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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In order to evaluate the genetic toxicity potential of the test substance one in vitro test according to OECD guideline 471 (i.e. Ames test) and one in vivo test according to OECD guideline 474 (i.e. MNT) were performed. Both tests were negative in result.

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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)

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

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'.

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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The mutagenic effects of the test item were determined in a reverse mutation assay with Salmonella typhimurium. Test systems were the strains TA97a, TA 98, TA 100 and TA 102 with (+) and without (-) the metabolic activation system S9 (from male Wistar rats). Concentrations tested were 0.005 - 0.05 - 0.16 - 0.5 - 1.6 and 5 mg/plate. Three replicates per concentration level and control were performed. Based on the results of this study the test item was found to have no mutagenic effects on Salmonella typhimurium strains TA 97a, TA 100, TA 98, and TA 102 with (+) and without (-) the metabolic activation system S9 from Wistar rats at concentrations up to 5 mg/plate.

In order to study the induction of micronuclei in bone marrow cells Hostacor 4323 was tested in an in vivo micronucleus test in erythrocytes according to OECD 474 guideline performed in NMRI mice. The test substance was administered to ten mice (5/ sex) twice at an interval of 24 hours orally by gavage to the test animals at a dose of 2000 mg/kg bw. The vehicle, sesame oil, was administered in the same way to the negative control groups. Cyclophosphamide was used as positive substance and was administered once orally at a dose of 50 mg/kg bw.The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Hostacor 4323. Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system.

Justification for classification or non-classification

Based on the available data no classification is warranted according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).