Oils, vegetable, deodorizer distillates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of ‘Oils, vegetable, deodorizer distillates’ can be deduced based on information available for its individual constituents. Studies conducted on glycerides, fatty acids, tocopherols and sterols, either in vitro or in vivo, indicated that these substances are not mutagenic. Moreover, predictions from the Toxtree (v.1.6) model revealed no evidence for mutagenic activity of any of the constituents.

Most of these substances have a long history of safe use in nutritional, cosmetic and/or industrial applications. Some (e.g. certain fatty acids and tocopherols) are listed as GRAS and/or demonstrate antitumourigenic activity under certain conditions (e.g. tocopherols and squalene; see Section 5.8 of the CSR).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

4, 20, 100, 500 and 2,500 µg/plate

Vehicle / solvent:

Water with Tween 80

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

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:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

None

Conclusions:

Under the test conditions, the substance fatty acids, C8-18 and C18-unsatd. was found to be non-mutagenic in Salmonella typhimurium bacteria reverse mutation assay.

Executive summary:

A study was conducted to determine the mutagenic potential of the constituent ‘Fatty acids, C8 -18 and C18 -unsatd.’ in a bacterial gene mutation assay with Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The test was conducted on agar plates in the absence or presence of S-9 mix. Suspensions of the substance were freshly prepared in water with Tween 80 before use at concentrations of 4, 20, 100, 500 and 2,500 µg/plate. Under the test conditions, ‘the substance fatty acids, C8 -18 and C18 -unsatd. was found to be non-mutagenic in Salmonella typhimurium bacterial reverse mutation assay (Gloxhuber and Wallat, 1982).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 97, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not reported

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

10 and 30% Aroclor 1254-induced S9 from male Syrian hamster liver and male Sprague Dawley rat liver

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333 and 10000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Tested on TA100 and TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Tested on TA97

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

Tested on TA 98, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Pre-incubation. The detailed protocol of Zeiger et al., 1988 was followed.

NUMBER OF REPLICATIONS: Three

Evaluation criteria:

Not reported

Statistics:

Not reported

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 97, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

None

Conclusions:

Under the test conditions, the substance can be considered to be non-mutagenic in Salmonella typhimurium mutation assay.

Executive summary:

A study was performed to investigate the potential mutagenicity of constituent ‘glycerides, C16 and C18-unsatd. and C18-unsatd. hydroxy’ (as castor oil) through the reverse mutation assay using Salmonella typhimurium strains TA 1535, TA 97, TA 98 and TA 100, along with Aroclor-induced 10 and 30% liver fraction for metabolic activation (S9-mix). Salmonella strains were exposed to five different concentrations (0, 100, 333, 1,000, 3,333 and 10,000 µg/plate) of the substance and vehicle control (DMSO), in both absence and the presence of S9-mix. Three parallel plates were used for each dose. Concurrent positive controls, 2-aminoanthracene (for all strains, with metabolic activation), 4-nitro-o-phenylenediamine (on TA 98, without metabolic activation), sodium azide (TA100 and TA1535) and 9-aminoacridine (TA97) were also included in the assays. In both absence and presence of the S9-mix, the substance did not cause a significant increase in the number of revertant colonies in comparison to the control. Both positive and vehicle control groups were also valid. Hence, under the test conditions, the substance was considered to be non-mutagenic in Salmonella typhimurium mutation assay (Irwin, 1992 (1)).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Not reported

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix: Aroclor 1254­induced liver extracts of Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 1600, 3000 and 5000 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation; number of cells evaluated at 0.0625, 0.2500 µg/mL = 200 and 50 respectively Migrated to IUCLID6: at 0.0625, 0.2500 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation; number of cells evaluated at 2.50 and 7.50 µg/mL = 200 and 50 respectively Migrated to IUCLID6: at 2.50 and 7.50 µg/mL

Details on test system and experimental conditions:

INCUBATION, HARVEST DETAILS: Protocol of Galloway et al., 1985; 1987 was followed.
- In the absence of S9: Cells were incubated with test material or solvent for 10 h at 37°C. Cells were then washed and fresh medium containing colcemid was added for an additional 3 h followed by harvest.
- In the presence of S9: Cells were incubated with test material or solvent for 2 h at 37°C. Cells were then washed, medium without test material was added, and incubation was continued for 10 h. Colcemid was added for the last 3 h of incubation before harvest.


SPINDLE INHIBITOR: Colcemid
STAIN (for cytogenetic assays): 6% Giemsa


NUMBER OF CELLS EVALUATED: 200

Evaluation criteria:

Not reported

Statistics:

Statistics were performed on % cells with aberrations

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

None

Conclusions:

Under the test conditions, the substance was considered to be non-genotoxic to Chinese hamster ovary cells in chromosomal aberration assay.

Executive summary:

A study was performed to investigate the genotoxic potential of the constituent ‘glycerides, C16 -18 and C18-unsatd.’ (as castor oil) to induce chromosomal aberrations in Chinese hamster ovary cells. Chinese hamster ovary cells were incubated with 0, 1,600, 3,000 and 5,000 µg/mL of the substance or solvent (DMSO). Cells were arrested in first metaphase by addition of colcemid and harvested by mitotic shake off, fixed, and stained in 6% Giemsa. Where relevant S9 was from the livers of Aroclor 1254-induced male Sprague Dawley rats was added. No significant chromosome aberrations were observed when treated with concentrations up to 5,000 µg/mL of the substance with and without S9. Hence, under the test conditions, the substance was considered to be non-genotoxic to Chinese hamster ovary cells in chromosomal aberration assay (Irwin, 1992 (2)).

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Not reported

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix: Aroclor 1254­induced liver extracts of Sprague-Dawley rats

Test concentrations with justification for top dose:

160, 500, 1600 and 5000 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation; number of cells evaluated at 0.10 and 0.60 µg/mL = 50 and 10 respectively Migrated to IUCLID6: at 0.10 and 0.60 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation; number of cells evaluated at 0.0005 and 0.0050 µg/mL = 50 and 10 respectively Migrated to IUCLID6: at 0.0005 and 0.0050 µg/mL

Details on test system and experimental conditions:

BRIEF DESCRIPTION OF METHOD: Protocol presented by Galloway et al. (1985, 1987) was followed.
- In the absence of S9, cells were incubated with test material or solvent for 2 h at 37°C. Then BrdU was added and incubation was continued for 24 h. Cells were washed, fresh medium containing BrdU and colcemid was added, and incubation was continued for 3 h.
- In the presence of S9, cells were incubated with test material or solvent for 2 h at 37°C. The cells were then washed, and medium containing BrdU without test material was added. Cells were incubated for a further 26 h, with colcemid present for the final 2-3 h.

DURATION
- Exposure duration: 2 h at 37°C
- Selection time (if incubation with a selection agent): 26 and 24 h with and without metabolic activation respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 30-31 and 29 h with and without metabolic activation respectively


SELECTION AGENT (mutation assays): Bromodeoxyuridine (BrdU)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid


NUMBER OF CELLS EVALUATED: 50

Evaluation criteria:

Not reported

Statistics:

Statistics performed on SCE/chromosome values.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

None

Conclusions:

Under the test conditions, the substance was considered to be non-genotoxic to Chinese hamster ovary cells in sister-chromatid exchange assay.

Executive summary:

A study was performed to investigate the genotoxic potential of the constituent ‘glycerides, C16 -18 and C18 -unsatd.’ (as castor oil) to induce sister-chromatid exchanges in Chinese hamster ovary cells. Chinese hamster ovary cells were incubated with 160, 500, 1,600 and 5,000 µg/mL of the substance or solvent (DMSO). Cells were then collected by mitotic shake-off, fixed, air-dried and stained. Where relevant S9 was from the livers of Aroclor 1254-induced male Sprague Dawley rats was added. No significant sister-chromatid exchange was observed in Chinese hamster ovary cells treated with concentrations up to 5,000 µg/mL of the substance with and without S9. Hence, under the test conditions, the substance was considered to be non-genotoxic to Chinese hamster ovary cells in sister-chromatid exchange assay (Irwin, 1992 (3)).

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Remarks:

Lab name not reported in the IUCLID dataset

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

No data

Test concentrations with justification for top dose:

50, 130, 500, 1,500 and 5,000 µg/plate

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

None

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

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

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No toxicity was observed up to the highest dose level

No

Conclusions:

Under the test conditions, the substance was considered to be non-mutagenic.

Executive summary:

A study was conducted to determine the mutagenic potential of the constituent ‘glycerides, C16 -18 and C18 -unsatd.’ (as palm oil) according to OECD guideline 471. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were treated with the substance using the Ames plate incorporation method at five dose levels (up to 5,000 µg/plate), with and without metabolic activation. Two independent assays were conducted. Under the conditions of the study, the substance was considered to be non-mutagenic (IUCLID DS, 2000).

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Remarks:

Lab name not reported in the publication

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomal enzyme homogenate

Test concentrations with justification for top dose:

10, 33, 100, 333 and 1,000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

for TA 1535

Positive control substance:

sodium azide

Remarks:

5 µg/plate dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

for TA 98

Positive control substance:

2-nitrofluorene

Remarks:

20 µg/plate dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

for TA 100

Positive control substance:

methylmethanesulfonate

Remarks:

650 µg/plate dissolved in DMSO

Untreated negative controls:

yes

Remarks:

ethanol

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

for WP2 uvrA

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

10 µg/plate dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

For the bacterial test with metabolic activation: TA1535, TA98 and TA100

Positive control substance:

other: 1 µg/plate 2-aminoanthracene dissolved in DMSO

Remarks:

none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

For the bacterial test with metabolic activation: TA1537 and TA100

Positive control substance:

other: 2.5 µg/plate 2-aminoanthracene dissolved in DMSO

Remarks:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

For the bacterial test with metabolic activation: TA1537

Positive control substance:

other: 5 µg/plate 2-aminoanthracene dissolved in DMSO

Remarks:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

For the bacterial test with metabolic activation: WP2uvrA

Positive control substance:

other: 10 µg/plate 2-aminoanthracene dissolved in DMSO

Remarks:

None

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

Evaluation criteria:

The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain. The positive control chemicals, sodium azide for TA1535; 9-aminoacridine for TA1537, 2-nitrofluorene for TA98, methylmethanesulphonate for TA100 and 4-nitroquinoline N-oxide for WP2uvrA should produce positive responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group means.
A test substance was considered negative (not mutagenic) in the test if (a) The total number of revertants in tester strain TA100 was not greater than two times the concurrent control, and the total number of revertants in the tester strains TA1535, TA1537, TA98 or WP2uvrA was not greater than three times the concurrent control, (b) The negative response was reproducible in at least one independently repeated
experiment.

Statistics:

Not applicable

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges.

None

Conclusions:

Under the conditions of this test, the substance was considered to be non-mutagenic.

Executive summary:

A study was conducted to determine the potential mutagenicity of the constituent ‘glycerides, C16 -18 and C18 -unsatd.’ (as pine nut oil) according to the OECD guideline 471. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the substance using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 3 to 5,000 μg/plate. The experiment was repeated using the same dose range as the range-finding test. The substance precipitated at the two highest concentrations tested and therefore it was tested up to the third dose level of 1,000 μg/plate. No precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the substance, either with or without metabolic activation. Under the conditions of this test, the substance was considered to be non-mutagenic (Speijers, 2009).

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method followed unknown, data from handbook

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

S. typhimurium, other: strains not reported

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Metabolic activation system:

no data

Test concentrations with justification for top dose:

5000 µg/plate

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Key result

Species / strain:

S. typhimurium, other: strains not reported

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

None

Conclusions:

The test substance was found to be non-mutagenic in Salmonella typhimurium reverse mutation assay.

Executive summary:

A study was conducted to determine the mutagenicity potential of the constituent ‘glycerides, C8 -18 and C18 -unsatd.’ (as coconut oil) according to an unspecified method. Under the study conditions, the substance was found to be non-mutagenic in Salmonella typhimurium reverse mutation assay at a concentration of 5,000 µg/plate (Biotech Index, 1970).

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mutagenecity potential of the constituent coconut oil was determined in Salmonella typhimurium gene mutation assay with and without metabolic activation.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

50, 150, 500, 1,500 and 5,000 µg/plate

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

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

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

None

Conclusions:

The test substance was found to be non-mutagenic in Salmonella typhimurium gene mutation assay.

Executive summary:

A study was conducted to determine the mutagenicity potential of the constituent ‘glycerides, C8 -18 and C18 -unsatd.’ (as coconut oil) in a bacterial reverse mutation assay using Salmonella typhimurium strains. The substance was tested at concentrations of 50, 150, 500, 1,500 and 5,000 µg/plate with Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100. Under the study conditions, the substance was found to be non-mutagenic in the Salmonella typhimurium gene mutation assay (IUCLID DS, 2000).

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

Lab name not reported in the ANZFA risk analysis report

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: Fresh human lymphocyte

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

≤200 μg/mL

Vehicle / solvent:

No data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

Similar negative results has been obtained in other chromosome aberration tests cited in the ANZFA risk analysis report. As additional weight of evidence, the results has been summarised in the below table:

Test material	Concentration	Test object	Results	Reference
Phytosterol esters (Roche)	≤ 100 μg/ml (+/- S9)	Fresh human lymphocyte	Negative	Akhurst LC and Taylor K, Feb 03, 1997
Plant sterols	≤ 160 μg/ml (+/- S9)	Fresh human lymphocyte	Negative	Akhurst LC and Taylor K (Feb. 12 1997)

Conclusions:

Under the test conditions, the substance was considered to be non-genotoxic to human lymphocytes in chromosome aberration assay.

Executive summary:

A study was performed to investigate the genotoxic potential of plant sterols in human lymphocytes. No significant chromosome aberrations were observed when treated with concentrations up to 200 µg/mL of the substance, with and without metabolic activation (S9). Under the test conditions, the substance was considered to be non-genotoxic to human lymphocytes in a chromosomal aberration assay (ANZFA, 2001).

Similar negative results were obtained in two studies of Akhurst LC and Taylor K (Feb 03, 1997) and Akhurst LC and Taylor K (Feb 12, 1997) conducted with  fresh human lymphocyte at concentration of  ≤100 μg/ml (phytosterol ester) and  ≤160 μg/ml (plant sterol) respectively in a chromosome aberration assay. These studies have been cited in the ANZFA risk analysis report (ANZFA, 2001).

Reference 10

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The constituent β-sitosterol at concentration of 1000 µM was incubated with gamma DNA for 1 h at 37 degree celsius and DNA strand breaks were identified by electrophoresis under neutral conditions.

GLP compliance:

not specified

Type of assay:

other: acellular assay (DNA damage in gamma DNA)

Target gene:

Not applicable

Species / strain / cell type:

other: gamma DNA

Details on mammalian cell type (if applicable):

Not reported

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

1000 µM

Vehicle / solvent:

No data

Details on test system and experimental conditions:

TEST SYSTEM: gamma DNA

EXPOSURE DURATION: 1 h

Evaluation criteria:

No data

Statistics:

No data

Key result

Species / strain:

other: gamma DNA

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

None

Conclusions:

Under the test conditions, the substance did not induce DNA damage.

Executive summary:

A study was performed to investigate the genotoxic potential of β-sitosterol. The test substance at concentration of 1000 µM was incubated with gamma DNA for 1 h at 37 degree Celsius and DNA strand breaks were identified by electrophoresis under neutral conditions. Under the test conditions, the substance did not induce DNA damage (Tice, 1997).

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA1538, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

No data

Test concentrations with justification for top dose:

Up to 5000 µg/plate (12 µmol/plate)

Vehicle / solvent:

No data

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA1535, TA1537 and TA1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

None

Conclusions:

Under the test conditions, the substance was to be non-mutagenic in Salmonella mutation assay (Ames test).

Executive summary:

A study was performed to investigate the potential mutagenicity of the constituent β –sitosterol (at concentrations upto 5000 µg/plate (12 µmol/plate)) through the reverse mutation assay using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 without metabolic activation. The substance did not cause a significant increase in the number of revertant colonies. Under the test conditions, the substance was considered to be non-mutagenic in Salmonella mutation assay (Ames test) (Tice, 1999).

Similar negative results were obtained in two studies of Lawson et al., 1989 and Malaveille et al., 1982  conducted with Salmonella typhimurium strains T98 and TA100 with and without metabolic activation. Lawson et al used concentrations 150, 300, and 600 µg/plate (0.36, 0.72, and 1.4 mol/plate) and Malaveille et al used 1000 µg/plate (2.4 µmol/plate). These studies have been cited in the Integrated Laboratory Systems (ILS) review of toxicological literature.

Reference 12

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Remarks:

Lab name not reported in the ANZFA risk analysis report

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

≤ 5000 μg/plate

Vehicle / solvent:

No data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

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:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

Similar negative results has been obtained in other Ames tests cited in the ANZFA risk analysis report. As additional weight of evidence, the results has been summarised in the below table:

Test material	Concentration	Test object	Results	Reference
Phytosterol esters (Roche)	≤5000 µg/plate (+/- S9)	S. typhimurium TA98, TA100, TA1535, TA1537	Negative	Gant RA, 1996
Plant sterols	≤5000 µg/plate (+/- S9)	S. typhimurium TA98, TA100, TA1535, TA1537; E. coli CM891 WP2	Negative	Kitching J and Anderson DH, 1998

Conclusions:

Under the test conditions, the substance was considered to be non-mutagenic in Salmonella mutation assay (Ames test).

Executive summary:

A study was performed to investigate the potential of phytosterols to induce mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 in Ames test. No mutations were observed when treated with concentrations up to 5000 µg/mL of the substance, with and without metabolic activation (S9).

Under the test conditions, the substance was considered to be non-mutagenic in Ames test (ANZFA, 2001).

Similar negative results were obtained in two studies of Gant RA (1996) and Kitching J and Anderson DH (1998) conducted with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E. coli CM891 WP2 respecively with and without metabolic activation at concentration of 5000 µg/plate. These studies have been cited in the ANZFA risk analysis report

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Mutagenic effects of the constituent alpha-tocopherol was assessed in bacterial reverse mutation assay.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Not reported

Species / strain / cell type:

S. typhimurium, other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not reported

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not reported

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Not reported

Test concentrations with justification for top dose:

0.033–10 mg/plate

Vehicle / solvent:

Not reported

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

Not reported

Evaluation criteria:

Not reported

Statistics:

Not reported

Key result

Species / strain:

S. typhimurium, other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Not reported

Similar negative results have been obtained in other Ames tests cited in the CIR report. As additional weight of evidence, the results has been summarised in the below table:

Form of alpha- tocopherol	Dose	Protocol	Results	Reference
dl-alpha	1,000 µg/plate	S. typhimurium TA98 and TA100 without and with metabolic activation	Negative	Wood, 1998
d-alpha	10,000 µg/plate	S. typhimurium TA1535, TA1537, TA1538, TA98, and TA 100 without and with metabolic activation; Escherichia coli WP2 (uvrA) without and with metabolic activation	Negative	SRI International, Inc., 1979
dl-alpha	≤5 mg/plate	S. typhimurium TA92, TA1535, TA100, TA1537, TA94, and TA98	Negative	Ishidate et al., 1984
dl-alpha	1 mg/plate	S. typhimurium TA102	Negative	Anderson et al., 1995

Conclusions:

Under the test conditions, the substance was found to be non-mutagenic in Salmonella typhimurium reverse mutation assay.

Executive summary:

According to the CIR review report, d-alpha tocopherol was found to be negative in a Salmonella typhimurium reverse mutation assay at a concentration of 0.033–10 mg/plate (Fiume, 2002).

Reference 14

Endpoint:

genetic toxicity in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Antigenotoxic effects of dl-alpha tocopherol at 10 µM concentrations was evaluated by observing the incidences of chromosomal breaks induced by 7,12-dimethylbenz(a)anthracene in leucocyte cultures.

GLP compliance:

not specified

Type of assay:

other: determination of chromosomal breakage

Target gene:

Not applicable

Species / strain / cell type:

other: leucocyte culture

Details on mammalian cell type (if applicable):

Not reported in the JECFA evaluation report

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Metabolic activation system:

Not applicable

Test concentrations with justification for top dose:

10 µM

Vehicle / solvent:

Not reported in the JECFA evaluation report

Details on test system and experimental conditions:

Not reported in the JECFA evaluation report

Evaluation criteria:

Not reported in the JECFA evaluation report

Statistics:

Not reported in the JECFA evaluation report

Key result

Species / strain:

other: leucocyte culture

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

The addition of dl-alpha-tocopherol to leucocyte cultures at a concentration of 10 µM reduced by 63% the number of chromosome breaks induced by 1.6 µM 7,12-dimethylbenz(a)anthracene.

Conclusions:

Under the test conditions, the substance was considered to be non-genotoxic.

Executive summary:

Antigenotoxicity effects of the constituent dl-alpha tocopherol at 10 µM concentrations was evaluated by observing the reduction of chromosomal breaks induced by 7,12-dimethylbenz(a)anthracene in leucocyte cultures. The addition of the substance to leucocyte cultures reduced the number of chromosome breaks induced by 1.6 µM 7,12 -dimethylbenz(a)anthracene. Similar effects were observed in another anti-genotoxicity study of Shamberger et al., 1979 cited in the JECFA evaluation, where dl-alpha-tocopherol markedly reduced the mutagenic effect of malonaldehyde and ß-propiolactone in five strains of Salmonella typhimurium, which mutated with a frameshift mechanism. Under the test conditions, the substance was considered to be non-genotoxic (JECFA, 1987).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The mutagenic potential of ‘Oils, vegetable, deodorizer distillates’ can be deduced based on information available for its individual constituents. Studies conducted on glycerides, fatty acids, tocopherols and sterols, eitherin vitroorin vivo, indicated that these substances are not mutagenic.Moreover, predictions from the Toxtree (v.1.6) model revealed no evidence for mutagenic activity of any of the constituents.

Most of these substances have a long history of safe use in nutritional, cosmetic and/or industrial applications. Some (e.g. certain fatty acids and tocopherols) are listed as GRAS and/or demonstrate antitumourigenic activity under certain conditions (e.g. tocopherols and squalene; see Section 5.8 of the CSR).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

FDA Good Laboratory Practices Regulations (21 CFR 58)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: 6 wk
- Fasting period before study: No
- Housing: Individually caged in polycarbonate cages lined with heat-treated hardwood chips and covered with polyester filter sheets. The cages were stored on stainless steel racks equipped with an automatic watering system
- Diet: NIH 07; available ad libitum
- Water: Ad libitum
- Acclimation period: 15 d
- Other: Feed hoppers in the animal cages were changed twice weekly


ENVIRONMENTAL CONDITIONS
- Temperature: 68-76°F
- Humidity: 42-72%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light



- Age when killed: 19 wk

Route of administration:

oral: feed

Vehicle:

Plain diet


- Vehicle(s)/solvent(s) used: None

Details on exposure:

DIET PREPARATION
- Method of mixing: Formulated diets were prepared by blending the appropriate amount of castor oil with a small quantity of feed to prepare a premix. The premix then was layered between the required amounts of feed in a twin-shell blender and blended for 15 min to achieve a uniform mix.
- Mixing appropriate amounts with (Type of food): 10% (100 mg/g) determined by gravimetric analysis, and blends at 0.5% (5 mg/g) determined by HPLC analysis.
- Storage temperature of food: Stored for no longer than 3 weeks at 5°C
- Stability under test conditions: 0.5% dose level is stable for at least 21 d when stored in the dark at 5°C and for 3 d when stored open to air and light in a rodent cage.

Duration of treatment / exposure:

13 wk or 90 d

Frequency of treatment:

Daily

Post exposure period:

At termination of 13 wk study

Remarks:

Doses / Concentrations:
0, 0.6, 1.3, 2.5, 5.0 and 10.0% in feed
Basis:
actual ingested

No. of animals per sex per dose:

10 mice per sex per dose

Control animals:

yes, plain diet

Positive control(s):

- Positive control: Urethane
- Dose: 0.2%
- Brief description: Male mice treated for 4 weeks with urethane in the drinking water (0.2%) were used as positive control. These animals were not part of the 13-week study, but were added as a measure of quality control for the assay.

Tissues and cell types examined:

Peripheral blood samples were examined for frequency of polychromatic and normochromatic micronucleated erythrocytes (PCE and NCE)

Details of tissue and slide preparation:

- Method of sampling: Cardiac puncture
- Stain used: Hoechst 33258/pyronin Y (MacGregor et al., 1983)
- Number of cells scored for micronuclei: 2,000 PCE and 10,000 NCE from each animal

Evaluation criteria:

Not reported

Statistics:

Not reported

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Not reported

No significant elevation in the frequency of micronucleated erythrocytes was observed in either male or female mice administered castor oil in dosed feed.

Conclusions:

Under the test conditions, the substance was considered to be non-mutagenic in the micronucleus test in B6C3F1 mice.

Executive summary:

A study was performed to investigate the genotoxic potential of the constituent ‘glycerides, C16 and C18 -unsatd. and C18 -unsatd. hydroxy’ (as castor oil) to induce micronuclei in polychromatic erythrocytes (PCE) in the peripheral blood of the mouse. Groups of 10 mice/sex were exposed to 0, 0.6, 1.3, 2.5, 5.0 and 10.0% concentrations of the substance mixed in diet for 13 weeks. At termination, smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals. Slides were stained with Hoechst 33258/pyronin Y. About 2,000 PCE and 10,000 NCE from each animal were scored for frequency of micronuclei. No significant elevation in the frequency of micronucleated erythrocytes was observed in either male or female mice administered test substance in dosed feed. Therefore, under the test conditions, the substance was considered to be non-genotoxic in the micronucleus test in B6C3F1 mice (Irwin, 1992).

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A study was conducted to evaluate if the constituent palm oil have any clastogenic effect in Balb/c mice. 10 mice were used per group, one treated with palm oil, one with red palm oil and one control group treated with corn oil. The exposure period lasted 5 d and the dose was 4500 mg/kg bw. Analyses were made on bone marrow metaphase cells isolated 24 h post application for chromosome abberations and mitotic index.

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals or test system and environmental conditions:

No data

Route of administration:

oral: gavage

Vehicle:

No data

Details on exposure:

No data

Duration of treatment / exposure:

5 d

Frequency of treatment:

No data

Post exposure period:

No data

Remarks:

Doses / Concentrations:
4500 mg/kg bw
Basis:
no data

No. of animals per sex per dose:

10 female mice per group

Control animals:

yes

Positive control(s):

No data

Tissues and cell types examined:

Bone marow cells

Details of tissue and slide preparation:

No data

Evaluation criteria:

No data

Statistics:

No data

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No statistically significant difference in the frequency of chromosomal aberrations and mitotic index between either the treated groups and the control group

None

Conclusions:

Under the conditions of the test, the substance did not show any clastogenic effects in mice bone marrow cells.

Executive summary:

A study was conducted to evaluate if the constituent ‘glycerides, C16-18 and C18-unsatd.’ (as palm oil) have any clastogenic effects in Balb/c mice. 10 mice were used per group. The exposure period lasted 5 d and the dose was 4500 mg/kg bw. Analyses were made on bone marrow metaphase cells isolated 24 h post application for chromosome abberations and mitotic index. The study showed no statistically significant difference in the frequency of chromosomal abberations and mitotic index between either of the treated groups and the control group. Under the conditions of the test, the substance did not show any clastogenic effects in mice bone marrow cells (IUCLID, 2000).

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

Lab name not reported in the ANZFA risk analysis report

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

not specified

Details on test animals or test system and environmental conditions:

No data

Route of administration:

oral: gavage

Vehicle:

No data

Details on exposure:

No data

Duration of treatment / exposure:

2 d

Frequency of treatment:

Daily

Post exposure period:

No data

Remarks:

Doses / Concentrations:
≤ 2000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

No data

Control animals:

yes

Positive control(s):

Yes but details not available

Tissues and cell types examined:

No data

Details of tissue and slide preparation:

No data

Evaluation criteria:

No data

Statistics:

No data

Key result

Sex:

not specified

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

None

Conclusions:

Under the test conditions, the substance is considered to be non-genotoxic in micronucleus test in rats.

Executive summary:

A study was performed to investigate the genotoxic potential of the constituent plant sterol ester to induce micronuclei in polychromatic erythrocytes (PCE) in the peripheral blood of the Wistar rat at concentration 2,000 mg/kg bw/day when administered for 2 d. No significant elevation in the frequency of micronucleated erythrocytes was observed. Under the test conditions, the substance was considered to be non-genotoxic in the micronucleus test in rats (ANZFA, 2001).

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Antigenotoxic effect of β-sitosterol at 150 mg/kg bw/day was evaluated by inhibition of DMBA (2-methyl-4- dimethylaminoazobenzene at 25 mg/kg bw, by intraperitoneal route) induced micronuclei formation in bone marrow polychromatic erythrocytes in a micronucleus assay.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

female

Details on test animals or test system and environmental conditions:

No data

Route of administration:

intraperitoneal

Vehicle:

No data

Details on exposure:

No data

Duration of treatment / exposure:

2 d

Frequency of treatment:

Daily

Post exposure period:

No data

Remarks:

Doses / Concentrations:
≤ 150 mg/kg bw/day (0.36 mmol/kg/day)
Basis:
nominal conc.

No. of animals per sex per dose:

No data

Control animals:

not specified

Positive control(s):

No data

Tissues and cell types examined:

No data

Details of tissue and slide preparation:

No data

Evaluation criteria:

No data

Statistics:

No data

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

None

Conclusions:

Under the test conditions, the substance was considered to be non-genotoxic.

Executive summary:

A study was performed to investigate the antigenotoxic potential of the constituent β–sitosterol by inhibition of DMBA induced micronuclei induction in bone marrow polychromatic erythrocytes in a micronucleus assay. The substance (150 mg/kg bw/day (0.36 mmol/kg bw/day)) was injected in B6C3F1 female mice intraperitoneally for 2 d before injecting 25 mg/kg bw DMBA (2-methyl-4- dimethylaminoazobenzene at 25 mg/kg bw, by the intraperitoneal route). The substance inhinited micronuclei formation by 60%. Under the test conditions, the substance was considered to be non-genotoxic substance (Tice, 1997).

 

Reference 5

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Clatogenic effects of the constituent alpha-tocopherol were assessed in Chromosomal aberration assay.

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

other: albino

Sex:

male

Route of administration:

oral: unspecified

Duration of treatment / exposure:

6 months

Frequency of treatment:

Daily

Post exposure period:

Not applicable

Remarks:

Doses / Concentrations:
100, 300 mg/kg bw in olive oil
Basis:
actual ingested

No. of animals per sex per dose:

6-7 animals in total

Control animals:

other: yes, concurrent vehicle (olive oil or water)

Tissues and cell types examined:

Cells were examined for chromosomal aberrations

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Similar negative results have been obtained in other of chromosomal aberration assays studies cited in the CIR review report. As additional weight of evidence, the results have been summarised in the below table:

Form of alpha- tocopherol	Dose	Protocol	Results	Reference
dl-alpha	Not stated	Rat bone marrow cells for chromosomal aberration in vivo	Negative	Kawachi et al., 1980
alpha	1000 mg/kg twice weekly	Groups of 5 mice were dosed IP with Tocopherol twice weekly for 1, 2, 3, or 4 weeks	No significant difference from control values	Kod´ytkov´a, Madar, and Sra´m, 1980

Conclusions:

Under the test conditions, the substance did not show any clastogenic effect in rats.

Executive summary:

A chromosomal aberration assay was conducted in rats (cells not specified) to evaluate the clastogenic effects of alpha-tocopherol. For the assay, 6–7 male albino rats were dosed orally with 100 and 300 mg/kg bw/day of the substance in olive oil for 6 months, and then examined for chromosomal aberrations. The control groups were simultaneously administered water or olive oil. The substance did not produce chromosomal aberrations; animals of the 300 mg/kg bw group had a decrease in aberrant cells and the number of pulverized cells. Hence, under the test conditions, the substance did not show any clastogenic effect in rats (Zondlo, 2002). 

Reference 6

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Clastogenic effects of the constituent alpha-tocopherol were assessed in Micronucleus assay.

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male

Route of administration:

oral: unspecified

Duration of treatment / exposure:

Single dose study

Frequency of treatment:

Single

Post exposure period:

Not applicable

Remarks:

Doses / Concentrations:
75 mg/kg bw in corn oil
Basis:
actual ingested

No. of animals per sex per dose:

Not reported

Control animals:

not specified

Tissues and cell types examined:

Number of micronucleus

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

None

Conclusions:

Under the test conditions, the substance did not produce any clastogenic effect in micronucleus assay conducted in mice.

Executive summary:

An micronucleus assay was conducted in mice to evaluate the clastogenic effects of the constituent dl-alpha-tocopherol. For the assay, male CD-1 mice were dosed singly with 75 mg/kg bw of the substance in corn oil. Under the test conditions, the substance did not produce any clastogenic effect in the micronucleus assay conducted in mice (Fiume, 2002).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro data

There is no published information on the in vitro mutagenicity of ‘Oils, vegetable, deodorizer distillates’ in animals. However, data is available for its constituents (see also Table above):

·        Glycerides with chain lengths varying between C8 -18 or C16 -18, including C18 -unsatd. and C18 hydroxy did not exhibit mutagenic activity in bacterial reverse mutation (Ames) assays, an in vitro mammalian chromosome aberration test with Chinese hamster ovary cells and/or a sister chromatid exchange assay in Chinese hamster ovary cells (Biotech Index, 1970c; IUCLID, 2000a and d; Irwin, 1992; Speijers et al., 2009).

·        A bacterial reverse mutation (Ames) assay conducted with mixed fatty acids of chain lengths varying between C8 -18, including C18 -unsatd. suggested absence of mutagenic potential (Gloxhuber and Wallat, 1982). A range of individual fatty acids of chain lengths varying between C10 and C22 have also been tested in Ames assays and shown to be negative in mutagenicity (Opdyke, 1981; CIR, 1987; HERA, 2002). Also, fatty acids from C12 to C19 demonstratec anticlastogenic effects in the chromosome abberation test (HERA, 2002).

·        Tests in bacterial and human cells in vitro suggest that tocopherols are not mutagenic and may in some cases have mutagenicity-inhibiting effects (Tomassi and Silano, 1986; JECFA, 1987).

·        Phytosterols and their esters were found to be negative in a battery of in vitro bacterial and mammalian mutagenicity tests at doses up to 200 µg/mL (ANZFA, 2001; Tice, 1999).

·        No specific data was found for squalene but the substance is a constituent of cells in most higher animals and may be synthesised by animal tissues from acetate (CIR, 1982). There are no indications for mutagenic potential.

In vivo data

There is no published information on the in vivo mutagenicity of ‘Oils, vegetable, deodorizer distillates’ in animals. However, data is available for some of its constituents (see also Table above):

·        Chromosomal aberration testing in mice with glycerides containing fatty acids of chain lengths in the range of C16 -18, including C18 -unsatd. and C-18 hydroxy were negative in mutagenicity (IUCLID, 2000d; Irwin, 1992).

·        Tocopherols, phytosterols and their esters were found to be negative in a battery of in vivo genotoxicity tests (Fiume, 2002; ANZFA, 2001; Tice, 1997).

·        No specific in vivo data was found for fatty acids and squalene.

Moreover, predictions for ‘Oils, vegetable, deodorizer distillates’ (based on the main constituents) from the Toxtree (v.1.6) model revealed no evidence for mutagenic activity.

Overall, the above evidence suggests that ‘Oils, vegetable, deodorizer distillates’ does not have a mutagenic potential.

Justification for classification or non-classification

The mutagenic potential of ‘Oils, vegetable, deodorizer distillates’ can be deduced based on information available for its individual constituents. Studies conducted with glycerides, fatty acids, tocopherols and sterols, either in vitro or in vivo, indicated that these substances are not mutagenic. Moreover, predictions from the Toxtree (v.1.6) model revealed no evidence for mutagenic activity of any of the constituents.

These substances all have a long history of safe use in nutritional, cosmetic and/or industrial applications. Some (e.g. certain fatty acids and tocopherols) are listed as GRAS and/or demonstrate antitumourigenic activity under certain conditions (e.g. tocopherols and squalene; see Section 5.8 of the CSR).

Based on the above information, the substance does not warrant any classification according to CLP Regulation EC/1272/2008.