Castor oil, oxidized

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro Bacterial Reverse Mutation Assay (Ames, OECD 471): not mutagenic, study with Blown castor oil In vitro Chromome aberration Assay (OECD 473): not cytogenic, RA to studies with Blown linseed oil and Blown rapeseed oil In vitro Gene mutation assay in mammalian cells (OECD 476): RA to studies with Blown linseed oil and Blown rapeseed oil. In one MLA some indication for a mutagenic potential at high, precipitating concentrations were noted. In contrast concentrations showing no or only slight precipitation did not reveal any indication for a mutagenic potential in both tests. Thus, the effect noted at high test concentrations are considered artificial and most likely of no biological relevance. Further in vitro tests are foreseen to confirm the lack of mutagenic effects in vitro. No mutagenic effects were observed in the study with Blown rapeseed oil.

Link to relevant study records

Reference

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:

other: The study was performed in accordance with OECD guidline 471 and under GLP conditions.

Justification for data waiving:

other:

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 440/2008

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

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

Additional strain / cell type characteristics:

not applicable

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

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

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: none for preliminary toxocity test, triplicate for main test

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)

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test item precipitate (globular in appearance) was noted at 5000 µg/plate. The observation did not prevent scoring of revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA:
A history profile of vehicle/untreated and positive control values (reference items) for 2010 and 2011 are presented in Appendix 2 of the reoprt.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In a bacterial reverse mutation test that was performed according to OECD 471 and under GLP conditions, Blown castor oil was considered to be notmutagenic under the conditions of this test.

Executive summary:

In a bacterial reverse mutation test that was performed according to OECD 471 and under GLP conditions, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the testing material, Blown castor oil, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, 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 toxicologically 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 testing material,Castoroil, oxidized, was considered to be non‑mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames test

In a bacterial reverse mutation test that was performed according to OECD 471 and under GLP conditions, Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and Escherichia colistrain WP2uvrAwere treated with the testing material, Blown castor oil, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, with and without the addition of a rat liver homogenate metabolizing system (S9). The dose range for the first experiment was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate.

No toxicologically 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 testing material, Castoroil, oxidized, was considered to be non‑mutagenic under the conditions of this test.

 

Chromosome aberration test

Chromosome aberration tests are available for two substances in the category blown oils: Blown linseed oil and Blown rapeseed oil. The results of these tests are read across to Blown castor oil.

 

The ability of Blown linseed oil 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.

 

The ability of Blown rapeseed oil to induce chromosome aberrations was evaluated in a chromosome aberration study that was performed in accordance with OECD 473 and under GLP conditions. 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. Blown rapeseed 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. It was concluded that Blown rapeseed oil is not clastogenic in human lymphocytes under the experimental conditions described in the report.

 

Based on the results of the chromosome aberration assays for Blown linseed oil and Blown rapeseed oil, Blown castor oil is considered to be not clastogenic.

 

Mouse lymphoma assay

 

Mouse lymphoma assays (MLA) are available for two substances in the category blown oils: Blown linseed oil and Blown rapeseed oil. The results of these tests are read across to Blown castor oil.

 

For Blown linseed oil, two MLA-studies are available. 

In the first MLA available Blown linseed oil 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 a second confirmation assay acetone as solvent was used, as Blown linseed oil is better soluble in this solvent based on practical experience.

The substance 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 second MLA.

No toxicity was observed at this dose level in the absence and presence of S9-mix. However, the test item precipitated already in the culture medium at this dose level.

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. An increase of the mutation frequencies was observed for the positive control substances in the presence and in the absence of a metabolic activation system.

The test item did not induce a significant increase in the mutation frequency neither in the presence nor in the absence of a metabolic activation system after a 3 and 24 hour treatment period, respectively.

 

Taken all data together no final evaluation of Blown linseed oil with regard to its mutagenic potential in vitro is possible. The biological relevance of the findings in one MLA at very high test concentrations (based on the very limited solubility of the test item) is highly questionable for the following reasons:

The concentration of the test item in the DMSO stock solution as well as in the test was inappropriate and is considered to cause an inhomogeneous solution and precipitation under the given test conditions for the concentration at which relevant effects were observed (cloudy culture media are reported).

This evaluation is supported by detailed knowledge in handling the substance under technical conditions as well as solubility results reported e.g. in the context of the chromosomal aberration or the Ames test. E.g. in the chromosomal aberration test pre-experiments indicated that 100 mg/mL of the test item in DMSO clearly forms a suspension and pronounced precipitation was seen at 100 µg/mL.

An inhomogeneous solution can result in much higher local concentrations and consequently cause massive local cytotoxicity compared to the overall cytotoxicity noted for the entire culture.

The fact, that precipitations of test items can cause artificial findings is the reason that in the OECD guidelines for in vitro mutagenicity tests both cytotoxicity and/or precipitation/solubility define the maximum test concentration to be used.

Based on these considerations, the findings noted at the high test concentrations in one MLA are considered to be artificial and does not allow a final conclusion.

 

This evaluation is further supported by the other in vitro mutagenicity tests at hand.

 

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, even up to high test concentrations where clear precipitation was noted. In all assays the very limited solubility of the test item causing precipitation and not cytotoxicity was the limiting factor for the maximum test concentration.

 

Furthermore a potential of gross chromosome aberrations in mammalian cells as indicated by the proportion of small colonies in the MLA at the high test concentrations were not seen by the chromosome aberration test in vitro.

 

As the current data set does not allow a final conclusion on the in vitro mutagenic potential of Blown linseed oil no animal test are currently proposed.

Instead and to support the weight of evidence approach an in vitro mammalian cell gene mutation test according to OECD TG 476 (HPRT) and an in vitro mammalian cell micronucleus test according to OECD TG 487 will be performed after confirmation by ECHA. If those tests do not reveal any indication for mutagenic potential and consequently support the evaluation that the effects noted at high test concentrations in one MLA are artificial, there is no need for any further in vivo mutagenicity test.

 

Additionally, a MLA test with Blown rapeseed oil is available.

The test with Blown rapeseed oil was performed in accordance with OECD 476 and under GLP-conditions. In this test, hexane was used as a vehicle. 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, Blown rapeseed 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, Blown rapeseed oil did not induce a significant increase in the mutation frequency. In conclusion, Blown rapeseed oil is not mutagenic in the TK mutation test system under the experimental conditions described in the report.

 

Based on the results of the MLA-tests with Blown linseed oil and Blown rapeseed oil, Blown castor oil is considered to be not mutagenic in mammalian cells. Further in vitro tests with Blown linseed oil will be taken into account when available.

Justification for selection of genetic toxicity endpoint
The selected study is the only available key study for this endpoint which was performed with Blown castor oil. The other key studies were performed with read across substances Blown linseed oil and Blown rapeseed oil.

Justification for classification or non-classification

Based on the in vitro data available no final evaluation of Blown castor oil with regard to its mutagenic potential is possible, based on the equivocal effects observed for Blown linseed oil. However, the data set available does not trigger a C&L with regard to mutagenic effects.