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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic or genotoxic

Link to relevant study records

Referenceopen allclose all

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
Justification for type of information:
Guideline study under GLP
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
Specific details on test material used for the study:
As a result of increasingly rigorous criteria being applied to the analysis of commercial material used in physical property/toxicity testing, the identity of the material has been modified to reveal a more accurate and precise depiction of the commercial substance. This enhancement is reflected in changes in chemical identifiers such as EC and/or CAS numbers from those noted in earlier versions of data records or in study reports.
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:
rat liver S9 fraction, 5%
Test concentrations with justification for top dose:
0.5, 0.75, 1.0, 1.58, 2.5, 5.0, 7.5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycin, 60 ug/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); with confirmatory tests using the preincubation method.
Rat liver S9 fraction was obtained from Molecular Toxicology, Inc., and used at 5%. The prepared S9 mix contained the following sterile cofactors (Maron & Ames, 1983): 8 mM MgCl2, 33 mM KCl, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP.

The test substance was formulated as a solution in DMSO (0.0050, 0.0075, 0.01, 0.0158, 0.025, 0.05, 0.075, 0.158, 0.5, 1.58, 5, 15.8 and 50 mg/mL) to provide corresponding dose levels of up to 5000 µg/plate. The solutions were vortexed prior to use.

The confirmatory test employed the pre-incubation modification of the plate incorporation test. The test or control substances, bacteria suspension, and S9/substitution buffer were incubated under agitation for approximately 30 minutes at 37±2°C prior to mixing with the overlay agar and pouring onto the minimal agar plates before proceeding as described for the initial test. The study design for the confirmatory test, including strains, dose levels etc. was as described above for the initial (main) test.

A supplemental test (plate incorporation and pre-incubation) was performed to adequately interpret assay results, due to the findings of toxicity (incomplete bacterial lawn) and contamination in the original studies. The study was performed with strains TA 1535 and 1538, at 8 doses ranging from 0.5 to 15.8 µg/plate. Appropriate vehicle and positive controls were included. This supplemental data and the data collected during original phase of testing are presented in body of the report.

NUMBER OF REPLICATIONS:

The background lawn for vehicle control plates should appear normal (i.e., slightly hazy with abundant microscopic non-revertant bacterial colonies). The mean revertant colony counts for each strain treated with the vehicle should lie close to or within the expected range taking into account the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012). The positive controls (with S9 where required) should produce substantial increases in revertant colony numbers with the appropriate bacterial strain as specified in the Evaluation of Mutagenicity Section below.
In the case where part of the study is invalid based on these criteria, detailed results for that part of the study will not be reported and the affected part of the study would normally be subjected to an automatic repeat as described in an amendment, if appropriate.

Toxic effects of the test substance are indicated by the partial or complete absence of a background lawn of non-revertant bacteria (colony counts, if any, should not be reported) or a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle control taking into account the laboratory historical control range. Where precipitation obscures observations on the condition of the background lawn, the lawn can be considered normal and intact if the revertant colony counts are within the expected range based on results for lower dose levels and historical control counts for that strain.


Evaluation criteria:
For each experimental point, the Mutation Factor (MF) was calculated by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. The mutagenic activity of the test item was assessed by applying the following criteria:

The results were considered positive (i.e., indicative of mutagenic potential) if:

The results for the test item showed a substantial increase in revertant colony counts, i.e., response MF ≥ 2 for strains TA98, TA100, and WP2 uvrA or MF ≥ 3 for strains TA1535 and TA1537, with mean value(s) outside the laboratory historical control range. Otherwise, results were considered negative.
The above increase must be dose related and/or reproducible, i.e., increases must be obtained at more than one experimental point (at least one strain, more than one dose level, more than one occasion or with different methodologies).

If the second criterion is not met, the results may be classified as equivocal, and further testing may be appropriate.
A test substance that produces neither a concentration related increase in the number of revertant colonies nor a reproducible substantial increase in revertant colonies is considered to be non-mutagenic in this test system.

Statistics:
The testing laboratory calculated means and standard deviations for all quantitative data collected.
Key result
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
Remarks:
incomplete bacterial lawn was observed; additional data was generated.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012). The positive control substances caused the expected substantial increases in revertant colony counts in both the absence and presence of S9 in each phase of the test confirming the sensitivity of the test and the activity of the S9 mix. Therefore, each phase of the test is considered valid.

Signs of precipitation were observed at 5000 µg/plate for all strains tested, for both methods, and with and without metabolic activation. Contamination was noted with strain TA1535 at dose levels of 500 and/or 1580 µg/plate in both the absence and presence of S9 using either method.

Evidence of toxicity was observed by presence of an incomplete lawn for strain TA98 at 500 µg/plate and with strains TA1535 and TA1537 at doses ≥ 50 µg/plate. To further investigate toxicity, supplemental testing was performed for strains TA1535 and TA1537 using eight dose levels in a range of 0.5 to 15.8 µg/plate.

Single plate contamination, that did not obscure plate counts, was observed for strain TA1357 at 0.5 µg/plate, without the presence of S9. No evidence of toxicity, contamination or precipitation was observed in the supplemental testing performed for stains TA1535 or TA1537.

For all strains, at least five non-toxic dose levels without precipitation or plate contamination were evaluated, therefore bacterial mutagenicity was adequately assessed.

There was no concentration-related or substantial test substance related increases in the number of revertant colonies observed with strains TA1535, TA1537, TA98 , TA100 or E. coli WP2 uvrA in both the absence and presence of S9 using either the plate incorporation or the pre-incubation method. Based on these findings and on the evaluation system used, n-Octadecenyl Succinic Anhydride (n-ODSA), CAS# 67066-88-0, did not elicit evidence of bacterial mutagenicity in the Ames assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

n-Octadecenyl Succinic Anhydride (n-ODSA) was not mutagenic in the Ames assay.
Executive summary:

The Ames test was conducted with n-Octadecenyl Succinic Anhydride (n-ODSA), CAS# 67066-88 0 at levels of 0.5, 0.75, 1.0, 1.58, 2.5, 5.0, 7.5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate, with the high level being the standard limit for this test. The main test was conducted using the plate incorporation method in both the absence and presence of metabolic activation (chemically-induced rat liver S9 mix). The results of the test were confirmed using a similar study design but employing the pre-incubation modification of the Ames test.

Signs of precipitation were observed at 5000 µg/plate for all strains tested, for both methods, and with and without metabolic activation. Contamination was noted with strain TA1535 at dose levels of 500 and/or 1580 µg/plate in both the absence and presence of S9 using either method. In addition, individual plate contamination was noted with strain TA1537 in the 0.5 µg/plate dose level without metabolic activation; however this contamination did not obscure the counts.

Evidence of toxicity was observed by presence of an incomplete lawn for strain TA98 at 500 µg/plate and with strains TA1535, TA1537 at doses ≥ 50 µg/plate. To further investigate toxicity supplemental testing was performed for strains TA1535, TA1537 using eight dose levels in a range of 0.5 to 15.8 µg/plate.

Single plate contamination, that did not obscure plate counts, was observed for strain TA1357 at 0.5 µg/plate, without the presence of S9. No other evidence of toxicity, contamination or precipitation was observed in the supplemental testing performed for stains TA1535, TA1537. For all strains, at least five non-toxic dose levels without precipitation or plate contamination were evaluated, therefore bacterial mutagenicity was adequately assessed.

In conclusion, based on these findings and on the evaluation system used, n-Octadecenyl Succinic Anhydride (n-ODSA), CAS# 67066 -88 -0 did not elicit evidence of bacterial mutagenicity in the Ames assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This study was conducted on 2,5-furandione, dihydro-, mono-C15- 20-alkenyl derivatives (CAS 68784-12-3), an analogue substance used as the source of information for the assessment of the target substance through read-across. Therefore, this study is informative for evaluation of the environmental fate and toxicity of the target substance, Reaction products of furan-2,5-dione and octadec-1-ene (known here as n-ODSA EC 701-338-8; no CASRN available), and it is adequate for classification and risk assessment.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
primary culture, other: lymphocytes
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a female donor (35 years old) for the first and second experiment and from a 46 year-old male donor for Experiment II. Blood samples were drawn by venous puncture and collected in heparinized tubes. Short-term cultures of human lymphocytes were stimulated to divide by the addition of a phytohaemagglutinin, PHA) to the culture medium. Mitotic activity began at about 40 hours after PHA stimulation and reached a maximum at around 3 days. The
chromosome constitution remained diploid during short-term culture.
Treatments commenced 50 - 80 hours after culture initiation when the cells were actively proliferating At preparation time 22 hours, a minimum of three cultures treated with separated concentrations of the test item were evaluated for the potential to induce structural chromosomal aberrations.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Experiment 1
DMSO 0.5 % without S9 4 / 18 hrs
Pos. control 825.0 µg without S9 4 / 18 hrs
ASA 22.7 µg without S9 4 / 18 hrs
ASA 39.8 µg without S9 4 / 18 hrs
ASA 213.2 µg without S9 4 / 18 hrs
ASA 373.2 µg without S9 4 / 18 hrs
DMSO 0.5 % with S9 4 / 18 hrs
Pos. control 22.5 µg with S9 4 / 18 hrs
ASA 22.7 µg with S9 4 / 18 hrs
ASA 39.8 µg with S9 4 / 18 hrs
ASA 121.9 µg with S9 4 / 18 hrs
ASA 213.2 µg with S9 4 / 18 hrs

Experiment 2
DMSO 0.5 % without S9 22 / 0 hrs
Pos. control 770.0 µg without S9 22 / 0 hrs
ASA 13.0 µg without S9 22 / 0 hrs
ASA 22.7 µg without S9 22 / 0 hrs
ASA 69.6 µg without S9 22 / 0 hrs
ASA 121.9 µg without S9 22 / 0 hrs
DMSO 0.5 % with S9 4 / 18 hrs
Pos. control 30.0 µg with S9 4 / 18 hrs
ASA 29.8 µg with S9 4 / 18 hrs
ASA 52.2 µg with S9 4 / 18 hrs
ASA 279.9 µg with S9 4 / 18 hrs
ASA 489.8 µg with S9 4 / 18 hrs
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; ASA was added to the test medium dissolved in DMSO. Cultures were treated with test medium.

DURATION
- Preincubation period: 72 hours
- Exposure duration: 4 hours or 18 hours or 22 hours, respectively
- Expression time (cells in growth medium): 18 hours, 4 hours or 0 hours, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, for 3 hours prior fixation
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 per concentration, each with and without S9 and experiment I and II

NUMBER OF CELLS EVALUATED: 2x100 metaphases per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (main test) and cloning efficiency (range finder)

OTHER EXAMINATIONS:
- Determination of polyploidy: visual (100x microscope) counting of centromers. If multiple copies of the haploid chromosome number (other than diploid) are scored then the count is recorded and the cell classified as polyploid.
- Determination of endoreplication: visual (100x microscope). If the chromosomes are arranged in closely apposed pairs, i.e. 4
chromatids instead of 2, the cell is scored as endoreduplicated.

Evaluation criteria:
Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in
mitosis) was determined

A test item is classified as non-mutagenic if the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding gaps). and no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding gaps) and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if the number of induced numerical aberrations is not in the range of our historical control data
(0.0 – 0.8 % polyploid cells).
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Key result
Species / strain:
primary culture, other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant increase in the pH value was observed (e.g. Exp. I: solvent control: pH 7.4 versus pH 7.5 at 3500 µg/mL).
- Effects of osmolality: No relevant increase in the osmolarity was observed (e.g. Exp. I: solvent control: 378 mOsm, 330 mOsm at 3500 µg/mL).
- Evaporation from medium: not expected due to very low vapor pressure
- Water solubility: The test material is uvcb. Water solubility is not defined.
- Precipitation: In Experiment I, visible precipitation of the test item in the culture medium was observed at 39.8 µg/mL and above in the absence of S9 mix and at 69.6 µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix at 22.7 µg/mL and above and at 52.2 µg/mL and above in the presence of S9 mix.

- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test item and a solvent and positive control. All cell cultures were set up in duplicate. Exposure times was 4 hrs (with and without S9 mix). The preparation interval was 22 hrs after start of the exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: Comparison with historical control is a prerequisite for acceptance of the study. To be valid, the study has to meet the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of historical laboratory control data range: 0.0 - 4.0 % aberrant cells, excluding gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

In Experiment I, visible precipitation of the test item in the culture medium was observed at 39.8 µg/mL and above in the absence of S9 mix and at 69.6 µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix at 22.7 µg/mL and above and at 52.2µg/mL and above in the presence of S9 mix. Cytotoxic effects were observed in all experimental parts of this study. In detail, clearly reduced mitotic indices occurred at the highest evaluated concentrations in Experiment I (54.3 % of control in the absence of S9 mix, 50.4 % of control in the presence of S9 mix) and in Experiment II (21.1 % of control in the absence of S9 mix, 45.9 % of control in the presence of S9 mix). In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.5 – 3.0 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data: 0.0 - 4.0 % aberrant cells, excluding gaps. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. In both experiments, either EMS (770 or 825µg/mL) or CPA (22.5 or 30.0µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test item, the structural analogue C15-20 ASA, did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, C16/18 Alkenyl succinic anhydride is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and precipitating concentrations. This study is informative for evaluation of the toxicity of the registered substance, n-ODSA EC 701-338-8, and is adequate for filling the data requirement for the registration of this substance. It is valid for hazard classification and risk assessment.
Executive summary:

C16/18 Alkenyl succinic anhydride, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments in vitro, with and without S9 mix. In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations. The highest applied concentration in the pre-test on toxicity (3500 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation. In the absence and presence of S9 mix, cytotoxicity was observed at the highest evaluated concentrations being far in the range of test item precipitation. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

This study is informative for evaluation of the toxicity of the registered substance, n-ODSA EC 701-338-8, and is adequate for filling the data requirement for the registration of this substance. It is valid for hazard classification and risk assessment.

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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This study was conducted on 2,5-furandione, dihydro-, mono-C15- 20-alkenyl derivatives (CAS 68784-12-3), an analogue substance used as the source of information for the assessment of the target substance through read-across. Therefore, this study is informative for evaluation of the environmental fate and toxicity of the target substance, Reaction products of furan-2,5-dione and octadec-1-ene (known here as n-ODSA EC 701-338-8; no CASRN available), and it is adequate for classification and 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
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: selected in HAT medium to reduce the number of mutants before the test started
Metabolic activation:
with and without
Metabolic activation system:
S9 supernatant and S9 cofactor solution (below) to result in a final protein concentration of 0.75 mg/mL in the cultures:
8 mM MgCl2
33 mM KC
l 5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
Test concentrations with justification for top dose:
The test concentrations below are in µg/mL
Experiment I
without S9 mix* 6.3 12.5 25.0 50.0 600.0 800.0 1200.0
with S9 mix* 6.3 12.5 25.0 50.0 600.0 800.0 1200.0
Experiment II
without S9 mix** 10.0 20.0 40.0 80.0 320.0 480.0 640.0
With S9 mix* 10.0 20.0 40.0 80.0 640.0 960.0 1280.0
Experiment III
without S9 mix** 100.0 200.0 400.0 600.0 800.0 1000.0
* 4 hours treatment
** 24 hours treatment
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The study was performed to investigate the potential of ASA to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The assay was performed in three 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. Since the recommended cytotoxic range of a relative cloning efficiency of approximately 10 - 20% was not covered in the second experiment without metabolic activation a repeat experiment was performed at higher concentrations (experiment III). The treatment time of experiment III was 24 hours without metabolic activation.
The highest concentration of the test item in the pre-experiment was 5 µL/mL. The concentration range of the main experiments was limited by cytotoxic effects and the solubility of the test item in aqueous medium.
Appropriate reference mutagens, used as positive controls, were used to demonstrate the sensitivity of the test item and the activity of the metabolic activation system.

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (+/- S9) 1st experiment / 4 hours (+S9) and 24 hours (-S9) 2nd experiment / 24 hours (-S9) 3rd experiment
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): about 15 days

SELECTION AGENT (mutation assays): 6-Thioguanine
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 3 - 5 x10^5 cells per flask (concentration, +/-S9, experiment, replicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: determined with a separate assay

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other:

OTHER:
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-muta¬genic 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 fre¬quency at least at one of the concen¬trations 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.
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.
experimental group p-value
experiment I, culture I without S9 mix 0.254
experiment I, culture II without S9 mix 0.079
experiment I, culture I with S9 mix 0.670
experiment I, culture II with S9 mix 0.955
experiment II, culture I without S9 mix 0.308
experiment II, culture II without S9 mix 0.094
experiment II, culture I with S9 mix 0.853
experiment II, culture II with S9 mix 0.582
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none. The pH was checked in the solvent control (pH 7.45) and in the highest concentration (pH 7.04)
- Effects of osmolality: none. Osmolarity was checked in the solvent control (391) and in the highest concentration (322)
- Evaporation from medium: none: Vapor pressure is very low
- Water solubility: ASA is a uvcb substance. There are at least some water-insoluble fractions at the higher test concentrations.
- Precipitation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed by the unaided eye down to the lowest concentration of 0.039 µL/mL with and without metabolic activation following 4 hours treatment. After 24 hours treatment (without metabolic activation) precipitation occurred at 0.078 µL/mL and above.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency

COMPARISON WITH HISTORICAL CONTROL DATA: The parameters of this study are all in the range of the historical controls

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first and second experiment with and without metabolic activation the cultures at the lowest concentration with metabolic activation and at the two lowest concentrations without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. The maximum concentration with metabolic activation was not continued in both experiments due to exceedingly severe cytotoxic effects. In experiment III the three highest concentrations were not continued for the same reason.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information): negative

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the analogue C15-20 ASA is considered to be non-mutagenic in this HPRT assay. This study is informative for evaluation of the toxicity of the target substance, n-ODSA EC 701-338-8, and is adequate for filling the data requirement for the registration of this substance. It is valid for hazard classification and risk assessment.
Executive summary:

The test item C15 -20 ASA was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in three independent experiments, using identical general experimental procedures. In the main experiments of this study (with and without S9 mix) the range of the solvent controls was from 2.6 up to 30.1 mutants per 106cells; the range of the groups treated with the test item was from 0.0 up to 50.8 mutant colonies per 106cells.

EMS(0.15 mg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. 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, C15 -20 ASA is considered to be non-mutagenic in this HPRT assay.

This study is informative for evaluation of the toxicity of a category substance, n-ODSA EC 701-338-8, and is adequate for filling the data requirement for the registration of this substance. It is valid for hazard classification and risk assessment.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study in compliance with GLP and guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
primary culture, other: lymphocytes
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a female donor (35 years old) for the first and second experiment and from a 46 year-old male donor for Experiment II. Blood samples were drawn by venous puncture and collected in heparinized tubes. Short-term cultures of human lymphocytes were stimulated to divide by the addition of a phytohaemagglutinin, PHA) to the culture medium. Mitotic activity began at about 40 hours after PHA stimulation and reached a maximum at around 3 days. The
chromosome constitution remained diploid during short-term culture.
Treatments commenced 50 - 80 hours after culture initiation when the cells were actively proliferating At preparation time 22 hours, a minimum of three cultures treated with separated concentrations of the test item were evaluated for the potential to induce structural chromosomal aberrations.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Experiment 1
DMSO 0.5 % without S9 4 / 18 hrs
Pos. control 825.0 µg without S9 4 / 18 hrs
ASA 22.7 µg without S9 4 / 18 hrs
ASA 39.8 µg without S9 4 / 18 hrs
ASA 213.2 µg without S9 4 / 18 hrs
ASA 373.2 µg without S9 4 / 18 hrs
DMSO 0.5 % with S9 4 / 18 hrs
Pos. control 22.5 µg with S9 4 / 18 hrs
ASA 22.7 µg with S9 4 / 18 hrs
ASA 39.8 µg with S9 4 / 18 hrs
ASA 121.9 µg with S9 4 / 18 hrs
ASA 213.2 µg with S9 4 / 18 hrs

Experiment 2
DMSO 0.5 % without S9 22 / 0 hrs
Pos. control 770.0 µg without S9 22 / 0 hrs
ASA 13.0 µg without S9 22 / 0 hrs
ASA 22.7 µg without S9 22 / 0 hrs
ASA 69.6 µg without S9 22 / 0 hrs
ASA 121.9 µg without S9 22 / 0 hrs
DMSO 0.5 % with S9 4 / 18 hrs
Pos. control 30.0 µg with S9 4 / 18 hrs
ASA 29.8 µg with S9 4 / 18 hrs
ASA 52.2 µg with S9 4 / 18 hrs
ASA 279.9 µg with S9 4 / 18 hrs
ASA 489.8 µg with S9 4 / 18 hrs
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; ASA was added to the test medium dissolved in DMSO. Cultures were treated with test medium.

DURATION
- Preincubation period: 72 hours
- Exposure duration: 4 hours or 18 hours or 22 hours, respectively
- Expression time (cells in growth medium): 18 hours, 4 hours or 0 hours, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, for 3 hours prior fixation
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 per concentration, each with and without S9 and experiment I and II

NUMBER OF CELLS EVALUATED: 2x100 metaphases per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (main test) and cloning efficiency (range finder)

OTHER EXAMINATIONS:
- Determination of polyploidy: visual (100x microscope) counting of centromers. If multiple copies of the haploid chromosome number (other than diploid) are scored then the count is recorded and the cell classified as polyploid.
- Determination of endoreplication: visual (100x microscope). If the chromosomes are arranged in closely apposed pairs, i.e. 4
chromatids instead of 2, the cell is scored as endoreduplicated.

Evaluation criteria:
Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in
mitosis) was determined

A test item is classified as non-mutagenic if the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding gaps). and no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding gaps) and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if the number of induced numerical aberrations is not in the range of our historical control data
(0.0 – 0.8 % polyploid cells).
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Key result
Species / strain:
primary culture, other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant increase in the pH value was observed (e.g. Exp. I: solvent control: pH 7.4 versus pH 7.5 at 3500 µg/mL).
- Effects of osmolality: No relevant increase in the osmolarity was observed (e.g. Exp. I: solvent control: 378 mOsm, 330 mOsm at 3500 µg/mL).
- Evaporation from medium: not expected due to very low vapor pressure
- Water solubility: The test material is uvcb. Water solubility is not defined.
- Precipitation: In Experiment I, visible precipitation of the test item in the culture medium was observed at 39.8 µg/mL and above in the absence of S9 mix and at 69.6 µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix at 22.7 µg/mL and above and at 52.2 µg/mL and above in the presence of S9 mix.

- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test item and a solvent and positive control. All cell cultures were set up in duplicate. Exposure times was 4 hrs (with and without S9 mix). The preparation interval was 22 hrs after start of the exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: Comparison with historical control is a prerequisite for acceptance of the study. To be valid, the study has to meet the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of historical laboratory control data range: 0.0 - 4.0 % aberrant cells, excluding gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

In Experiment I, visible precipitation of the test item in the culture medium was observed at 39.8 µg/mL and above in the absence of S9 mix and at 69.6 µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix at 22.7 µg/mL and above and at 52.2µg/mL and above in the presence of S9 mix. Cytotoxic effects were observed in all experimental parts of this study. In detail, clearly reduced mitotic indices occurred at the highest evaluated concentrations in Experiment I (54.3 % of control in the absence of S9 mix, 50.4 % of control in the presence of S9 mix) and in Experiment II (21.1 % of control in the absence of S9 mix, 45.9 % of control in the presence of S9 mix). In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.5 – 3.0 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data: 0.0 - 4.0 % aberrant cells, excluding gaps. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. In both experiments, either EMS (770 or 825µg/mL) or CPA (22.5 or 30.0µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test item (C15-20 ASA) did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, C16/18 Alkenyl succinic anhydride is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and precipitating concentrations.
Executive summary:

C16/18 Alkenyl succinic anhydride, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments in vitro, with and without S9 mix. In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations. The highest applied concentration in the pre-test on toxicity (3500 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation.

In the absence and presence of S9 mix, cytotoxicity was observed at the highest evaluated concentrations being far in the range of test item precipitation. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
GLP- and guideline compliant.
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
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: selected in HAT medium to reduce the number of mutants before the test started
Metabolic activation:
with and without
Metabolic activation system:
S9 supernatant and S9 cofactor solution (below) to result in a final protein concentration of 0.75 mg/mL in the cultures 8 mM MgCl2 33 mM KCl 5 mM glucose-6-phosphate 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
Test concentrations with justification for top dose:
The test concentrations below are in µg/mL
Experiment I
without S9 mix* 6.3 12.5 25.0 50.0 600.0 800.0 1200.0
with S9 mix* 6.3 12.5 25.0 50.0 600.0 800.0 1200.0
Experiment II
without S9 mix** 10.0 20.0 40.0 80.0 320.0 480.0 640.0
With S9 mix* 10.0 20.0 40.0 80.0 640.0 960.0 1280.0
Experiment III
without S9 mix** 100.0 200.0 400.0 600.0 800.0 1000.0
* 4 hours treatment
** 24 hours treatment
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The study was performed to investigate the potential of ASA to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The assay was performed in three 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. Since the recommended cytotoxic range of a relative cloning efficiency of approximately 10 - 20% was not covered in the second experiment without metabolic activation a repeat experiment was performed at higher concentrations (experiment III). The treatment time of experiment III was 24 hours without metabolic activation.
The highest concentration of the test item in the pre-experiment was 5 µL/mL. The concentration range of the main experiments was limited by cytotoxic effects and the solubility of the test item in aqueous medium.
Appropriate reference mutagens, used as positive controls, were used to demonstrate the sensitivity of the test item and the activity of the metabolic activation system.

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (+/- S9) 1st experiment / 4 hours (+S9) and 24 hours (-S9) 2nd experiment / 24 hours (-S9) 3rd experiment
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): about 15 days

SELECTION AGENT (mutation assays): 6-Thioguanine
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 3 - 5 x10^5 cells per flask (concentration, +/-S9, experiment, replicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: determined with a separate assay

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other:

OTHER:
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-muta¬genic 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 fre¬quency at least at one of the concen¬trations 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.
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.
experimental group p-value
experiment I, culture I without S9 mix 0.254
experiment I, culture II without S9 mix 0.079
experiment I, culture I with S9 mix 0.670
experiment I, culture II with S9 mix 0.955
experiment II, culture I without S9 mix 0.308
experiment II, culture II without S9 mix 0.094
experiment II, culture I with S9 mix 0.853
experiment II, culture II with S9 mix 0.582
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none. The pH was checked in the solvent control (pH 7.45) and in the highest concentration (pH 7.04)
- Effects of osmolality: none. Osmolarity was checked in the solvent control (391) and in the highest concentration (322)
- Evaporation from medium: none: Vapor pressure is very low
- Water solubility: ASA is a uvcb substance. There are at least some water-insoluble fractions at the higher test concentrations.
- Precipitation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed by the unaided eye down to the lowest concentration of 0.039 µL/mL with and without metabolic activation following 4 hours treatment. After 24 hours treatment (without metabolic activation) precipitation occurred at 0.078 µL/mL and above.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency

COMPARISON WITH HISTORICAL CONTROL DATA: The parameters of this study are all in the range of the historical controls

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first and second experiment with and without metabolic activation the cultures at the lowest concentration with metabolic activation and at the two lowest concentrations without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. The maximum concentration with metabolic activation was not continued in both experiments due to exceedingly severe cytotoxic effects. In experiment III the three highest concentrations were not continued for the same reason.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information): negative

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore,C15-20 ASA is considered to be non-mutagenic in this HPRT assay. T
Executive summary:

The test item ASA was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in three independent experiments, using identical general experimental procedures. In the main experiments of this study (with and without S9 mix) the range of the solvent controls was from 2.6 up to 30.1 mutants per 106cells; the range of the groups treated with the test item was from 0.0 up to 50.8 mutant colonies per 106cells.

EMS(0.15 mg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. 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, ASA is considered to be non-mutagenic in this HPRT assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

This information is from the substance 2,5-furandione, dihydro-,mono-C15-20-alkenylderivatives (CAS 68784-12-3, a mixture of a hexadecenyl- and octadecenyl succinic anhydrides), an analogue used for the assessment of several endpoints through read-across. The hypothesis for read-across between the substance being registered (Reaction products of furan-2,5-dione and octadec-1-ene; known here as n-ODSA EC 701-338-8; no CASRN available), and the analogue substance is a common functional group: a 2,5-furandione, dihydro- structure, also known as a succinic anhydride, to which is attached a long-chain monounsaturated olefin. In the environment, the anhydride moiety is quickly hydrolysed to form a dioic acid.  When the substance to be registered and the analogue substance are compared, changes in the purity of the starting olefin stock, or small differences in the length (between sixteen and twenty) or arrangement (linear or branched) of the carbon chain are not anticipated to significantly affect the environmental fate properties or the toxicity of the substances. For each endpoint study based upon read-across, the analogue approach is substantiated by an evaluation provided in the Analogue Approach Report Format (AARF) attached to the endpoint study summary file. The AARF allows the read-across information to fulfil the information requirements of the REACH Annexes VII-X, to be the basis for classification and labelling decisions, and for risk assessment.

n-ODSA EC 701-338-8 was specifically tested and found to be non-mutagenic in the Ames assay. In vitro testing of C15 -20 ASA was similarly found to be non-mutagenic in a mammalian mutation assay (hprt) and non-clastogenic in a chromosome aberration assay. The absence of activity in these in vitro assays indicates that the structurally similar substance, n-ODSA EC 701-338-8, is likely not genotoxic.


Justification for selection of genetic toxicity endpoint
Experimental data on the registered substance

Short description of key information:

The substance is not mutagenic in the Ames assay; similarly, a read-across substance is inactive in mammalian in vitro genotoxicity tests and so it is expected that the target substance will not be genotoxic in vitro.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The read-across substance, and hence the target, n-ODSA EC 701-338-8, is not genotoxic according to the criteria of Regulation EC No. 1272/2008.