Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In two separate in vitro laboratory studies, succinic anhydride was non-mutagenic in the Ames assay with and without metabolic activation at doses of up to 10000 ug/plate. No evidence of in vitro mutagenicity was seen when succinic anhydride was tested for chromosomal aberrations and sister chromatid exchanges in two additional laboratory studies conducted in vitro.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
18 February 2010 to 7 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Fully GLP-compliant. The gene mutation assay with maleic acid is considered appropriate for read-across to address the potential for succinic anhydride to induce point mutations in mammalian cell lines. There is no evidence of mutagenic potential from the remaining genotoxicity and carcinogenicity studies. The rapid hydrolysis of succinic anhydride to the human endogenous metabolite succinic acid, which acts as a ubiquitous substrate for ATP generation via the Kreb's cycle, would suggest that mutagenicity is unlikely. The structural and functional similarity of maleic acid and succinic acid justify the use of read-across information.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus of V79 cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium.Thawed stock cultures are propagated at 37 °C in 80 cm2 plastic flasks (GREINER, 72632 Frickenhausen, Germany). About 5×10E5 cells are seeded into each flask with 15 mL of MEM (minimal essential medium; SEROMED, 12247 Berlin, Germany) supplemented with 10 % fetal bovine serum (FBS; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % neomycin. The cells are sub-cultured twice weekly. The cell cultures are incubated at 37 °C in a 4.5 % carbon dioxide atmosphere (95.5% air).For the selection of mutant cells the medium is supplemented with 11 µg/mL 6-thioguanine (6-TG, SIGMA GmbH, 82041 Deisenhofen, Germany).For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hanks salts, Neomycin (5 µg/mL) and Ampho-tericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 µg/mL thioguanine. All cultures were incubated at 37 °C in a humidified atmos-phere with 1.5 % CO2 (98.5 % air).- Properly maintained: yes- Periodically checked for karyotype stability: yes- Periodically checked for Mycoplasma contamination: yes- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
First experiment (doses applied, but not evaluated: in parentheses), without and with metabolic activation, 4 h treatment:(37.5), 75.0, 150, 300, 600, 1200 µg/mLSecond experiment (doses applied, but not evaluated: in parentheses), without metabolic activation, 24 h treatment:(75.0), 150, 300, 600, 900, 1200 µg/mLSecond experiment (doses applied, but not evaluated: in parentheses), with metabolic activation, 4 h treatment:(75.0), 150, 300, 600, 900, 1200 µg/mLIn experiment I and II the cultures at the lowest concentrations of 37.5 µg/mL in experiment I and 75.0 µg/mL in experiment II with and without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: On the day of the experiment (immediately before treatment), the test item was dissolved in deionised water. The final concentration of deionised water in the culture medium was 10 % v/v.- Justification for choice of solvent/vehicle: Common solvent for this type of studies.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activationMigrated to IUCLID6: for cultures without metabolic activation. Dissolved in: Nutrient medium. Final concentration: 0.225 mg/mL = 1.8 mM (experiment I), 0.150 mg/mL = 1.2 mM (experiment II).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activationMigrated to IUCLID6: for cultures with metabolic activation. Dissolved in: DMSO. Final concentration in nutrient medium 0.5 %. Final concentration: 1.1 µg/mL = 4.3 µM.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Preincubation period: yes- Exposure duration: 4 hours (first experiment with/without metabolic activation and second experiment with metabolic activation) and 24 hours (second experiment without metabolic activation)- Expression time (cells in growth medium): 48 hoursSELECTION AGENT (mutation assays): medium is supplemented with 11 µg/mL 6-thioguanine (6-TG, SIGMA GmbH, 82041 Deisenhofen, Germany).NUMBER OF REPLICATIONS: 2DETERMINATION OF CYTOTOXICITYby the assessment of the relative cloning efficiency (mean number of colonies per flask divided by the mean number of colonies per flask of the corresponding control) × 100
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.A positive response is described as follows:A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 900 µg/mL and above in experiment II without metabolic activation following 24 hours of exposure
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: noRANGE-FINDING/SCREENING STUDIES: yes, see aboveCOMPARISON WITH HISTORICAL CONTROL DATA: yes, see also attachment
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

For Tables See Attachment.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix: 37.5; 75.0; 150; 300; 600; and 1200 µg/mL

with S9 mix: 37.5; 75.0; 150; 300; 600; and 1200 µg/mL

Experiment II:

without S9 mix: 75.0; 150; 300; 600; 900; and 1200 µg/mL

with S9 mix: 75.0; 150; 300; 600; 900; and 1200 µg/mL

The maximum concentration of the test item in the main experiments is equal to molar concentration of approximately 10 mM.

No precipitation of the test item was observed up to the maximal concentration in all ex-periments.

Cytotoxic effects as indicated by a relative cloning efficiency of less than 50 % in both parallel cultures solely occurred at 900 µg/mL and above in experiment II without metabolic activation following 24 hours of exposure.

No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of the second experiment without metabolic activation at 900 and 1200 µg/mL. This effect, however, was not reproduced in the parallel culture performed under identical conditions. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 statistics software. A significant trend of the mutation frequency was solely determined in the first culture of the first experiment without metabolic activation. This trend was judged as irrelevant fluctuation however, since it actually was reciprocal, going down versus increasing concentrations.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 10.7 up to 28.5 mutants per 10E6 cells; the range of the groups treated with the test item was from 9.2 up to 59.1 mutants per 10E6 cells.

EMS (225 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Maleic acid is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of Maleic acid to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest applied concentration (1200 µg/mL) was equal to a molar concentration of approximately 10 mM - the limit for this assay.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

Conclusion

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Maleic acid is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro assays

A number of published studies investigating the invitro mutagenicity of succinic anhydride were summarized in a review by the US National Toxicology Program. Succinic anhydride showed no mutagenic activity when tested in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 and TA1537 with and without Aroclor 1254 -induced male Sprague Dawley rat or Syrian hamster liver S9 at doses of up to 10000 ug/plate. Succinic anhydride did not induce sister chromatid exchanges or chromosomal aberrations in cultured CHO cells in the presence or absence of exogenous metabolic activation.

A gene mutation HRPT assay was completed with the structural and functional analogue of succinic anhydride (or the metabolic product succinic acid), maleic acid. Maleic acid under the experimental conditions reported did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Maleic acid, and by read-across, succinic anhydride is considered to be non-mutagenic in this HPRT assay.

Given the negative response in the in vitro battery of tests, no in vivo assays are required in accordance with stipulations in Column 2 of Annexes VII to X of Regulation 1907/2006.

A negative result (no genotoxicity) was also reported in the in vivo screening tests of Kawachi et al (1980) for the bone marrow test in rats (in vivo, chromosome aberreations) and silk worms (mutations).

Justification for classification or non-classification

The results of the available studies do not trigger classification according to current EU criteria.