C10-13-alkyl(branched, unsaturated)-succinic acid-4-(3-methyl-butyl)-ester

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:

199-06-01 to 1999-06-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline and GLP conform well documented scientific study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

921-522-3

EC Number:

921-522-3

IUPAC Name:

921-522-3

Constituent 2

Reference substance name:

Tetrapropylene succinic acid monoisobutylester

IUPAC Name:

Tetrapropylene succinic acid monoisobutylester

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): Hostacor 4323
- Chemical name: Tetrapropenyl succinic acid monoisobutylester
- Physical state: liquid, light brown
- Density: 0.969 g/cm3 (at 20°C)
- Analytical purity: n.a.
- Lot/batch No.: 101253281
- Expiration date of the lot/batch: June 2000
- Storage condition of test material: at room temperature (app. 20°C) protected from lighr and moisture
- water solubility: insoluble

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

The genotypes of the tested strains were checked at each study:
Histidine auxotrophy, ampicillin resistance, tetracycline resistance, UV-sensitivity and growth inhibition by crystal violet.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation system: (S9) fraction from livers of Wistar male rats, which were induced with Phenobarbital intraperitoneal and beta-Naphtoflavone orally.

Test concentrations with justification for top dose:

1. Study (+/- S9):
All strains: 0.005, 0.05, 0.5 (mg/plate)
Based on the reults of the 1. study concentrations of the second study were selected.

2. Study (+/- S9):
TA97a, TA98, TA 100: 0.5, 0.16, 0.5, 1.6, 5 (mg/plate)
TA102: 0.016, 0.05, 0.16, 0.5, 1.6 (mg/plate)

3. Study:
TA102 (- S9): 0.5, 1.6, 5 (mg/plate)
TA102 (+ S9): 5 (mg/plate)

Vehicle / solvent:

Dimethy sulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

other: ICR 191; 4-Nitro-o-phenylen-diamine; Nitrofurantoine; 2-Aminoanthracen, Danthron

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

First study was conducted with 3 concentration levels of the test substance from 0.005 – 0.5 mg/plate in geometrical series of factor 10.
Based on the results of the first study concentrations in a logarithmic series with a factor of √ 10 were tested in the second and third study as specified. Plates for confirmation of genotypes were incubated for 24 h and plates of mutagenicity test for 48 h, respectively, at 37 ± 1 °C. Genotypes were evaluated for each study.
Plates of the mutagenicity test were inspected for present and reduced background lawn after an incubation time of 48 h, respectively. Colonies per plate (revertants) were counted if no reduced background lawn was observed.

Evaluation criteria:

Evaluation:
Since the reduced background lawn is regarded to be a cytotoxic effect, plates with reduced background lawn were not included into evaluation procedures. Arithmetic mean values and standard deviations were calculated out of colonies per plate of three replicates.

The test substance is to be interpreted mutagenic if there is a concentration effect relationship and the induction rate is ≥2.

Validity criteria:
Spontaneous revertants (negative controls) had to be within the following ranges:
- TA 97a ± S9: 60- 300 revertants/plate
- TA98 ±S9: 15- 50 revertants/plate
- TA 100 ± S9: 60 - 200 revertants/plate
- TA 102 ± S9: 240 - 460 revertants/plate

The induction rates of the positive controls had to be ≥2

Results and discussion

Test results

Species / strain:

other: TA 97a, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of Mutagenic and cytotoxic effects of test substance

S. typhimurium strain	S9	Tested concentration [mg/plate]	Lowest mutagenic concentration[mg/plate]	Lowest cytotoxic concentration[mg/plate]
TA97a	—	0.005 - 5.0	none	5
	+	0.005 - 5.0	none	none
TA98	—	0.005 - 5.0	none	none
	+	0.005 - 5.0	none	none
TA100	—	0.005 - 5.0	none	5
	+	0.005 - 5.0	none	none
TA102	—	0.005 - 5.0	none	none
	+	0.005 - 5.0	none	none

 

Table 2: Mutagenicity test (1. Study)

		No. Revertants (mean of 3 plates)
Concentration		TA97a		TA98		TA100		TA102
[mg/plate]		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Negative Control		189	226	22	23	100	88	379	418
Test substance	0.5	158	141	13	27	71	87	cytotoxic	390
	0.05	208	175	18	20	99	76	349	401
	0.005	172	193	19	23	102	79	349	364

 

Table 3: Mutagenicity test (2. Study)

		No. Revertants (mean of 3 plates)
Concentration		TA97a		TA98		TA100		TA102
[mg/plate]		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Negative Control		196	204	18	16	177	131	365	335
Test substance	5.0	cytotoxic	144	13	10	cytotoxic	115	317	405
	1.6	126	170	9	10	94	96	302	450
	0.5	181	201	15	18	111	125	298	415
	0.16	198	208	13	29	145	104	392	404
	0.05	179	177	18	19	154	111	424	443

 

  Table 4: Mutagenicity test (3. Study)

		No. Revertants (mean of 3 plates)
Concentration		TA102
[mg/plate]		- S9	+ S9
Negative Control		330	339
Test substance	5	225	293
	1.6	309	Nt
	0.5	311	Nt

Nt = Not tested

Table 5: Induction rates of positive controls

strain	Reference substance	(µg/plate)	(S9)	Induction rate/study
				1.	2.	3.
TA 97a	ICR 191 acridine mutagen dihydrochloride	0.5	—	>5.3	>5.1	Nt
	2-Aminoanthracene	2.0	+	>4.4	>4.9	Nt
TA 98	4-nitro-1,2-phenylenediamine	0.5	—	3.2	5.5	Nt
	2-Aminoanthracene	2.0	+	>43.5	>62.5	Nt
TA 100	Nitrofrantoine	0.2	—	4.4	2.7	Nt
	2-Aminoanthracene	2.0	+	11.3	>7.7	Nt
TA 102	Cumene hydroperoxide	100	—	>2.6	>2.7	>3.0
	Danthron	30	+	>2.4	>3.0	>3.0

Nt = Not tested

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the results of this test, the test substance is regarded to be not mutagenic.

Executive summary:

In order to assess the mutagenic effects of the test substance it were determined in a reverse mutation assay according to OECD test guideline 471 in three independent studies. The test system were the Salmonella typhimurium strains TA97a, TA 98, TA100 and TA102 with and without metabolic activation system S9. Positive and negative controls were included in each study. Duration of each study was 48 hours. The test substance was applied once at test intitiation for the 1. study for all strains at the concentrations (with and without S9) of 0.005, 0.05 and 0.5 (mg/plate) dissolved in vehicle DMSO. Based on the results of the 1. study, concentrations of 0.5., 0.16, 0.5 and 5 mg/plate (with and wihtout S9) were selected for the second study for strains TA97a, TA98 and TA 100. For the strain TA102 concentrations of 0.016, 0.05, 0.16, 0.5, 1.6 (mg/plate) with and without S9 were selected. For the third study the strain TA102 was tested without S9 at concentrations of 0.5, 1.6 and 5 (mg/plate) and with S9 at concentration of 5 mg/plate. The validity of the test system was approved with sufficient positive controls.

Based on the results of this test the test substance is regarded to be not mutagenic.