Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ can be deduced based on information available for its major constituents. Studies conducted on glycerides, fatty acids and unsaponifiable matters (including tocopherols, sterols, squalene and hydrocarbons) either in vitro or in vivo, indicated that these substances are not genotoxic. 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).

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 gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

Modified Ames assay

GLP compliance:

not specified

Type of assay:

other: Modified Ames assay

Target gene:

No data

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with

Vehicle / solvent:

DMSO

Evaluation criteria:

A Mutagenicity Index (MI), a ranking for relative mutagenic potency, was determined the assay. The MI is the slope of the dose response for mutagenesis. The assay was judged to be positive if the Mutagenicity Index was greater than 1.

Statistics:

No data

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

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

Roy described the mutagenicity results for a range of petroleum-derived materials, 28 of which were lubricating oil base stocks. A Mutagenicity Index (MI) was determined for each test substance and this was compared to the PAC content and to a carcinogenicity index that had also been determined for each material. The results for the technical white oil were as follows. 

Sample	MI*	%PAC**	%T***	%T/LP****
Technical white oil	0	0.3	0	0
* MI denotes Mutagenicity index.
** %PAC is weight% of 3-7 ring PNAs in the oil.
*** %T is the percentage of mice with tumors in skin carcinogenicity  studies reported elsewhere.
**** %T/LP is the percentage of mice with tumors multiplied by the reciprocal of the latency period. The author  describes this as a carcinogenic potency index.

Conclusions:

Under the study conditions, the substance was considered to be non-mutagenic in an in vitro modified Ames test.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance techincal white oil using the modified Ames test according to the Blackbum et al (1984, 1986) protocol. Salmonella typhimurium strains TA98 in the presence of metabolic activation (Hamster liver induced S9-Aroclor 1254) was treated with the DMSO extract of the test substance using range of doses that provided a definition of the response. A tangent to the dose-response curve at zero dose (Mutagenicity Index) was computed (Blackburn el al., 1986) and used as a measure of mutagenic potency. The assay was judged to be positive if the Mutagenicity Index was greater than 1. In the study, the Mutagenicity Index was 0 for the technical white oil. Under the study conditions, the test substance was considered to be non-mutagenic in the modified Ames test, with metabolic activation (Roy, 1988).

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Principles of method if other than guideline:

Each of the bacterial strains has a mutation that prevents it from making an amino acid (a protein building block) required for normal cell growth. The S. typhimurium strains are unable to manufacture histidine; the E. coli strain cannot manufacture tryptophan. The tester strains will not be able to grow without amino acid supplementation, unless the mutated gene is changed back to the correct DNA sequence (reversion mutation). Being exposed to a mutagenic chemical increases the chance that the mutant gene will be restored to the correct sequence, which allows the bacteria to grow and form colonies. Each substance under study is tested in a variety of bacterial strains with different types of altered DNA sequences in order to provide a comprehensive assessment of the mutagenic potential of the substance.

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver

Test concentrations with justification for top dose:

1, 3.3, 10, 33, 100, 333, 1000 (TA 98, TA 100)
10, 33, 100, 333, 1000, 3333 (TA 35, TA97)

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

not specified

Details on test system and experimental conditions:

Positive controls
0.4 ug/Plate 2-Aminoanthracene (TA 100, TA 35, TA 98, +S9)
0.5 ug/Plate Sodium Azide (TA 100, TA 35, -S9)
0.75 ug/Plate 2-Aminoanthracene (TA 100, TA 97, +S9)
1.0 ug/Plate 2-Aminoanthracene (TA 100, TA 35, TA 98, +S9)
2.0 ug/Plate 2-Aminoanthracene (TA 100, TA 35, TA 97, +S9)
24.0 ug/Plate 9-Aminoacridine (TA 97, -S9)
1.0 ug/Plate 4-Nitro-O-Phenylenediamine (TA 98, -S9)

Evaluation criteria:

To validate the test, a trained investigator compared the number of colonies on the chemical-treated plates to the number of colonies on the negative control plates. If the substance under test is mutagenic, the chemical-treated plates will have a much greater number of colonies than the negative control plates.

A positive response is a reproducible, dose-related increase in mutant colonies in any single strain, with or without the addition of S9 metabolic enzymes. While there is no minimum percentage of increase required for a result to be considered positive, a twofold increase in mutant colonies in a treated plate is usually considered to be a positive (mutagenic) response.
An equivocal response is any increase that is not reproducible, not dose-related, or not high enough in magnitude to be considered positive.
A negative response occurs when no increases in mutant colonies are seen in the cultures treated with the test chemical, compared with the control.

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, but tested up to precipitating concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

For detailed results, kindly refer the attached background material section of the IUCLID.

Conclusions:

Under the study conditions, the substance was considered to be non-mutagenic in an in vitro Ames test.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance squalene using bacterial reverse mutation assay (Ames test) according to the National Toxicology Program protocol. Salmonella typhimurium strains TA1535, TA97, TA98 and TA100 were treated with the test substance at eight dose levels (i.e., 1, 3.3, 10, 33, 100, 333, 1000, 3333 μg/plate), in triplicate, both with and without the addition of a rat and hamster liver homogenate metabolizing system (10% and 30% liver S9 in standard co-factors). The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Test substance precipitate was observed on the plates at ≥33 μg/plate of the doses tested in either the presence or absence of S9 -mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9 -mix). Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (NTP, 1990).

Reference 11

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

Micronucleus preparation was performed according to the procedures of Fenech (2000) and Palus et al. (2003). Whole blood (0.2 mL) was added to 2.5 mL of Chromosome Medium B. The peripheral lymphocytes were incubated at 37 deg C for 72 h and exposed to squalene at 1250, 2500, 5000, 10,000 and 20,000 µg/mL concentrations during the last 48 h. 44 hfrom the initiation, cytochalasin B was added to block cytokinesis.

References:
Fenech, M., 2000. The in vitro micronucleus technique. Mutat. Res. 455, 81–95.

Palus, J., Rydzynski, K., Dziubaltowska, E., Wyszynska, K., Natarajan, A.T., Nilsson, R., 2003. Genotoxic effects of occupational exposure to lead and cadmium. Mutat. Res. 540, 19–28.

GLP compliance:

not specified

Remarks:

Research study

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human peripheral

Details on mammalian cell type (if applicable):

Peripheral blood was obtained from two healthy (1 male and 1 female)non-smoking donors (of ages 24–26 years), they had no medication within the prior 3 weeks and no radiological examination within the prior 3 months. Whole blood (0.2 mL) was added to 2.5 mL Chromosome Medium Bsupplemented with 10 µg/mL bromodeoxyuridine.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

The cells were treated with 1250, 2500, 5000, 10,000, 20,000 ug/mL concentrations of squalene for 24 h and 48 h.

Untreated negative controls:

yes

Remarks:

Physiological saline

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

The cultures were incubated at 37 deg C for 72 h.

Evaluation criteria:

Totally two thousands binucleated cells (1000 cells per donor) for each treatment of squalene were examined blindly, following the scoring criteria adopted
by the Human Micronucleus Project (Bonassi et al., 2001 ). Five hundred cells per donor (totally 1000 cells) were scored to evaluate the nuclear division index
(NDI). NDI was calculated using the following formula: [(1 N1) +(2 N2) +(3 (N3 + N4))]/N; where N1 to N4 represent the number of cells with one to four nuclei and N is the total number of viable cells scored (Surrales et al., 1995 ).

Statistics:

For data evaluation, the z-test was used for the percentage of abnormal cells, CA/cell, MI, RI, NDI, MN assays. The t-test was applied for SCEs and comet assay results to determine the statistical difference between treated and untreated samples. Dose–response relationships were determined from the correlation and regression coefficients for the percentage of abnormal cells, CA/cell, SCE, mean MN and DNA.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Squalene did not affect the frequency of binucleate cells with micronucleus in all treatment groups compared to the negative control. Squalene also decreased the nuclear division index (NDI) insignificantly.

For result tables, kindly refer to the attached background material section of the IUCLID.

Conclusions:

Under the test conditions, the substance was considered to be non-genotoxic to human lymphocytes in an in vitro mammalian cell micronucleus test.

Executive summary:

A study was conducted to evaluate the genotoxic potential of test substance, squalene in human lymphocytes using an in vitro mammalian cell micronucleus test. Micronucleus preparation was performed according to the procedures of Fenech (2000) and Palus et al. (2003). The human lymphocytes cells were treated with 1250, 2500, 5000, 10,000, 20,000 µg/mL concentrations of squalene for 24 h and 48 h without metabolic activation. Totally two thousand binucleated cells (1000 cells per donor) for each treatment of squalene were examined blindly, following the scoring criteria adopted by the Human Micronucleus Project (Bonassi et al., 2001). Five hundred cells per donor (totally 1000 cells) were scored to evaluate the nuclear division index (NDI). NDI was calculated using the following formula: [(1 N1) +(2 N2) +(3 (N3 + N4))]/N; where N1 to N4 represent the number of cells with one to four nuclei and N is the total number of viable cells scored (Surrales et al., 1995). Squalene did not affect the frequency of binucleate cells with micronucleus in all treatment groups compared to the negative control. Squalene also decreased the nuclear division index (NDI) insignificantly. Under the study conditions, the test substance was considered to be non-genotoxic to human lymphocytes in an in vitro mammalian cell micronucleus test (Yüzbaşıoğlu, 2013).

Reference 12

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

The methods of Evans (1984) and Perry and Thompson (1984) were followed in CA tests with minor modifications (Yüzbasıog˘ lu et al., 2006 ).

References:
Evans, H.J., 1984. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. In: Kilbey, B.J., Legator, M., Nichols, W., Ramel, C. (Eds.), Handbook of Mutagenicity Test Procedures. Elsevier Science Publishers, Amsterdam, pp. 405–427

Perry, P.E., Thompson, E.J., 1984. The methodology of sister chromatid exchanges. In: Kilbey, B.J., Legator, M., Nichols, W., Ramel, C. (Eds.),Handbook of
Mutagenicity Test Procedures, second ed. Elsevier Science Publishers, BV, Amsterdam, pp. 495–529.

Yüzbasıog˘lu, D., Çelik, M., Yılmaz,S., Ünal,F., Aksoy, H., 2006. Clastogenicity of the fungicide afugan in cultured human lymphocytes. Mutat. Res. 604, 53–59.

GLP compliance:

not specified

Remarks:

Research study

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human peripheral

Details on mammalian cell type (if applicable):

Peripheral blood was obtained from two healthy (1 male and 1 female)non-smoking donors (of ages 24–26 years), they had no medication within the prior 3 weeks and no radiological examination within the prior 3 months. Whole blood (0.2 mL) was added to 2.5 mL Chromosome Medium Bsupplemented with 10 µg/mL bromodeoxyuridine.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

The cells were treated with 1250, 2500, 5000, 10,000, 20,000 ug/mL concentrations of squalene for 24 h and 48 h.

Untreated negative controls:

yes

Remarks:

Physiological saline

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

The cultures were incubated at 37 deg C for 72 h.

Evaluation criteria:

To evaluate the chromosomal aberrations, 200 well spread metaphases (100 metaphases from each donor) were scored blindly, for each treatment. The mean frequency of abnormal cells and the number of CAs per cell (CAs/cell) were calculated. The mitotic index (MI) was determined by scoring of 1000 cells from each donor.

Statistics:

For data evaluation, the z-test was used for the percentage of abnormal cells, CA/cell, MI, RI, NDI, MN assays. The t-test was applied for SCEs and comet assay results to determine the statistical difference between treated and untreated samples. Dose–response relationships were determined from the correlation and regression coefficients for the percentage of abnormal cells, CA/cell, SCE, mean MN and DNA.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

At 24 h treatment, while 1250, 2500, 10,000 and 20,000 µg/mL concentrations reduced the frequency of abnormal cell, 5000 µg/mL concentration increased this frequency. However, neither of them was significant. Similarly, squalene decrease d the frequency of CA/cell compared to negative control but these reductions were not significant. At 48 h treatment, squalene increased the frequency of aberrations and CA/cell but neither of them were significant compared to control. Squalene did not affect MI significantly at all concentrations and treatment periods (24 h and 48 h) compared to negative control.

For result tables, kindly refer to the attached background material section of the IUCLID.

Conclusions:

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

Executive summary:

A study was conducted to evaluate the genotoxic potential of test substance, squalene in human lymphocytes using an in vitro chromosomal aberration (CA) test. The methods of Evans (1984) and Perry and Thompson (1984) were followed in CA test with minor modifications (Yüzbasıog˘ lu et al., 2006). The human lymphocytes cells were treated with 1250, 2500, 5000, 10,000, 20,000 µg/mL concentrations of squalene for 24 h and 48 h without metabolic activation. To evaluate the chromosomal aberrations, 200 well spread metaphases (100 metaphases from each donor) were scored blindly, for each treatment. The mean frequency of abnormal cells and the number of CAs per cell (CAs/cell) were calculated. The mitotic index (MI) was determined by scoring of 1000 cells from each donor. At 24 h treatment, while 1250, 2500, 10,000 and 20,000 µg/mL concentrations reduced the frequency of abnormal cell, 5000 µg/mL concentration increased this frequency. However, neither of them was significant. Similarly, squalene decrease d the frequency of CA/cell compared to negative control but these reductions were not significant. At 48 h treatment, squalene increased the frequency of aberrations and CA/cell but neither of them were significant compared to control. Squalene did not affect MI significantly at all concentrations and treatment periods (24 h and 48 h) compared to negative control. Under the study conditions, the test substance was considered to be non-genotoxic to human lymphocytes in an in vitro mammalian chromosome aberration test (Yüzbaşıoğlu, 2013).

Reference 13

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

The methods of Evans (1984) and Perry and Thompson (1984) were followed in SCE tests with minor modifications (Yüzbasıog˘ lu et al., 2006 ). For the SCE assay, the slides were stained with Giemsa, according to Speit and Houpter’s (1985) method, with some modifications (Yüzbasıog˘ lu et al., 2006 )

References:
Evans, H.J., 1984. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. In: Kilbey, B.J., Legator, M., Nichols, W., Ramel, C. (Eds.), Handbook of Mutagenicity Test Procedures. Elsevier Science Publishers, Amsterdam, pp. 405–427

Perry, P.E., Thompson, E.J., 1984. The methodology of sister chromatid exchanges. In: Kilbey, B.J., Legator, M., Nichols, W., Ramel, C. (Eds.),Handbook of
Mutagenicity Test Procedures, second ed. Elsevier Science Publishers, BV, Amsterdam, pp. 495–529.

Speit, G., Houpter, S., 1985. On the mechanisms of differential giemsa staining of bromodeoxyuridine-substituted chromosomes. II. Differences between the
demonstrations of sister chromatid differentiation and replication patterns. Human. Genet. 70, 126–129.

Yüzbasıog˘lu, D., Çelik, M., Yılmaz,S., Ünal,F., Aksoy, H., 2006. Clastogenicity of the fungicide afugan in cultured human lymphocytes. Mutat. Res. 604, 53–59.

GLP compliance:

not specified

Remarks:

Research study

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human peripheral

Details on mammalian cell type (if applicable):

Peripheral blood was obtained from two healthy (1 male and 1 female)non-smoking donors (of ages 24–26 years), they had no medication within the prior 3 weeks and no radiological examination within the prior 3 months. Whole blood (0.2 mL) was added to 2.5 mL Chromosome Medium Bsupplemented with 10 µg/mL bromodeoxyuridine.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

The cells were treated with 1250, 2500, 5000, 10,000, 20,000 ug/mL concentrations of squalene for 24 h and 48 h.

Untreated negative controls:

yes

Remarks:

Physiological saline

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

The cultures were incubated at 37 deg C for 72 h.

Evaluation criteria:

In order to score SCEs, 25 second-division metaphases (M2) were analyzed blindly for each donor. In addition to SCEs, cells were analyzed for the relative frequency of firstdivision metaphases (M1),second-division metaphases (M2),and third and subsequent division metaphases (M3). Replication index (RI) is the average number of replications completed metaphase cells and is calculated as follows: [(1 M1) +(2 M2) +(3 M3)]/N (N = number of observed cells)(Schneider and Lewis, 1981 ).

Statistics:

For data evaluation, the z-test was used for the percentage of abnormal cells, CA/cell, MI, RI, NDI, MN assays. The t-test was applied for SCEs and comet assay results to determine the statistical difference between treated and untreated samples. Dose–response relationships were determined from the correlation and regression coefficients for the percentage of abnormal cells, CA/cell, SCE, mean MN and DNA.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Squalene significantly increased SCE/cell at all concentrations (except 10,000 µg/mL) at 24 h treatment compared to negative control. However, this increase was poorly concentration-dependent. At 48 h treatment, however, squalene did not increase SCE/cell compared to control. 10,000 µg/mL concentration induced SCE/cell just the same amount to control group. SCE/cell in all the other concentrations was lower than that of negative control group. Other side, squalene did not affect RI significantly at all concentrations and treatment periods (24 h and 48 h) compared to negative control.

For result tables, kindly refer to the attached background material section of the IUCLID.

Conclusions:

Under the test conditions, the substance was considered to be non-genotoxic to human lymphocytes in an in vitro sister chromatid exchange assay in mammalian cells.

Executive summary:

A study was conducted to evaluate the genotoxic potential of test substance, squalene in human lymphocytes using an in vitro sister chromatid exchange (SCE) assay in mammalian cells. The methods of Evans (1984) and Perry and Thompson (1984) were followed in SCE tests with minor modifications (Yüzbasıog˘ lu et al., 2006). For the SCE assay, the slides were stained with Giemsa, according to Speit and Houpter’s (1985) method, with some modifications (Yüzbasıoglu et al., 2006). The human lymphocytes cells were treated with 1250, 2500, 5000, 10,000, 20,000 µg/mL concentrations of squalene for 24 h and 48 h without metabolic activation. In order to score SCEs, 25 second-division metaphases (M2) were analyzed blindly for each donor. In addition to SCEs, cells were analyzed for the relative frequency of first division metaphases (M1), second-division metaphases (M2) and third and subsequent division metaphases (M3). Replication index (RI) is the average number of replications completed metaphase cells and was calculated. Squalene significantly increased SCE/cell at all concentrations (except 10,000 µg/mL) at 24 h treatment compared to negative control. However, this increase was poorly concentration-dependent. At 48 h treatment, however, squalene did not increase SCE/cell compared to control. 10,000 µg/mL concentration induced SCE/cell just the same amount to control group. SCE/cell in all the other concentrations was lower than that of negative control group. Other side, squalene did not affect RI significantly at all concentrations and treatment periods (24 h and 48 h) compared to negative control. Under the study conditions, the test substance was considered to be non-genotoxic to human lymphocytes in an in vitro sister chromatid exchange assay in mammalian cells (Yüzbaşıoğlu, 2013).

Reference 14

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study the alkaline version of the comet assay was performed according to Singh et al. (1988) with a slight modification(Mamur et al., 2010 ).

References:
Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. Asimple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191

Mamur, S., Yüzbasıog˘lu, D., Ünal,F., Aksoy, H., 2012. Genotoxicity of food preservative sodium sorbate in human lymphocytes in vitro . Cytotechnology 64 (5), 553–562.

GLP compliance:

not specified

Remarks:

Research study

Type of assay:

comet assay

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human peripheral

Details on mammalian cell type (if applicable):

Peripheral blood was obtained from two healthy (1 male and 1 female)non-smoking donors (of ages 24–26 years), they had no medication within the prior 3 weeks and no radiological examination within the prior 3 months.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

1250, 2500, 5000, 10,000, 20,000 ug/mL

Untreated negative controls:

yes

Remarks:

Physiological saline

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 100mM H2O2

Details on test system and experimental conditions:

Lymphocytes were isolated using Biocoll separating solution. To detect the viability of cells, trypan blue exclusion test was used. Cell viability was >98%. The slides were incubated for 20 min ice-cold electrophoresis solution (0.3M NaOH, 1mM EDTA, pH >13), followed by electrophoresis at 25 V, 300 mA for 20 min. Each slide was stained with 50 µL of 20 µg/mL ethidium bromide. The slides were examined using a fluorescent microscope (Olympus)equipped with an excitation filter of 546 nm and a barrier filter of 590 nm at 400 magnification.

Evaluation criteria:

The tail lenght (µm) and tail intensity (%)of 100 comets on each slide (atotal of 200 comets per concentration) were examined, using specialized Image Analysis System (‘‘Comet Assay IV’’, Perceptive Instruments Ltd., UK).

Statistics:

For data evaluation, the z-test was used for the percentage of abnormal cells, CA/cell, MI, RI, NDI, MN assays. The t-test was applied for SCEs and comet assay results to determine the statistical difference between treated and untreated samples. Dose–response relationships were determined from the correlation and regression coefficients for the percentage of abnormal cells, CA/cell, SCE, mean MN and DNA.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Squalene decreased comet tail length (CTL) at 1250 and 2500 µg/mL concentrations but this decrease was significant at only 2500 µg/mL concentration compared to negative control. Squalene increased the CTL at the other three concentrations in isolated lymphocytes but this increase was not statistically significant. Also, squalene increased the comet tail intensity (CTI) at 1250, 2500, 5000 and 20,000 µg/mL concentrations but decreased the CTI at 10,000 µg/mL concentration. However, neither of them was statistically significant.

For result tables, kindly refer to the attached background material section of the IUCLID.

Conclusions:

Under the study conditions, the test substance was considered to be non-genotoxic to human lymphocytes in an in vitro alkaline comet assay in mammalian cells.

Executive summary:

A study was conducted to evaluate the genotoxic potential of test substance, squalene in human lymphocytes using an in vitro alkaline comet assay in mammalian cells. The alkaline comet assay was performed according to Singh et al. (1988) with a slight modification (Mamur et al., 2010). The human lymphocytes cells were treated with 1250, 2500, 5000, 10,000, 20,000 µg/mL concentrations of squalene for 1 h at 37 deg C without metabolic activation. The slides were incubated for 20 min ice-cold electrophoresis solution, followed by electrophoresis at 25 V, 300 mA for 20 min. Each slide was stained with 50 µL of 20 µg/mL ethidium bromide. The slides were examined using a fluorescent microscope equipped with an excitation filter of 546 nm and a barrier filter of 590 nm at 400 magnification. The tail lenght (µm) and tail intensity (%) of 100 comets on each slide (a total of 200 comets per concentration) were examined, using specialized Image Analysis System. Squalene decreased comet tail length (CTL) at 1250 and 2500 µg/mL concentrations but this decrease was significant at only 2500 µg/mL concentration compared to negative control. Squalene increased the CTL at the other three concentrations in isolated lymphocytes but this increase was not statistically significant. Also, squalene increased the comet tail intensity (CTI) at 1250, 2500, 5000 and 20,000 µg/mL concentrations but decreased the CTI at 10,000 µg/mL concentration. However, neither of them was statistically significant. Under the study conditions, the test substance was considered to be non-genotoxic to human lymphocytes in an in vitro alkaline comet assay in mammalian cells (Yüzbaşıoğlu, 2013).

Reference 15

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 16

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 17

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 18

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 19

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 20

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:

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 the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ can be deduced based on information available for its major constituents. Studies conducted on glycerides, fatty acids and unsaponifiable matter (including tocopherols, sterols, squalene and hydrocarbons) either in vitro or in vivo, indicated that these substances are not genotoxic. 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).)

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:

mammalian bone marrow chromosome aberration test

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not applicable

Principles of method if other than guideline:

The in vivo mutagenicity of test substance was evaluated by the mouse micronucleus assay. Groups of four male and four female mice per time period were dosed intraperitoneally with 25 mL/kg test substance given as a 10% solution in corn oil. Mice were killed at 48 and 72 h after dosing. The numbers of micronuclei per 1000 erythrocytes were counted.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Route of administration:

intraperitoneal

Vehicle:

Corn oil

Details on exposure:

No data

Duration of treatment / exposure:

2 d

Frequency of treatment:

Daily

Post exposure period:

48 and 72 h

Remarks:

Doses / Concentrations: 25 mL/kg as 10% solution in corn oil
Basis: nominal conc.

No. of animals per sex per dose:

Four animal each sex

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:

The numbers of micronuclei per 1000 erythrocytes were counted.

Statistics:

No data

Key result

Sex:

male/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:

Test substance did not produce any clastogenic activity at either time period.

None

Conclusions:

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

Executive summary:

A study was conducted to determine the genotoxicity potential of test substance using in vivo mammalian micronucleus assay. Groups of four male and four female mice per time period were dosed intraperitoneally with 25 mL/kg test substance given as a 10% solution in corn oil. Mice were killed at 48 and 72 h after dosing. The numbers of micronuclei per 1000 erythrocytes were counted. Test substance did not produce any clastogenic activity at either time period. Under the test conditions, the substance was not considered to be genotoxic (Mullin, 1990).

Reference 4

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Deviations:

not applicable

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Route of administration:

inhalation

Details on exposure:

No data

Duration of treatment / exposure:

6 h per day

Frequency of treatment:

Five days consecutively

Post exposure period:

Starting on the last treatment day (at least 2 h after exposure), males were cohoused with untreated virgin Sprague-Dawley rats (1 male: 2 females) for 7 days. The mating procedure continued for six consecutive weeks. Females were removed from the male rats and sacrificed 18 days after cohousing (Days 11-18 of gestation).

Remarks:

Doses / Concentrations: 0, 300 or 900 ppm vapor
Basis: nominal conc.

No. of animals per sex per dose:

10 male per dose and 20 females per dose

Control animals:

not specified

Positive control(s):

No data

Tissues and cell types examined:

Females were examined for implantation sites, early and late resorptions and viable fetuses.

Details of tissue and slide preparation:

At the end of the study, males were sacrificed and the seminal vesicles, epididymides, prostate and testes were preserved.

Statistics:

No data

Key result

Sex:

male/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:

There were no differences from controls that were considered to be treatment-related in the pregnancy rate, number of implantations or numbers of early or late fetal deaths.

None

Conclusions:

Under the study conditions, the test substance was not considered to be mutagenic by the dominant lethal test.

Executive summary:

A study was conducted to determine the germ cell mutagenicity potential of the test substance using in vivo rodent dominant lethal test. Groups of 10 each, proved fertile male Sprague-Dawley rats were exposed to 0, 300 or 900 ppm test substance vapor for 6 h per day for five consecutive days. Starting on the last treatment day (at least 2 h after exposure), males were cohoused with untreated virgin Sprague-Dawley rats (1 male: 2 females) for 7 days. The mating procedure continued for six consecutive weeks. Females were removed from the male rats and sacrificed 18 days after cohousing (Days 11-18 of gestation). Females were examined for implantation sites, early and late resorptions and viable fetuses. At the end of the study, males were sacrificed and the seminal vesicles, epididymides, prostate and testes were preserved. There were no differences from controls that were considered to be treatment-related in the pregnancy rate, number of implantations or numbers of early or late fetal deaths. Under the study conditions, the test substance was not considered to be mutagenic by the dominant lethal test (Mullin, 1990).

Reference 5

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Comet assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

The in vivo DNA damaging potential of squalene was evaluated in rats following sub-cutaneous injection and quantification of tail length and tail intensity.

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Sex:

not specified

Details on test animals or test system and environmental conditions:

Wistar rats (10–12weeks old) were procured from RefikSaydam National Public Health Agency, Experimental Animal Unit (Ankara, Turkey).Rats were kept in separate cages in an experimental room under controlled conditions of temperature (22±2 C) and humidity (50– 60%) with feed and water being available ad libitum. Lighting was controlled to provide 12 h artificial light followed by 12 h darkness.

Route of administration:

subcutaneous

Vehicle:

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

Duration of treatment / exposure:

Group 1 was the treatment group studied 1day after the squalene injection.
Group 2 was studied 14 days after squalene injection

Frequency of treatment:

Single

Post exposure period:

1 and 14 days

Remarks:

Doses / Concentrations: 0, 0.07, 0.14, 0.28, 0.56, 1.12 mg/kg bw
Basis: nominal concentration

No. of animals per sex per dose:

5 animals per dose

Control animals:

yes, concurrent no treatment

Positive control(s):

yes, with mitomycin-C 2 mg/Kg

Evaluation criteria:

Quantification of DNA breakage was realized using Comet Image Analysis System (‘‘Comet Assay IV’’, Perceptive Instruments Ltd., UK).At least 300 comets for each experimental group were recorded as tail length and tail intensity.

Statistics:

For data evaluation, the z-test was used for the percentage of abnormal cells, CA/cell, MI, RI, NDI, MN assays. The t-test was applied for SCEs and comet assay results to determine the statistical difference between treated and untreated samples. Dose–response relationships were determined from the correlation and regression coefficients for the percentage of abnormal cells, CA/cell, SCE, mean MN and DNA.

Key result

Sex:

not specified

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DNA damage was seen in lymphocytes of rats one day after injection of squalene in in vivo (Group 1). Squalene significantly increased Comet Tail Length (CTL) at 0.14, 0.28, 0.56 mg/kg doses however it significantly decreased CTL at 0.07 and 1.12 mg/kg doses. Whereas, squalene decreased comet tail intensity (CTI) at all doses (except 0.28 mg/kg), however, this decrease was significant only at 0.07 and 1.12 mg/kg doses compared to control. In contrast to the results obtained for group 1, in group 2, squalene significantly increased both CTL and CTI (except 0.07 mg/kg) at all doses used but 1.12 mg/kg.

For result tables, kindly refer to the attached background material section of the IUCLID.

Conclusions:

Under the test conditions, the substance was considered to be genotoxic in an in vivo comet assay in rat.

Executive summary:

A study was performed to investigate the genotoxic potential of the test substance, squalene using in vivo mammalian comet assay in rat. Five dose levels of squalene, determined based on the amount of squalene in human vaccines (0.07 mg/kg, 0.14 mg/kg, 0.28 mg/kg, 0.56 mg/kg, 1.12 mg/kg) were given to rats subcutaneously for each treatment groups (5 animals/groups). In addition, an untreated control and a positive control (mitomycin-C,2mg/kg) were also used to test the validity of the assay for all treatment groups. Rat blood samples taken from two different groups were used in in vivo Comet assay. Group 1 was the treatment group studied 1 day after the squalene injection. Group 2 was studied 14 days after squalene injection. For in vivo comet assay, approximately 100 µL whole blood was collected from rat’s tail vein into lithium–heparin tubes. Due to the intensity of cells, blood samples were diluted and suspended with phosphate buffer (pH:7.4) in 1:1 ratio and then centrifuged. Lymphocytes were isolated by Biocoll separating solution. After the isolation step, lymphocytes were resuspended in PBS (phosphate buffered saline). Afterwards, the protocol for in vitro comet assay was applied. Quantification of DNA breakage was realized using Comet Image Analysis System. At least 300 comets for each experimental group were recorded as tail length and tail intensity. DNA damage was seen in lymphocytes of rats one day after injection of squalene in in vivo (Group 1). Squalene significantly increased Comet Tail Length (CTL) at 0.14, 0.28, 0.56 mg/kg doses however it significantly decreased CTL at 0.07 and 1.12 mg/kg doses. Whereas, squalene decreased comet tail intensity (CTI) at all doses (except 0.28 mg/kg), however, this decrease was significant only at 0.07 and 1.12 mg/kg doses compared to control. In contrast to the results obtained for group 1, in group 2, squalene significantly increased both CTL and CTI (except 0.07 mg/kg) at all doses used but 1.12 mg/kg. Under the test conditions, the substance was considered to be genotoxic in an in vivo comet assay in rat (Yüzbaşıoğlu, 2013).

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

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 7

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 8

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 9

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:

mammalian erythrocyte micronucleus test

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 absence of a genotoxicity study with the test substance, ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ the endpoint has been assessed based on studies for substances representative of the main constituents, which can be categorised as glycerides, fatty acids or fatty acid methyl esters (which will eventually hydrolyse to fatty acids (Mattson and Volpenhein, 1972)) and unsaponifiable matter (including tocopherols, sterols, squalene and hydrocarbons). Asa large number of studies have been conducted on the individual constituents, particularly in the context of nutritional research, for practical reasons, only a limited number of studies are reported below:

vitro data

Glycerides:

• 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).

Fatty acids:

• 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 demonstrate anticlastogenic effects in the chromosome aberration test (HERA, 2002).

Unsaponifiable matter:

• Tests in bacterial and human cells in vitro suggest thattocopherolsare not mutagenic and may in some cases have mutagenicity-inhibiting effects (Tomassi and Silano, 1986; JECFA, 1987).
• Phytosterols and their esterswere 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).
• Squalene was found to be non-genotoxic in an in vitro Ames test (NTP, 1990) as well as in chromosomal aberration, sister chromatid exchange and comet assays conducted with human peripheral lymphocytes (Yüzbaşıoğlu, 2013).
• Results from in vitro Ames, chromosomal aberration and mouse lymphoma assays with C10-3 or C11-13 isoparaffins, white mineral oil and/or C14-C20 aliphatic (<2% aromatic) hydrocarbon solvents, suggest that hydrocarbons are not genotoxic (CIR, 2012; Mckee, 2015; Mullin, 1990; Roy, 1990; EFSA, 2012).

In vivo data

Glycerides:

• 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).

Fatty acids:

• No in vivo mutagenicity data was located. However, there is no association between the normal intake of large amounts of fatty acids in the diet and mutagenicity (HERA, 2002[1]).

Unsaponifiable matter:

• Tocopherols, phytosterolsand their esters were found to be negative in a battery of in vivo genotoxicity tests (Fiume, 2002; ANZFA, 2001; Tice, 1997).
• Squaleneinduced DNA damage in in vivo comet assay in rats, however in other genotoxicity tests squalene was tested negative (Yüzbaşıoğlu, 2013).
• Hydrocarbons were negative in an in vivo genotoxicity tests (CIR, 2012; Mullin, 1990).

Overall, the above evidence suggests that the test substance, ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ does not have a genotoxicity potential.

[1]Human & Environmental Risk Assessment on ingredients of European household cleaning products (HERA). Fatty Acid Salts. June, 2002

Justification for classification or non-classification

Based on the available weight of evidence information on the constituents, the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ does not warrant classification for genotoxicity according to EU CLP criteria (EC 1272/2008).