Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:
- Ames test (OECD 471, GLP): not mutagenic
- Chromosome Aberration (CA) test (OECD 473, GLP): not mutagenic
- Mammalian Cell Gene Mutation Test (OECD 476, GLP): not mutagenic

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
Remarks:
According to OEDC guideline 471, GLP comliant.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 440/2008
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine or tryptophan locus.
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
Test concentrations with justification for top dose:
0, 50, 150, 500, 1500 and 5000 µg/plate.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
tetrahydrofuran
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: non for preliminary toxocity test, triplicate for main test

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: reduction in number of spontaneous revertants (below a factor 0.5 fold under the concurrent solvent control value and/or bacterial lawn should exhibit evidence of thinning when viewed microscopically.
Evaluation criteria:
A minimun of four non-toxic test item dose levels is required for the assay to be acceptable

A test item will be considered non-mutagenic (negative) in the test system if one or more of the following criteria are not met:
A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby 1979)
A reproducible increase at one or more concentrations
Biological relevance against in-house historical control ranges
Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-gistorical range response)
Statistics:
Statistical analysis of data as determinded by UKEMS (Mahon et al 1989)
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

The testing material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the testing material, Soybeanoil, polymerized, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). The dose range for the first experiment was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

The test item,Soybeanoil, polymerized, was considered to be non‑mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28-January-2010 to 08-February-2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles. A reliability of 2 is assigned in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID, due to the read-across purpose.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate

Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
Experiment 2:
Without and with S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle:Test compound was soluble in tetrahydrofuran and tetrahydrofuran has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Positive control substance:
9-aminoacridine
Remarks:
without S9 Migrated to IUCLID6: 60 µg/plate in water for TA1537
Positive control substance:
2-nitrofluorene
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for TA98
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation of Standolized linseed oil was observed at 3330 and 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.


Conclusions:
Interpretation of results: negative

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

It is concluded that this test is valid and that // is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation of Standolized linseed oil was observed at 3330 and 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.


Conclusions:
Interpretation of results: negative

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

It is concluded that this test is valid and that // is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
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:
28-January-2010 to 08-February-2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles. A reliability of 2 is assigned in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID, due to the read-across purpose.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) (Invitrogen Corporation) and 30 U/ml heparin.

Lymphocyte cultures
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 3hr exposure; 24 hr fixation: 10, 33 and 100 µg/ml
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1, 3, 10, 33, 100 and 333 µg/ml
With S9-mix, 3hr exposure; 24 hr fixation: 10, 33 and 100 µg/ml
First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 5, 10 and 15 µg/ml
With S9-mix, 3 h exposure, 24 h fixation time: 5, 15 and 25 µg/ml
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 3, 7 and 15 µg/ml
Without S9-mix, 48 hr exposure; 48 hr fixation: 1, 3 and 7 µg/ml
With S9-mix, 3 hr exposure; 48 hr fixation: 20, 25 and 30 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle:Test compound was soluble in tetrahydrofuran and tetrahydrofuran has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Key result
Species / strain:
lymphocytes: human peripheral
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 33 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed up to and including the highest precipitating tested dose

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.
Conclusions:
Interpretation of results: negative

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Standolized linseed oil did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No effects of Standolized linseed oil on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Standolized linseed oil does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Standolized linseed oil is not clastogenic in human lymphocytes under the experimental conditions described in this report.

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
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Key result
Species / strain:
lymphocytes: human peripheral
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 33 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed up to and including the highest precipitating tested dose

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.
Conclusions:
Interpretation of results: negative

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Standolized linseed oil did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No effects of Standolized linseed oil on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Standolized linseed oil does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Standolized linseed oil is not clastogenic in human lymphocytes under the experimental conditions described in this report.

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:
02-Jun-2010 to 19-Jul-2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles. A reliability of 2 is assigned in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID, due to the read-across purpose.
Reason / purpose:
reference to same study
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
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Species strain
- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 0.3, 1, 3, 10 and 100 µg/mL
Without S9-mix, 24 hours treatment: 0.3, 1, 3, 10 and 100 µg/mL
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 33 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 33 µg/mL
With S9-mix, 3 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 33 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: Test compound was soluble in tetrahydrofuran and tetrahydrofuran has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 10 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest tested dose level

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to and including the highest tested dose level in both experiments.
Remarks on result:
other: strain/cell type: L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses

In the absence of S9-mix, Standolized linseed oil did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Standolized linseed oil did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

In conclusion, Standolized linseed oil is not mutagenic in the TK mutation test system under the experimental conditions described in the report.
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
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 10 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest tested dose level

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to and including the highest tested dose level in both experiments.
Remarks on result:
other: strain/cell type: L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses

In the absence of S9-mix, Standolized linseed oil did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Standolized linseed oil did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

In conclusion, Standolized linseed oil is not mutagenic in the TK mutation test system under the experimental conditions described in the report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

All three available in vitro genetic toxicity tests (Ames with Standolised Soybean oil and CA and gene mutation test with Standolised Linseed oil) indicate that Standolised Soybean oil has no mutagenic potential.


Justification for selection of genetic toxicity endpoint
No selection was made as all three available key studies are equally relevant for this endpoint (Ames, CA, gene mutation test) and all show the same negative result.

Justification for classification or non-classification

Based on the available information for mutagenicity, Standolised soybean oil does not need to be classified for this endpoint according to the criteria outlined in Annex I of CLP (1272/2008/EC) and Annex VI of DSD (67/548/EEC).