Registration Dossier
Registration Dossier
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EC number: 272-038-8 | CAS number: 68649-95-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Neither in the bacterial mutagenicity test nor in the chromosomal aberration test in mammalian cells any indication for point or chromosomal mutagenic effects were noted. In addition, three mammalian mutagenicity tests (mouse lymphoma assays) were performed. In one MLA the potential for a mutagenic potential at high, precipitating concentrations was noted. In contrast concentrations showing no or only slight precipitation did not reveal any indication for a mutagenic potential in both tests, however a third MLA indicated mutagenic potential in the absence of metabolic activation. A further in-vivo test is foreseen to discard or confirm the genotoxic potential of Linseed Oil, Oxidized.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2012-11-16
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study reliable with restrictions because only limit test (causing already slight precipitation) was performed.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 21 July 1997
- Deviations:
- yes
- Remarks:
- limit test
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 31 May 2008
- Deviations:
- yes
- Remarks:
- limit test
- Qualifier:
- according to guideline
- Guideline:
- other: The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT)
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- thymidine kinase (TK) locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - 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.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital and ß-naphthoflavone-induced rat liver S9
- Test concentrations with justification for top dose:
- +/- S9: 33 µg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Stability and solubility in vehicle - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.2 % (v/v) in the exposure medium
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 5 and 15 µg/mL
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.2 % (v/v) in the exposure medium
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 10 µg/mL
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 hrs with and without S9-mix and 24 hour without S9-mix
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 day
SELECTION AGENT: 5 μg/ml trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, rel. total growth, growth rate - Evaluation criteria:
- In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT 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. 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: The tested concentration don’t reach a mutation frequency of MF(controls) + 126. - Statistics:
- not applicable
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no 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: not analysed
- Effects of osmolality: not analysed
- Evaporation from medium: no
- Water solubility: yes
- Precipitation: yes
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.
Mutation frequencies in cultures treated with positive control chemicals were increased by 15- and 10-fold for MMS in the absence of S9-mix, and 14-fold for CP in the presence of S9-mix compared to control.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed in the absence and presence of S9-mix. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results
negative with and without metabolic activation
In conclusion, Linseed oil, oxidized did not induce significant increase of mutation frequency neither in the presence nor in the absence of a metabolic activation system. Thus, Linseed oil, oxidized at a concentration of 33 µg/mL is not mutagenic in the TK mutation test system under the experimental conditions described. - Executive summary:
This report describes the effects of Linseed oil, oxidized on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The study procedures described in this report were based on OECD TG 476 (adopted July 21, 1997) and (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.17.
Batch S12/161 + FMS 12 -002 of Linseed oil, oxidized was a clear brown liquid. Acetone was used as solvent as the test substance is better soluble in this solvent based on practical experience.
Linseed oil, oxidized was tested at the dose level of 33 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
A single (limit) concentration, causing already precipitation was considered sufficient in this experiment as another mouse lymphoma assay was already available showing no mutagenic effects at several concentrations even exceeding the test concentration tested in the present MLA. No toxicity was observed at this dose level in the absence and presence of S9-mix.
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.
Mutation frequencies in cultures treated with positive control chemicals were increased 15- and 10-fold for MMS in the absence of S9-mix, and 14-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.
In the absence of S9-mix, Linseed oil, oxidized did not induce a significant increase in the mutation frequency both after 3 and 24 hours treatment time.
In the presence of S9-mix, Linseed oil, oxidized did not induce a significant increase in the mutation frequency in the first experiment.
It is concluded that Linseed oil, oxidized is not mutagenic in the mouse lymphoma L5178Y test system at a concentration of 33 µg/mL 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:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22-jan-2010 to 26-feb-2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- 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, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) 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, 24/48hr exposure; 24/48 hr fixation: 1, 3, 10, 33, 100, 333 and 1000 µg/mL
Combined Dose range finding/First cytogenetic test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 10, 33 and 100 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 33, 100 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 10, 33, 100 µg/mL
With S9-mix, 3 hr exposure; 48 hr fixation: 10, 33, 100 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
Blown linseed oil was suspended in dimethyl sulfoxide at concentrations of 100 mg/ml and above. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. At concentrations of 33 mg/ml and below the test substance was dissolved in dimethyl sulfoxide.
- Justification for choice of solvent/vehicle: In DMSO a homogeneous suspension could be obtained and DMSO has been accepted and approved by authorities and international guidelines - Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- 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:
- without 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, 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 100 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:
- Precipitation was seen at the highest tested dose level selected for scoring. - Conclusions:
- Interpretation of results (migrated information):
negative
The positive and negative controls were within the historical control data.
Blown 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.
Finally, it is concluded that this test is valid and that Blown linseed oil is not clastogenic in human lymphocytes under the experimental conditions described in the report. - Executive summary:
The ability of the test item to induce chromosome aberrations was evaluated in cultured peripheral human lymphocytes in the presence and in the absence of a metabolic activation system. Based on pre-tests the test substance was evaluated as soluble in DMSO at concentrations of 33 mg/mL and below but clearly formed a suspension at concentrations of >= 100 mg/mL.
In the first cytogenetic assay the test item was tested up to 100 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8 % (v/v) S9-fraction.
In the second cytogenetic assay, the test item was tested up to 100 µg/mL for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix the test item was also tested up to 100 µg/mL for a 3 h exposure time with a 48 h fixation time. Precipitation was obvious at 100 µg/mL.
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. The test item 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 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 the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- March 7, 2006 - March 20, 2006
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study was conducted in accordance with OECD/EU guidelines and GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Gene coding for histidine (Salmonella strains) or tryptophane (E. coli) synthesis).
- Species / strain / cell type:
- other: TA-1535, TA-1537, TA-98, TA-100 and WP2 uvrA
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 tissue fraction from livers of male SD rats induced with Phenobarbital and betanaphtoflavone.
- Test concentrations with justification for top dose:
- 313, 625, 1250, 2500 and 5000 µg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO; water
- Justification for choice of solvent/vehicle: DMSO was used for the test substance and several positive controls, water for the remaining positive controls. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Without S9: sodium azide (TA 1535 and TA 100), 9-aminoacridine (TA 1537, methylmethanesulphonate (WP2uvrA), 2-nitrofluorene (TA 98), . With S9: 2-aminoanthracene (all strains).
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) for the first assay; preincubation (37°C, 30 minutes) followed by plate incorporation for the second assay.
DURATION
- Preincubation period: only for 2nd assay: 30 minutes (37°C)
NUMBER OF REPLICATIONS: 3 plates per concentration
DETERMINATION OF CYTOTOXICITY
other: decline in the number of spontaneous revertants, thinning of the background lawn or microcolony formation
OTHER EXAMINATIONS: number of revertant colonies
OTHER: - Evaluation criteria:
- For the test item to be considered mutagenic, two-fold or more increases in mean revertant numbers must be observed at two consecutive dose-levels or at the highest practicable dose-level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose-levels.
- Statistics:
- Mean and standard deviation of the number of revertant colonies per plate were calculated.
- 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
- Vehicle controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- See below.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation
In an Ames test according to OECD 471 with independent repeat, the test material was not mutagenic in the presence and absence of metabolic activation in strains TA-1535, TA-1537, TA-98, TA-100 and E. coli WP2uvrA. - Executive summary:
The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation. The test item was used as a solution/suspension in dimethylsulphoxide (DMSO). In Main Assay I, using the plate incorporation method, the test item was assayed at the maximum dose-level of 5000 μg/plate and at the four dose-levels of 2500, 1250, 625 and 313 μg/plate. As no increases in revertant numbers were observed, all treatments of Main Assay II included a pre-incubation step and used the same dose-levels employed in Main Assay I. In both experiments clear precipitation of the test item was observed at the three higher concentrations, both in the absence and presence of S9 metabolism. No toxicity was observed in any experiment, with any tester strain, in the absence and presence of S9 metabolic activation. The test item did not induce a biologically relevant increase in the number of revertant colonies in the plate incorporation or pre-incubation assay in the absence or presence of S9 metabolism at any dose-level in any tester strain.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- April 26, 2006 - May 25, 2006
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Precipitation of the test item was reported indicating an inhomogenous suspension of the test item at evaluated concentrations. The study results were considered to be not assignable as the concentrations tested and evaluated by far exceeded the solubility and might have caused artificial effects due to precipitation.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Gene coding for the enzyme thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Complete medium 5% (95% Minimal medium A and 5% heat –inactivated horse-serum).
- 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:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 tissue fraction from livers of male SD rats induced with Phenobarbital and beta-naphtoflavone.
- Test concentrations with justification for top dose:
- 23.4, 46.9, 93.8, 188, 375 and 563 µg/mL (without S9)
93.8, 188, 375, 750 and 1500 µg/mL (with S9) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: none (but this is a commonly used vehicle). - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Methyl methane sulphonate (without S9), 10 µg/mL; Benzo(a)pyrene (with S9), 2.0 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days
SELECTION AGENT (mutation assays): trifluorothymidine
NUMBER OF REPLICATIONS: 2 tubes per concentration containing 5x10^5 cells/mL were treated (one tube for the positive controls)
NUMBER OF CELLS EVALUATED: 7.7x10^5 cells per replicate tube for selection of mutants (i.e. 2000 cells in each well of four 96-well plates);
DETERMINATION OF CYTOTOXICITY: survival following treatment on 307 cells per replicate tube (i.e. a 0.2 mL aliquot of a culture diluted to 8 cells/mL was placed into each well of two 96-well plates);
- Method: after incubation for 8 days, wells containing viable clones were identified by the eye and counted
OTHER EXAMINATIONS:
- plating efficiency following expression was determined on 307 cells per replicate tube (i.e. 1.6 cells/well were plated into each well of two 96-well plates). After incubation for 14 days, wells containing viable clones were identified by the eye and counted.
- In addition to the number of wells containing viable clones, the number of wells containing large colonies and the number containing small colonies were scored. - Evaluation criteria:
- The assay was considered valid if the following criteria were met: (1) The cloning efficiencies at Day 2 in the untreated control cultures in the absence of S9 metabolism fell within the range of 65-120%. (2) The untreated control growth factor over 2 days fell within the range of 8-32. (3) The mutant frequencies in the untreated control cultures fell within the range of 50-200 x 10^6 viable cells. (4) The positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and the negative control mutant frequencies was greater than half the historical mean value).
For a test item to be considered mutagenic, it was required that: (1) The mutant frequency at one or more doses is statistically significantly greater than that of the negative control. (2) There is a significant dose-relationship as indicated by the linear trend analysis. Any increase in mutant frequency should lie outside the historical control range to have biological relevance. - Statistics:
- For comparison of the treatment with the control, the one tailed Dunnett's test was performed on log mutant frequencies. The evaluation of a linear trend in mutant frequency was performed using weighted regression. The weighting factor was 1/X^2, where X is the variance of the log mutant frequencies. The slope b and its variance var(b) were calculated to form the statistics variable b^2/var(b), which was compared with tabulated critical values of Chi^2 with 1 degree of freedom.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- 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: determined at end of test in post-treatment medium: no effect of test material
- Effects of osmolality: determined at end of test in post-treatment medium: no effect of test material
- Evaporation from medium: no data
- Water solubility: no data in report (water solubility however is known to be <1 mg/L)
- Precipitation: upon addition of the test item to the cultures and at the end of the treatment period, cloudy appearance of the treatment medium was observed at dose levels between 375 and 1500 µg/mL.
- Other confounding effects: no data
RANGE-FINDING/SCREENING STUDIES: The test concentrations of the mutagenicity study were based on a preliminary cytotoxicity test
COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data from 115 tests were provided. The induced mutant frequencies at the two highest analyzable concentrations exceeded the historical control range.
ADDITIONAL INFORMATION ON CYTOTOXICITY: see table below. - Conclusions:
- Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation
The test material is mutagenic at the TK locus of L5178 mouse lymphoma cells, both in the presence and absence of S9. The proportion of small versus large colonies had shifted towards the small colonies at the concentrations producing a mutagenic effect. This may indicate the induction of gross chromosomal aberrations. - Executive summary:
Within this mouse lymphoma assay (MLA) the test item was investigated using DMSO as solvent.
Based on the report, the test item was considered to be soluble in DMSO up to 300 mg/mL and a concentration of 3000 μg/mL was selected as the highest dose level to be used in the cytotoxicity pre-test.
Based purely on cytotoxicity results, the mutagenic potential was evaluated in a concentration range between 23.4 and 563 µg/mL in the absence and between 93.8 and 1500 µg/mL in the presence of S9 metabolic activation system in the main test, although cloudy appearance of the culture media was reported at least for concentrations >= 375 µg/mL, indicating an inhomogeneous suspension/distribution of the test item at those concentrations.
In the absence of S9 metabolic activation, severe toxicity was observed at the highest concentration. At 375 μg/mL the relative survival (%RS) and the relative total growth (RTG) values were reduced to 12% and 7% of the concurrent negative control value, respectively indicating massive cytotoxicity too. Pronounced toxicity was still observed at the next lower dose level, reducing %RS and RTG to 54% and 51%, respectively. In the presence of S9 metabolic activation, a dose-related cytotoxicity effect was observed. The top concentration tested (1500 μg/mL) yielded 22% relative survival and 13% relative total growth, indicating massive cytotoxicity.
Vehicle and positive control treatments were included in the mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the vehicle control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.
In the absence of S9 metabolic activation, a statistically significant and biological relevant increase in mutant frequency was observed at 188 µg/mL and in the presence of S9 mix at 750 and 1500 µg/mL. After treatment with the test item, the proportion of small versus large colonies was shifted towards the small colonies at the concentration producing an increase of mutagenic events both in the absence and presence of S9 metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation (HPRT)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 07 January 2014 to 11 June 2014
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study reliable with restrictions because only limit test (causing already slight precipitation) was performed.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 31 May 2008
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Department of Toxicology, Agricultural University of Wageningen, The Netherlands (1986). (strain CH-V79-4).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD and EC).
- Number of passages if applicable: not specified
- Maintenance: All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 – 100% (actual range 41 – 100%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 35.0 – 37.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on
each working day.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Basic medium
The basic medium consisted of Minimum Eagles Medium with Earle’s salts (MEME, Invitrogen Corporation), supplemented with 5 mL of MEM non-essential amino acids solution (Invitrogen Corporation), 0.11 g of sodium pyruvate (Invitrogen Corporation), 0.292 g of L-glutamine (Invitrogen Corporation) and 2.2 g of sodium bicarbonate (Merck) per litre. Final concentrations of streptomycin and penicillin G (Invitrogen Corporation) were 50 μg/mL and 50 U/mL, respectively.
Growth medium
Basic medium, supplemented with 10% (v/v) foetal calf serum.
Exposure medium
Cells were exposed to Blown Linseed Oil in basic medium supplemented with 5% (v/v) foetal calf serum (MEM5-medium) (Invitrogen Corporation).
Selection medium
Selection medium consisted of growth medium, containing 5 μg/mL 6-TG.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat S9 homogenate
- Test concentrations with justification for top dose:
- Dose range: 3, 10, 33, 50 and 100 µg/mL
Experiment 1 and 2: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (precipitation from 33 µg/mL and higher) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Stability and solubility in vehicle - Untreated negative controls:
- yes
- Remarks:
- Untreated culture
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone (vehicle)
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 10 µg/mL
- Positive control substance:
- 3-methylcholanthrene
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 hrs with and without S9-mix and 24 hour without S9-mix
- Expression time (cells in growth medium): 6 days
- Selection time (if incubation with a selection agent): 7 days
SELECTION AGENT: thioguanine 6-TG
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, rel. total growth, growth rate - Rationale for test conditions:
- The study procedures described in this report were based on the most recent OECD and EC guidelines.
- Evaluation criteria:
- A mutation assay is considered acceptable if it meets the following criteria:
a) The absolute colony forming efficiency of the vehicle controls should be between 60 and 130%
b) At least seven doses of the test substance should have an acceptable number of surviving cells (106) which could be analysed for expression of the HPRT mutation.
c) The spontaneous mutant frequency in the vehicle-treated control should be < 6 per 105 clonable cells.
d) The positive control substances induced significant (at least three-fold) increases in the mutant frequency.
e) The selected dose range has to include a clearly toxic concentration (10 to 20%) of the average
of vehicle controls or should exhibit limited solubility or should extend to 5 mg/mL 0.01 M. - Statistics:
- In addition to the criteria stated above, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
No formal hypothesis testing is done.
A test substance is considered positive (mutagenic) in the mutation assay if it induces at least a three-fold increase in the mutation frequency compared to the vehicle control 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 none of the tested concentrations show a mutation frequency of at least three-fold compared to the vehicle control.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Remarks:
- in the absence of S9-mix the mutation frequency was above the historical limit. However since all observed mutation frequencies of the vehicle control were within the acceptability criteria of this assay, it was considered valid.
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- 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: not analysed
- Effects of osmolality: not analysed
- Evaporation from medium: no
- Water solubility: yes
- Precipitation: yes
COMPARISON WITH HISTORICAL CONTROL DATA: Valid, however, in the absence of S9-mix for the untreated control (second experiment), this mutation frequency was above the limit of the historical control data range. However since all observed mutation frequencies of the vehicle control were within the acceptability criteria of this assay, the validity of the test was considered to be not affected.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed in the absence and presence of S9-mix. - Conclusions:
- Blown Linseed Oil shows mutagenic potential in the HPRT gene mutation test with V79 Chinese hamster cells under the experimental conditions described in this report. The mutagenicity was confined only to incubations without metabolic activation.
- Executive summary:
The mutagenic activity of Blown Linseed Oil was evaluated in an in vitro mammalian cell HPRT gene mutation test with V79 Chinese hamster cells, performed in accordance with the OECD guidance, Klimisch 1. The test was performed in two independent experiments in the absence and presence of S9-mix. The test substance was dissolved in acetone.
In the first experiment, Blown Linseed Oil was tested up to concentrations of 100 μg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. Blown Linseed Oil precipitated in the culture medium at dose levels of 33 μg/mL and upwards. The untreated, vehicle and positive control were considered valid.
Mutation frequencies in cultures treated with positive control chemicals were increased 53- and 16-fold for EMS in the absence of S9-mix, and by 20-fold for 3-MCA and 31-fold for DMBA in the presence of S9-mix.
In the absence of S9-mix, Blown Linseed Oil induced a 21-fold increase in the mutation frequency. Verification of this result in a repeat experiment showed a 5.5-fold increase in the mutation frequency compared to the vehicle control. In the second experiment, the increases in the mutation frequency were dose-related, starting at 3 μg/mL, and also observed below precipitating dose levels. Taken together, the mutation frequency was more than three-fold increased and above the historical data range in two independent experiments.
In the presence of S9-mix, Blown Linseed Oil did not induce a significant increase in the mutation frequency.
It is concluded that Blown Linseed Oil shows mutagenic potential in the gene mutation test with V79 Chinese hamster cells under the experimental conditions described in this report. The mutagenicity was confined only to incubations without metabolic activation.
Referenceopen allclose all
dose |
RSG |
CE (day 2) |
RS (day 2) |
RTG |
mutation frequency per 10^6 survivors |
µg/mL |
(%) |
(%) |
(%) |
(%) |
total |
without metabolic activation |
|
|
|
|
|
3 hours treatment |
|
|
|
|
|
SC1 |
100 |
98 |
100 |
100 |
53 |
SC2 |
100 |
79 |
100 |
100 |
52 |
33 (1) |
102 |
108 |
122 |
124 |
48 |
MMS |
66 |
45 |
51 |
34 |
777 |
without metabolic activation |
|
|
|
|
|
24 hours treatment |
|
|
|
|
|
SC1 |
100 |
101 |
100 |
100 |
52 |
SC2 |
100 |
93 |
100 |
100 |
56 |
33 (1) |
116 |
95 |
99 |
114 |
47 |
MMS |
92 |
66 |
68 |
63 |
516 |
with 12% (v/v) metabolic activation |
|
|
|
|
|
3 hours treatment |
|
|
|
|
|
SC1 |
100 |
91 |
100 |
100 |
65 |
SC2 |
100 |
108 |
100 |
100 |
51 |
33 (1) |
102 |
104 |
104 |
106 |
56 |
CP |
71 |
60 |
60 |
43 |
793 |
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth;
SC = Solvent control = acetone; MMS = Methylmethanesulfonate; CP = Cyclophosphamide
(1) = Linseed oil, oxidized precipitated in the exposure medium
No effects of Blown 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 Blown 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.
Both with and without metabolic activation, and for all strains tested, there was no dose-response related trend apparent in both assays. The highest mean number of revertants per plate for any test dose was at the most a factor of 1.6 higher than that of the control. The response for all strains is unambiguously negative in both assays, both with and without metabolic activation. The positive controls always gave the expected responses (the mean number of revertants per plate was a factor of 3.7-32 higher than that of the control).
A summary of the results as is shown in the table below.
Table 1. Detailed results for mouse lymphoma forward mutation test
| S9 | dose level (µg/mL) | % survival | % relative total growth | mean mutant frequency per 10^6 viable cells | stat. significance | proportion small colony mutants |
| without | 0 | 100 | 100 | 92.5 | - | 0.43 |
| 23.4 | 99 | 100 | 102.6 | NS | NR | |
| 46.9 | 86 | 89 | 137.4 | NS | NR | |
| 93.8 | 80 | 100 | 146.8 | * | 0.48 | |
| 188 | 54 | 51 | 400.1 | ** | 0.53 | |
| 375 | 12 | 7 | 935.6 | # | NR | |
| 563 | 1 | 0 | 2760 | # | NR | |
| MMS | 106 | 74 | 504.3 | - | 0.53 | |
| linear trend | *** | |||||
| with | 0 | 100 | 100 | 114.7 | NS | 0.32 |
| 93.8 | 101 | 107 | 108.5 | NS | NR | |
| 188 | 99 | 68 | 109.3 | NS | NR | |
| 375 | 54 | 34 | 137.5 | ** | NR | |
| 750 | 40 | 21 | 239.1 | ** | 0.55 | |
| 1500 | 22 | 13 | 434.6 | 0.58 | ||
| B(a)P | 15 | 3 | 1989 | 0.55 | ||
| linear trend | *** |
NS: not statistically significant
NR: not recorded
% survival: determined after the 3-hour exposure.
% relative total growth: based on total growth factor during the 2-day expression and cloning efficiency after the 2-day expression.
* Significantly different from negative control (p ≤0.05).
** Significantly different from negative control (p ≤0.01).
* Significantly different from negative control (p ≤0.001).
# Excluded from statistical analysis due to high cytotoxicity.
Genetic toxicity in vivo
Description of key information
Test proposal OECD TG 489
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay) in vivo mammalian cell study: DNA damage and/or repair
Principles of method if other than guideline
While the test item Linseed oil, oxidized did not induce gene mutations in bacteria (Ames test, OECD
471) or chromosome aberrations in mammalian cells (Chromosome Aberration Assay, OECD 473) , positive and negative outcomes of point mutations were observed in mammalian cells (Mouse Lymphoma Assay and HPRT Assay, OECD 476).
According to REACH requirements Annex VIII/IX, section 8.4 an appropriate in vivo gene mutation test shall be proposed by the registrant, when there is a positive result from an in vitro gene mutation study in bacteria (Ames test, OECD 471) or from an in vitro gene mutation study in mammalian cells (OECD 476).
Based on the available in vitro data, an alkaline in vivo Comet assay (OECD 489) is proposed to assess the mutagenic properties of the test substance in vivo. The in vivo Comet assay is considered to be the appropriate test system to investigate short-lived substances at the first site of contact.
Furthermore, historical control data are available for various tissues.
The test substance is proposed to be administered orally by gavage to rats. Doses will be based on data available for repeated dose toxicity and acute oral toxicity study. Based on the results of the repeated dose toxicity and acute oral toxicity study and taken the OECD requirements into consideration, a maximum dose of 2000 mg/kg bw/day, and two additional doses are proposed. Organ s proposed to be evaluated are the forestomach and the liver, as the site of first contact after oral administration and the site of metabolism, respectively. The tissues are proposed to be evaluated after two administration days.
Six animals in the dose groups and negative control group as well as 4 animals in the positive control groups are proposed to reach a minimum of 5 and 3 analysable animals per sex, respectively, as requested according to the Guideline. Since the available data did not demonstrate relevant differences between males and females, the use of males is proposed.
Type of assay
single cell gel/comet assay in rodents for detection of DNA damage mammalian cell study: DNA damage and/or repair
Species
Rat
Route of administration
oral: gavage
Link to relevant study records
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study planned
- Study period:
- to be planned
- Justification for type of information:
- TESTING PROPOSAL ON VERTEBRATE ANIMALS
NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out : BLO (Blown Linseed oil) – Linseed oil, oxidised (CAS 68649-95-6 ; EC 272-038-8)
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: 5 in vitro studies on the target substance (OECD TG 471, OECD TG 473, negative and three OECD TG 476, 2 positive and 1 negative)
OECD Guideline 471 (Bacterial Reverse Mutation Assay) – negative, no indications of point mutations or frameshifts
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) – negative, no indications for structural and numerical chromosome aberrations
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) (2006) – positive (DMSO as solvent)
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) (2012) – negative (acetone as solvent)
OECD Guideline 476 HPRT (In Vitro Mammalian Cell Gene Mutation Test) (2014) – mutagenic potential only to incubations without metabolic activation (acetone as solvent)
- Available non-GLP studies: none
- Historical human data: none
- (Q)SAR: not sufficiently reliable for the prediction of genetic toxicity
- In vitro methods: performed, and the study result provide sufficient base for further in vivo examination
- Weight of evidence: no data are available
- Grouping and read-across: A read-across is not sufficiently reliable for the prediction of this in vivo effect
- Substance-tailored exposure driven testing: not applicable
- Approaches in addition to above: not applicable
CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The provision of an in vivo study is a standard REACH requirement. Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII. It is not possible to meet this data requirement through any non-testing methods or any Column 2 or Annex XI adaptations.
• Neither in the bacterial mutagenicity test nor in the chromosomal aberration test in mammalian cells any indication for point or chromosomal mutagenic effects were noted.
• Three mammalian mutagenicity tests (mouse lymphoma assays) were performed.
o In one MLA the potential for a mutagenic potential at high, precipitating concentrations was noted.
o Concentrations showing no or only slight precipitation did not reveal any indication for a mutagenic potential in both tests, however a third MLA indicated mutagenic potential in the absence of metabolic activation.
o A further in-vivo test is foreseen to discard or confirm the genotoxic potential of Linseed Oil, Oxidized.
FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: For Linseed oil, Oxidized it is proposed by the registrant to perform an OECD TG 489 assay as a follow up study. This “In vivo alkaline single-cell gel electrophoresis assay for DNA strand breaks (Comet assay)” can provide conclusive information on both the clastogenicity as well as possible gene mutations of the test substance.
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
in vivo mammalian cell study: DNA damage and/or repair
Principles of method if other than guideline
While the test item Linseed oil, oxidized did not induce gene mutations in bacteria (Ames test, OECD 471) or chromosome aberrations in mammalian cells (Chromosome Aberration Assay, OECD 473), positive and negative outcomes of point mutations were observed in mammalian cells (Mouse Lymphoma Assay and HPRT Assay, OECD 476).
According to REACH requirements Annex VIII/IX, section 8.4 an appropriate in vivo gene mutation test shall be proposed by the registrant, when there is a positive result from an in vitro gene mutation study in bacteria (Ames test, OECD 471) or from an in vitro gene mutation study in mammalian cells (OECD 476).
Based on the available in vitro data, an alkaline in vivo Comet assay (OECD 489) is proposed to assess the mutagenic properties of the test substance in vivo. The in vivo Comet assay is considered to be the appropriate test system to investigate short-lived substances at the first site of contact. Furthermore, historical control data are available for various tissues. The test substance is proposed to be administered orally by gavage to rats. Doses will be based on data available for repeated dose toxicity and acute oral toxicity study. Based on the results of the repeated dose toxicity and acute oral toxicity study and taken the OECD requirements into consideration, a maximum dose of 2000 mg/kg bw/day, and two additional doses are proposed. Organs proposed to be evaluated are the forestomach and the liver, as the site of first contact after oral administration and the site of metabolism, respectively. The tissues are proposed to be evaluated after two administration days.
Six animals in the dose groups and negative control group as well as 4 animals in the positive control groups are proposed to reach a minimum of 5 and 3 analysable animals per sex, respectively, as requested according to the Guideline. Since the available data did not demonstrate relevant differences between males and females, the use of males is proposed.
Type of assay
single cell gel/comet assay in rodents for detection of DNA damage
mammalian cell study: DNA damage and/or repair
Species
Rat
Route of administration
oral: gavage - Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
- Version / remarks:
- current
- Deviations:
- no
Reference
Additional information
Ames test
The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation. The test item was used as a solution/suspension in dimethylsulphoxide (DMSO). In Main Assay I, using the plate incorporation method, the test item was assayed at the maximum dose-level of 5000 μg/plate and at the four dose-levels of 2500, 1250, 625 and 313 μg/plate. As no increases in revertant numbers were observed, all treatments of Main Assay II included a pre-incubation step and used the same dose-levels employed in Main Assay I. In both experiments clear precipitation of the test item was observed at the three higher concentrations, both in the absence and presence of S9 metabolism. No toxicity was observed in any experiment, with any tester strain, in the absence and presence of S9 metabolic activation. The test item did not induce a biologically relevant increase in the number of revertant colonies in the plate incorporation or pre-incubation assay in the absence or presence of S9 metabolism at any dose-level in any tester strain.
Chromosome aberration test
The ability of the test item to induce chromosome aberrations was evaluated in cultured peripheral human lymphocytes in the presence and in the absence of a metabolic activation system. Based on pre-tests the test substance was evaluated as soluble in DMSO at concentrations of 33 mg/mL and below but clearly formed a suspension at concentrations of >= 100 mg/mL.
In the first cytogenetic assay the test item was tested up to 100 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8 % (v/v) S9-fraction.
In the second cytogenetic assay, the test item was tested up to 100 µg/mL for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix the test item was also tested up to 100 µg/mL for a 3 h exposure time with a 48 h fixation time. Precipitation was obvious at 100 µg/mL.
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. The test item 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 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 the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.
Mouse lymphoma assay
Three mouse lymphoma assays (MLA) were performed.
In the Genetic toxicity in vitro_OECD476_2006_RTC available the test item was investigated using DMSO as solvent.
Based on the report, the test item was considered to be soluble in DMSO up to 300 mg/mL and a concentration of 3000 μg/mL was selected as the highest dose level to be used in the cytotoxicity pre-test.
Based purely on cytotoxicity results, the mutagenic potential was evaluated in a concentration range between 23.4 and 563 µg/mL in the absence and between 93.8 and 1500 µg/mL in the presence of S9 metabolic activation system in the main test, although cloudy appearance of the culture media was reported at least for concentrations >= 375 µg/mL, indicating an inhomogeneous suspension/distribution of the test item at those concentrations.
In the absence of S9 metabolic activation, severe toxicity was observed at the highest concentration. At 375 μg/mL the relative survival (%RS) and the relative total growth (RTG) values were reduced to 12% and 7% of the concurrent negative control value, respectively indicating massive cytotoxicity too. Pronounced toxicity was still observed at the next lower dose level, reducing %RS and RTG to 54% and 51%, respectively. In the presence of S9 metabolic activation, a dose-related cytotoxicity effect was observed. The top concentration tested (1500 μg/mL) yielded 22% relative survival and 13% relative total growth, indicating massive cytotoxicity.
Vehicle and positive control treatments were included in the mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the vehicle control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.
In the absence of S9 metabolic activation, a statistically significant and biological relevant increase in mutant frequency was observed at 188 µg/mL and in the presence of S9 mix at 750 and 1500 µg/mL. After treatment with the test item, the proportion of small versus large colonies was shifted towards the small colonies at the concentration producing an increase of mutagenic events both in the absence and presence of S9 metabolic activation.
In Genetic toxicity in vitro_OECD476_2012_CRL the second assay acetone as solvent was used, as the test item is better soluble in this solvent based on practical experience.
This report describes the effects of Linseed oil, oxidized on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).
Linseed oil, oxidized was a clear brown liquid. Acetone was used as solvent as the test substance is better soluble in this solvent based on practical experience.
Linseed oil, oxidized was tested at the dose level of 33 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
A single (limit) concentration, causing already precipitation was considered sufficient in this experiment as another mouse lymphoma assay was already available showing no mutagenic effects at several concentrations even exceeding the test concentration tested in the present MLA. No toxicity was observed at this dose level in the absence and presence of S9-mix.
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.
Mutation frequencies in cultures treated with positive control chemicals were increased 15- and 10-fold for MMS in the absence of S9-mix, and 14-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.
In the absence of S9-mix, Linseed oil, oxidized did not induce a significant increase in the mutation frequency both after 3 and 24 hours treatment time.
In the presence of S9-mix, Linseed oil, oxidized did not induce a significant increase in the mutation frequency.
It is concluded that Linseed oil, oxidized is not mutagenic in the mouse lymphoma L5178Y test system at a concentration of 33 µg/mL under the experimental conditions described in this report.
As the outcome of the Ames test, the chromosome aberration and the two MLA tests did not lead to a final conclusion, a third OECD TG 476 HPRT test was performed. In the third test Genetic toxicity in vitro_OECD476_2014_CRL, again acetone was used as solvent.
The mutagenic activity of Blown Linseed Oil was evaluated in an in vitro mammalian cell gene (HPRT ) mutation test with V79 Chinese hamster cells, performed in accordance with the OECD guidance, Klimisch 1. The test was performed in two independent experiments in the absence and presence of S9-mix. The test substance was dissolved in acetone.
In the first experiment, Blown Linseed Oil was tested up to concentrations of 100 μg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. Blown Linseed Oil precipitated in the culture medium at dose levels of 33 μg/mL and upwards. The untreated, vehicle and positive control were considered valid.
Mutation frequencies in cultures treated with positive control chemicals were increased 53- and 16-fold for EMS in the absence of S9-mix, and by 20-fold for 3-MCA and 31-fold for DMBA in the presence of S9-mix.
In the absence of S9-mix, Blown Linseed Oil induced a 21-fold increase in the mutation frequency. Verification of this result in a repeat experiment showed a 5.5-fold increase in the mutation frequency compared to the vehicle control. In the second experiment, the increases in the mutation frequency were dose-related, starting at 3 μg/mL, and also observed below precipitating dose levels. Taken together, the mutation frequency was more than three-fold increased and above the historical data range in two independent experiments.
In the presence of S9-mix, Blown Linseed Oil did not induce a significant increase in the mutation frequency.
It is concluded that Blown Linseed Oil shows HPRT mutagenic potential in the gene mutation test with V79 Chinese hamster cells under the experimental conditions described in this report. The mutagenicity was confined only to incubations without metabolic activation.
Overall genotoxicity evaluation
Based on the Ames test and the Chromosome aberration test, the test substance does not show mutagenic potential. However, in the first MLA (2006) mutagenicity was observed. The results obtained with the high test concentrations are questionable since BLO is very poorly soluble. Therefore, a limit MLA study (OECD 476) was performed in 2012 in which one concentration was tested (33 µg/mL in acetone). This study did not show any genotoxic potential. As this limit study alone is not sufficient to overrule the first MLA study (report nr. 52210, 2006), a new full MLA study (OECD 476, HPRT) was performed. As this full MLA-study resulted in a positive mutagenicity outcome in the absence of S9, an in vivo study should be performed to conclusively discard or confirm the genotoxic potential of Linseed Oil, Oxidized.
Justification for classification or non-classification
Based on the in vitro data available no final evaluation of the test item with regard to its mutagenic potential is possible.
As the MLA-studies indicated a positive mutagenicity outcome in the absence of S9, the substance is considered potentially mutagenic. An in vivo study should thus be performed to conclusively discard or confirm the genotoxic potential of Linseed Oil, Oxidized.
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