Physical state: white powder
- Analytical purity: no data
- Storage condition of test material: at ambient temperature in the dark

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 500 mg/kg bw Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 and 2: 61.7, 185.2, 555.6, 1666.7 and 5000 μg/plate; TA 1535, TA 1537, TA 1538, TA 98 and TA 100; with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the vehicle was shown during the range-finding test to be suitable for use with the test substance

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (1 μg/plate, -S9, TA 1535 and TA 100); 9-aminoacridine (80 μg/plate, -S9, TA 1537); 2-nitrofluorene (2 μg/plate, -S9, TA 1538 and TA 98); 2 -aminoanthracene (2 μg/plate, +S9, TA 1535, TA 1538, TA 98 and TA 100; 5 μg/plate, +S9, TA 1537)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 replications in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:
The ability of the S9-mix to activate compounds was shown with 4-amino-biphenyl and 2-aminoanthracene, using TA 98 as an indicator strain. The S9-mix was checked for sterility.

Evaluation criteria:

A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the vehicle, together with evidence of a dose-response effect.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, 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

Remarks:

precipitation observed from 1666.7 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 1666.7 and 5000 μg/plate, the test substance precipitated in the plates

RANGE-FINDING/SCREENING STUDIES:
A range-finding assay with 0.001, 0.01, 0.1, 1.0, 10, 100 mg test substance/plate and the vehicle (DMSO) as negative control was performed to assess the toxicity of the test substance for S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without metabolic activation. No mutagenic or cytotoxic effects were observed. At 1 and 10 mg/plate the test substance precipitated (slightly) in the plates. Based on these results, 5 mg/plate was chosen as the highest concentration in the main experiment, with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of experiment 1

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	TA 1538	TA 98	TA 1537
-S9	DMSO	128 ± 20	21 ± 3	27 ± 4	22 ± 11	9 ± 6
-S9	61.7	142 ± 14	27 ± 6	30 ± 6	28 ± 9	9 ± 3
-S9	185.2	186 ± 11	29 ± 2	25 ± 4	20 ± 3	8 ± 2
-S9	555.6	159 ± 13	30 ± 2	28 ± 9	24 ± 6	6 ± 4
-S9	1666.7	185 ± 5	31 ± 3	29 ± 6	32 ± 17	4 ± 3
-S9	5000	161 ± 6	31 ± 2	28 ± 7	31 ± 5	10 ± 3
Positive controls, –S9	Name	SA	SA	2-NF	2-NF	9-AA
	Concentrations (μg/plate)	2	2	2	2	80
		426 ± 43	289 ± 12	710 ± 25	542 ± 41	569 ± 56
+S9	DMSO	140 ± 17	15 ± 9	44 ± 8	45 ± 2	10 ± 1
+S9	61.7	154 ± 12	19 ± 4	47 ± 21	54 ± 12	12 ± 1
+S9	185.2	129 ± 6	18 ± 9	48 ± 6	51 ± 4	11 ± 5
+S9	555.6	132 ± 6	20 ± 2	46 ± 8	51 ± 10	14 ± 3
+S9	1666.7^1	165 ± 17	17 ± 3	49 ± 3	56 ± 11	8 ± 1
+S9	5000^1	132 ± 8	22 ± 2	57 ± 12	40 ± 6	12 ± 4
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2	2	2	2	5
		1136 ± 84	352 ± 25	893 ± 60	827 ± 59	338 ± 112

9-AA = 9-aminoacridine

2-NF = 2-nitrofluorene

SA = sodium azide

2-AA = 2 -aminoanthracene

¹precipitation

 

 

Table 2: Results of experiment 2

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	TA 1538	TA 98	TA 1537
-S9	DMSO	173 ± 5	21 ± 2	28 ± 5	30 ± 8	10 ± 1
-S9	61.7	158 ± 12	28 ± 3	19 ± 4	28 ± 9	9 ± 3
-S9	185.2	184 ± 12	27 ± 5	28 ± 8	29 ± 3	8 ± 3
-S9	555.6	188 ± 4	28 ± 4	26 ± 6	34 ± 4	10 ± 3
-S9	1666.7	175 ± 9	33 ± 3	27 ± 6	28 ± 12	7 ± 4
-S9	5000	167 ± 11	30 ± 3	33 ± 2	31 ± 3	11 ± 2
Positive controls, –S9	Name	SA	SA	2-NF	2-NF	9-AA
	Concentrations (μg/plate)	2	2	2	2	80
		489 ± 85	391 ± 79	584 ± 55	543 ± 48	984 ± 87
+S9	DMSO	126 ± 14	21 ± 4	56 ± 6	47 ± 7	10 ± 5
+S9	61.7	143 ± 33	21 ± 3	45 ± 13	57 ± 3	14 ± 3
+S9	185.2	145 ± 28	15 ± 2	55 ± 7	48 ± 4	15 ± 2
+S9	555.6	141 ± 34	22 ± 5	55 ± 4	53 ± 5	16 ± 1
+S9	1666.7^1	150 ± 4	20 ± 3	54 ± 11	55 ± 11	12 ± 2
+S9	5000^1	129 ± 19	21 ± 1	49 ± 7	43 ± 7	12 ± 2
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2	2	2	2	5
		1314 ± 25	382 ± 30	859 ± 37	882 ± 16	237 ± 21

9-AA = 9-aminoacridine

2-NF = 4-nitrofluorene

SA = sodium azide

2 -AA = 2 -aminoanthracene

¹precipitation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

From the above findings it is concluded that AF-366 did not show mutagenic activity in Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 either in the absence or in presence of the S-9 mix, under the conditions employed in this evaluation.