Registration Dossier
Registration Dossier
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EC number: 629-679-7 | CAS number: 42482-06-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The substance has been tested in a guideline bacterial reverse mutation assay and found to be negative (not mutagenic). Other members of the C8-12 Alkenyl Succinic Anhydride category have also been nonmutagenic in in vitro genotoxicity tests. The conclusion is that the substance is not genotoxic.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine
- 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:
- S9 liver microsomal fraction from male Sprague Dawley rats induced with PB and BNF, purchased from Trinova Biochem GmbH, Giessen, Germany.
- Test concentrations with justification for top dose:
- 3.16 to 5000 µg/plate for TA 98, TA 100, TA 1535, and E. coli WP2.
5.0 to 5000 µg/plate for TA 1537. - Vehicle / solvent:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- For all strains with S9
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine
- Remarks:
- TA 98 and TA 1537 without S9
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- E. coli WP2 without S9
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA 100 and TA 1535 without S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:in agar (plate incorporation); and preincubation;
DURATION
- Preincubation period: 60 minutes
- Exposure duration: in agar is 48 hours
DETERMINATION OF CYTOTOXICITY
- Method: Clearing of diminution of the background lawn
Colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgerate GmbH.) In addition, TA 1535 and TA 1537 were counted manually. - Evaluation criteria:
- The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occur at non-toxic doses of the test item, and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with our without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of revisions is at least three times higher
as compared to the reversion rate of the solvent control (5).
According to the 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.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. - Statistics:
- No data.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In both the plate incorporation and pre-incubation method there was no increase in the mutation rate in any strains with our without S9.
- Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Octenyl Succinic Anhydride was tested in an Ames Assay (OECD 471) using both a plate incorporation method and pre-incubation method. No increase in mutation rate was seen. The test substance is not mutagenic under the conditions of this assay.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: other: clastogenicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2011-05-19 to 2011-07-26
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- A category approach will be used for the hazard assessment of several endpoints. The hypothesis for the category of C8-12 Alkenyl Succinic Anhydrides is that data can be read-across among members of the category because their properties and behaviours are similar, based on common functional groups and similar breakdown products, and based on a constant pattern in changing of the potency of properties of the various carbon chain lengths. These features are in accordance with Annex XI, Section 1.5, of Regulation EC No. 1907/2006. Common functional groups are: a dihydro-2,5 -furandione (cyclic anhydride) ring, carbon chain of length 8 to 12 carbons, with or without branching methyl groups, and a single double bond in the carbon chain, location unspecified. There are no additional functional groups which would contribute incremental or different toxicity.
The breakdown products are the dioic acids of the corresponding anhydride; these also have common functional groups. A constant pattern may also be displayed in acute toxicity, dermal irritancy and biodegradation, with the lowest carbon chain length (C8) displaying the highest activity. Irritation, toxicity and degradation potential diminish with increasing carbon chain length. Read-across among the category members is substantiated by the common behaviour in physico-chemical and toxicity behaviours, as provided in the Chemical Category Report Format (CCRF) attached to the IUCLID file. It is adequate to fulfil the information requirements of Annex IX, to be the basis for classification and labelling decisions, and for risk assessment. - Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- 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
- 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. - Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation Migrated to IUCLID6: 400 and 600 µg/mL
- 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.
- 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
- Conclusions:
- Interpretation of results (migrated information):
negative
During the in vitro chromosomal aberration test and under the experimental conditions reported, the test item, a category member, tripropenyl succinic anhydride, 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.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2011-09-12 to 2011-11-24
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- A category approach will be used for the hazard assessment of several endpoints. The hypothesis for the category of C8-12 Alkenyl Succinic Anhydrides is that data can be read-across among members of the category because their properties and behaviours are similar, based on common functional groups and similar breakdown products, and based on a constant pattern in changing of the potency of properties of the various carbon chain lengths. These features are in accordance with Annex XI, Section 1.5, of Regulation EC No. 1907/2006. Common functional groups are: a dihydro-2,5 -furandione (cyclic anhydride) ring, carbon chain of length 8 to 12 carbons, with or without branching methyl groups, and a single double bond in the carbon chain, location unspecified. There are no additional functional groups which would contribute incremental or different toxicity.
The breakdown products are the dioic acids of the corresponding anhydride; these also have common functional groups. A constant pattern may also be displayed in acute toxicity, dermal irritancy and biodegradation, with the lowest carbon chain length (C8) displaying the highest activity. Irritation, toxicity and degradation potential diminish with increasing carbon chain length. Read-across among the category members is substantiated by the common behaviour in physico-chemical and toxicity behaviours, as provided in the Chemical Category Report Format (CCRF) attached to the IUCLID file. It is adequate to fulfil the information requirements of Annex IX, to be the basis for classification and labelling decisions, and for risk assessment. - Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: IWGT Recommendations
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: 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 I with and without metabolic activation: 0.1, 0.5, 2.5, 5.0, 7.5, 10.0 mM
Pre-experiment II without metabolic activation (24 h long-term exposure): 0.1, 0.5, 1.0, 2.0, 4.0, 6.0 mM
Experiment I
without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.5, 4.0 mM
with metabolic activation: 0.5, 1.0, 2.0, 3.5, 4.0, 4.5, 5.0, 5.5 mM
Experiment II
without metabolic activation: 0.1, 0.2, 0.5, 1.1, 1.4, 1.7, 2.3, 2.6 mM
with metabolic activation: 1.5, 2.5, 3.7, 4.4, 4.8, 5.2, 5.6, 6.0 mM - Vehicle / solvent:
- RPMI cell culture medium was used as vehicle (RPMI + 5% HS)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- Migrated to IUCLID6: 2.5 µg/ml
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Migrated to IUCLID6: 200 µg/mL and 300 µg/mL
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- Migrated to IUCLID6: 10 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 24 h (long-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days
SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG) - Evaluation criteria:
- The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative. - Statistics:
- The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- In this mutagenicity test under the experimental conditions reported, the test item, which is a category member, tripropenyl succinic anhydride, is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells. 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:
The test item Tripropenyl succinic anhydride was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I, 4.0 mM (without metabolic activation) and 5.5 mM (with metabolic activation) were selected as the highest concentrations. In experiment II, 2.6 mM (without metabolic activation) and 6.0 mM (with metabolic activation) were selected as the highest concentrations. Experiment I, without and with metabolic activation and experiment II, with metabolic activation, were performed as a 4 h short-term exposure assay. Experiment II, without metabolic activation was performed as a 24 h long-term exposure assay. The test item was suspended in cell culture medium (RPMI with 5% HS)
The test item was investigated at the following concentrations:
Experiment I
without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.5, 4.0 mM
and with metabolic activation: 0.5, 1.0, 2.0, 3.5, 4.0, 4.5, 5.0, 5.5 mM
Experiment II
without metabolic activation: 0.1, 0.2, 0.5, 1.1, 1.4, 1.7, 2.3, 2.6 mM
and with metabolic activation: 1.5, 2.5, 3.7, 4.4, 4.8, 5.2, 5.6, 6.0 mM
No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I and II without and with metabolic activation.
In experiment I, withoutmetabolic activation, the relative total growth (RTG) was 4.0% for the highest concentration (4.0 mM) evaluated. The highest concentration evaluated with metabolic activation was 5.5 mM with a RTG of 11.0%. In experiment II, without metabolic activation, the relative total growth (RTG) was 17.0% for the highest concentration (2.6 mM) The highest concentration evaluated with metabolic activation was 6.0 mM with a RTG of 9.6%. Dose groups showing extremely high toxicity (RTG<10%) were not considered for evaluation of mutagenicity.
The Global Evaluation Factor was not exceeded by the induced mutant frequencies at any concentration (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories). In experiment I, with metabolic activation, an increased mutation frequency was found in the highest concentration (5.5 mM). This effect was found only in one concentrations in the highly cytotoxic dose range (RTG of 11%) and was not confirmed in the second experiment. This increase in mutant frequency was considered as not biologically relevant.
No dose-response relationship was observed.
Additionally, in experiment I and II, colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation).
EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.
This study is classified as acceptable. This study satisfies the requirements for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: other: clastogenicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011-05-19 to 2011-07-26
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- 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
- 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. - Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation Migrated to IUCLID6: 400 and 600 µg/mL
- 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.
- 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
- Conclusions:
- Interpretation of results (migrated information):
negative
During the in vitro chromosomal aberration test and under the experimental conditions reported, the test item, a category member, tripropenyl succinic anhydride, 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.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011-09-12 to 2011-11-24
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: IWGT Recommendations
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: 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 I with and without metabolic activation: 0.1, 0.5, 2.5, 5.0, 7.5, 10.0 mM
Pre-experiment II without metabolic activation (24 h long-term exposure): 0.1, 0.5, 1.0, 2.0, 4.0, 6.0 mM
Experiment I
without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.5, 4.0 mM
with metabolic activation: 0.5, 1.0, 2.0, 3.5, 4.0, 4.5, 5.0, 5.5 mM
Experiment II
without metabolic activation: 0.1, 0.2, 0.5, 1.1, 1.4, 1.7, 2.3, 2.6 mM
with metabolic activation: 1.5, 2.5, 3.7, 4.4, 4.8, 5.2, 5.6, 6.0 mM - Vehicle / solvent:
- RPMI cell culture medium was used as vehicle (RPMI + 5% HS)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- Migrated to IUCLID6: 2.5 µg/ml
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Migrated to IUCLID6: 200 µg/mL and 300 µg/mL
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- Migrated to IUCLID6: 10 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 24 h (long-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days
SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG) - Evaluation criteria:
- The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative. - Statistics:
- The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- In this mutagenicity test under the experimental conditions reported, the test item, which is a category member, tripropenyl succinic anhydride, is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells. 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:
The test item Tripropenyl succinic anhydride was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I, 4.0 mM (without metabolic activation) and 5.5 mM (with metabolic activation) were selected as the highest concentrations. In experiment II, 2.6 mM (without metabolic activation) and 6.0 mM (with metabolic activation) were selected as the highest concentrations. Experiment I, without and with metabolic activation and experiment II, with metabolic activation, were performed as a 4 h short-term exposure assay. Experiment II, without metabolic activation was performed as a 24 h long-term exposure assay. The test item was suspended in cell culture medium (RPMI with 5% HS)
The test item was investigated at the following concentrations:
Experiment I
without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.5, 4.0 mM
and with metabolic activation: 0.5, 1.0, 2.0, 3.5, 4.0, 4.5, 5.0, 5.5 mM
Experiment II
without metabolic activation: 0.1, 0.2, 0.5, 1.1, 1.4, 1.7, 2.3, 2.6 mM
and with metabolic activation: 1.5, 2.5, 3.7, 4.4, 4.8, 5.2, 5.6, 6.0 mM
No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I and II without and with metabolic activation.
In experiment I, withoutmetabolic activation, the relative total growth (RTG) was 4.0% for the highest concentration (4.0 mM) evaluated. The highest concentration evaluated with metabolic activation was 5.5 mM with a RTG of 11.0%. In experiment II, without metabolic activation, the relative total growth (RTG) was 17.0% for the highest concentration (2.6 mM) The highest concentration evaluated with metabolic activation was 6.0 mM with a RTG of 9.6%. Dose groups showing extremely high toxicity (RTG<10%) were not considered for evaluation of mutagenicity.
The Global Evaluation Factor was not exceeded by the induced mutant frequencies at any concentration (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories). In experiment I, with metabolic activation, an increased mutation frequency was found in the highest concentration (5.5 mM). This effect was found only in one concentrations in the highly cytotoxic dose range (RTG of 11%) and was not confirmed in the second experiment. This increase in mutant frequency was considered as not biologically relevant.
No dose-response relationship was observed.
Additionally, in experiment I and II, colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation).
EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.
This study is classified as acceptable. This study satisfies the requirements for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.
Referenceopen allclose all
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 |
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 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Additional information from genetic toxicity in vitro:
Octenyl succinic anhydride (OSA) has been tested in a guideline bacterial reverse mutation assay (both plate incorporation method and preincubation method) and found to be negative (not mutagenic). Other members of the C8-12 Alkenyl Succinic Anhydride category (tetrapropenyl succinic anhydride, n-dodecenyl succinic anhydride, and tripropenyl succinic anhydride) have also been found to be nonmutagenic in the Ames assay. Tripropenyl succinic anhydride was tested in a guideline chromosomal aberrations assay and in a mammalian mutation assay (mouse lymphoma assay), and found to be negative. In the review by the World Health Organization of Cyclic Acid Anhydrides (CICAD 75, 2009), genotoxicity tests for a variety of category members were negative for genotoxicity. These data indicate that the alkenyl succinic anhydrides are not genotoxic.
A category approach is used for the hazard assessment of several endpoints. The hypothesis for the category of C8-12 Alkenyl Succinic Anhydrides is that data can be read-across among members of the category because their properties and behaviours are similar, based on common functional groups and similar breakdown products, and based on a constant pattern in changing of the potency of properties of the various carbon chain lengths. Functional groups include a dihydro-2,5 -furandione cyclic anhydride ring, a carbon chain of length 8 -12 carbons, and a single double-bond within the carbon chain. The primary functional group associated with toxicity is the succinic anhydride moiety, which quickly is hydrolysed to form a butanedioic acid. A constant pattern may also be displayed in acute toxicity, dermal irritancy and biodegradation, with the lowest carbon chain length (C8) displaying the highest activity. Irritation, toxicity and degradation potential diminish with increasing carbon chain length. Read-across among the category members is substantiated by the common behaviour in physico-chemical and toxicity behaviours, as provided in the Chemical Category Report Format (CCRF) attached to the IUCLID file. It is adequate to fulfill the information requirements of Annex IX, to be the basis for classification and labelling decisions, and for risk assessment.
Justification for selection of genetic toxicity endpoint
Guideline study under GLP
Justification for classification or non-classification
This substance does not meet the criteria for mutagenicity in Regulation EC No. 1272/2008.
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