3-(2-dodecenyl)succinic anhydride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: other: clastogenicity

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2011-05-19 to 2011-07-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Dihydro-3-(tripropenyl)furan-2,5-dione

EC Number:

295-556-6

EC Name:

Dihydro-3-(tripropenyl)furan-2,5-dione

Cas Number:

92077-08-2

IUPAC Name:

92077-08-2

Test material form:

other: liquid

Details on test material:

Name: Tripropenyl succinic anhydride
Product: Genopur ASA
CAS No.: 92077-08-2
Batch No.: ESD0010823
Chemical Name: dihydro-3-(tripropenyl)furan-2,5-dione
Molecular Weight: 220 g/mol (calculated based on the given chemical structure)
pH: 1-2
Physical State at RT: liquid
Density: 1.01 g/cm3 (20°C, DIN 51757)
Colour: yellow to brown
Expiry Date: 01.12.2012
Storage Conditions: at room temperature

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment:
with and without metabolic activation: 0.02, 0.039, 0.078, 0.16, 0.31, 0.63, 1.25, 2.5, 5 and 10 mM

Experiment I:
without metabolic activation: 2, 3 and 4 mM
with metabolic activation: 8, 9 and 10 mM


Experiment II:
without metabolic activation: 0.5, 1 and 2 mM
with metabolic activation: 8.5, 9.5 and 10 mM

Vehicle / solvent:

-Vehicle (s)/solvent(s) used: cell culture medium
-Justification for choice of solvent/vehicle: Due to the nature of the test item it was suspended in cell culture medium (MEM) followed by ultrasonic for 10 minutes. The solvent was compatible with the survival of the cells and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Migrated to IUCLID6: 400 and 600 µg/mL

Controls 2

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Migrated to IUCLID6: 0.83 µg/mL

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10^4 - 5 x 10^4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell density

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Results of chromosome analysis

Without metabolic activation

	Cytotoxicity	Chromatid aberrations				Isochromatid aberrations				rel. Mitotic index (%)	rel. Cell density (%)	Poly-ploidy	mean % aberrant cells
		gaps	breaks	inter-changes	other	gaps	breaks	inter-changes	other				incl. Gaps	excl. Gaps
Experiment I
negative control	-	5	2	1	0	0	0	0	0	100	100	2	3.5	1.5
2 mM	no	2	0	0	1	0	0	0	0	78	87	1	1.5	0.5
3 mM	yes	5	2	0	0	0	0	0	0	52	70	2	3.0	1.0
4 mM	yes	7	2	0	1	0	0	0	0	34	66	2	4.0	1.5
5 mM	yes	-	-	-	-	-	-	-	-	4	52	-	-	-
EMS (600 µg/mL)	-	17	18	2	2	1	0	1	5	95	93	3	15.5	11.5
Experiment II
negative control	-	3	1	0	0	0	0	0	0	100	100	2	2.0	0.5
0.5 mM	no	0	0	0	0	1	0	0	0	97	112	1	0.5	0.0
1.0 mM	no	0	1	1	0	1	0	0	0	95	97	1	1.5	1.0
2.0 mM	yes	2	2	0	0	0	0	0	0	50	91	0	2.0	1.0
3.0 mM	yes	-	-	-	-	-	-	-	-	21	63	-	-	-
EMS (400 µg/mL)	-	8	13	4	0	0	0	0	3	44	87	1	12.0	8.5

 

Table 2: Results of chromosome analysis

With metabolic activation

	Cytotoxicity	Chromatid aberrations				Isochromatid aberrations				rel. Mitotic index (%)	rel. Cell density (%)	Poly-ploidy	mean % aberrant cells
		gaps	breaks	inter-changes	other	gaps	breaks	inter-changes	other				incl. Gaps	excl. Gaps
Experiment I
negative control	-	3	5	0	0	1	0	0	0	100	100	0	4.5	2.5
8 mM	no	5	0	0	0	0	0	0	0	82	94	1	2.0	0.0
9 mM	no	2	1	0	1	0	0	0	0	80	86	2	2.0	1.0
10 mM	yes	3	4	2	0	0	0	0	0	56	94	5	4.5	3.0
CPA (0.83 µg/mL)	-	5	10	8	3	2	1	3	0	103	90	2	12.5	11.0
Experiment II
negative control	-	3	2	1	0	0	0	0	0	100	100	0	3.0	1.5
8.5 mM	no	3	1	0	0	0	0	0	0	90	73	0	2.0	0.5
9.5 mM	no	4	3	0	1	0	0	0	1	99	82	0	4.0	2.5
10.0 mM	no	3	3	0	0	1	0	0	0	76	85	2	3.0	1.5
CPA (0.83 µg/mL)	-	5	12	7	0	0	1	1	1	94	81	2	11.5	9.5

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

During the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item, which is a category member (Tripropenyl succinic anhydride (TSA)), did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item Tripropenyl succinic anhydride is considered to be non-clastogenic. Data can be read-across among members of the C8-12 Alkenyl Succinic Anhydrides Category, based on common functional groups, similar break-down products and potency patterns among carbon-chain length. This is adequate to fulfill the information requirements, to be the basis for classification and labelling decisions, and for risk assessment.

Executive summary:

To investigate the potential of Tripropenyl succinic anhydride to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.

Due to the nature of the test item it was suspended in cell culture medium (MEM) followed by ultrasonic for 10 minutes.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 2, 3 and 4 mM

with metabolic activation: 8, 9 and 10 mM

Experiment II:

without metabolic activation: 0.5, 1 and 2 mM

with metabolic activation: 8.5, 9.5 and 10 mM

Precipitation of the test item was observed at concentrations of 3 mM and above (with and without metabolic activation). In the presence of S9 mix, less toxicity of the test item, compared to the experiment without metabolic activation, was found. Consequently, in the experiments with metabolic activation precipitation was noted at all concentrations evaluated.

In experiment I without metabolic activation toxic effects of the test item were observed at concentrations of 3 mM and higher, with metabolic activation at the highest concentration of 10 mM.

In experiment II without metabolic activation, toxic effects of the test item were observed at concentrations of 2 mM and higher. With metabolic activation no toxic effects of the test item were noted.

In the experiments I and II no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups evaluated were within the historical control data of the negative control.

In both experiments with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data.