Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized, reaction products with ethylene glycol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of the substance was examined in an Ames test (OECD 471) and in an HPRT test (OECD 476). Both assays were negative.

No in vitro cytogenicity study with the substance is available, but an in vitro micronucleus study is  available on a structural homolog. No signs of mutagenicity are observed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals, July 22, 2010, Guideline No. 487 “In vitro Mammalian Cell Micronucleus Test".

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Stocks of the V79 cells (obtained from LMP; Technical University Darmstadt, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank. Before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

See "any other information on materials and methods"

Vehicle / solvent:

The final concentration of DMSO in the culture medium was 0.5 % (v/v). The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Griseofulvin

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

DOSE-SELECTION:
With respect to the solubility of the test item, 4000.0 µg/mL of Soybean oil, epoxidized, reaction products with methanol was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 7.8 and 4000.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. No precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of S9 mix, this preliminary test was designated Experiment IA. The experimental part without S9 mix was repeated with the same top concentration due to the low response of the positive control MMC and designated Experiment IB. Since no clear cytotoxicity and test item precipitation was observed in Experiment IA and IB, the same concentrations were applied in Experiment II.

EXPERIMENTAL PERFORMANCE:
- Culture Medium and Conditions: For seeding and treatment of the cell cultures the culture medium was MEM (minimal essential medium) containing Hank’s salts, glutamine, Hepes (25 mM) and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL). All cultures were incubated at 37°C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- Seeding of the Cultures: Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37°C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes, which contained microscopic slides. The cells were seeded into Quadriperm dishes, which contained microscopic slides. Into each chamber 1.0 x 10^5 – 1.5 x 10^5 cells were seeded with regard to the preparation time.

TREATMENT:
- Exposure period 4 hours: The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium was added. Concurrent solvent and positive controls were performed. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. Then the cells were cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.
- Exposure period 24 hours: The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item without S9 mix. The medium was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.

PREPARATION OF THE CULTURES:
For the micronucleus analysis, 24 hours after the start of the exposure, the cells were treated on the slides in the chambers of the quadriperm dishes with deionised water for 1 to 1.5 min at 37°C. Afterwards the cells were fixed twice with a solution containing 3 parts ethanol, 1 part acetic acid and 1.25 % (v/v) formaldehyde. After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.

ANALYSIS OF MICRONUCLEI AND CYTOTOXICITY:
Evaluation of the cultures was performed manually using NIKON microscopes with 40x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). Briefly the micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 1000 cells in two parallel cultures were scored for micronuclei, so that at least 2000 cells from clones with 2-8 cells were analysed per test group. The frequency of micronucleated cells was reported as % micronucleated cells. Cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3-4 cells (c4), and 5-8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represented the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated. Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee for a sufficient cell proliferation during treatment and recovery.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY:
The micronucleus assay is considered acceptable if it meets the following criteria:
a) The number of micronucleated cells carrying one or more micronuclei found in the solvent controls falls within the range of the historical laboratory control data range.
b) The positive control substances produce a significant increase (at least two-fold the respective control value) in the number of micronucleated cells exceeding the historical laboratory solvent control data range.

EVALUATION OF RESULTS:
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical control data and
- either a statistically significant concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.
A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated test groups is in the range of the historical control data and
- no statistically significant concentration-related increase in the number of micronucleated cells is observed.

Statistics:

Statistical significance was confirmed by means of the Chi square test

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment IA, IB and II in the presence of S9 mix at 125.0 µL/mL and above. In Experiment II in the absence of S9 mix phase separation was observed at 250.0 µg/mL and above. No relevant influence on pH value was observed. The osmolarity was slightly increased at the highest applied concentration in Experiment II. In the absence and presence of metabolic activation no clear cytotoxicity measured as reduced proliferation index was observed. No mutagenicity was observed at the concentrations evaluated. In Experiment IA and IB in the absence and presence of S9 mix two statistically significant increases were observed after treatment with 1000 µg/mL (1.20 and 1.30 %). The values are in the range of the laboratory historical control data (0.15-1.50 % and 0.05-1.70 % micronucleated cells, respectively) and therefore biologically irrelevant. In Experiment II in the presence of S9 mix two statistically significant increases were observed after treatment with 62.5 and 2000.0 µg/mL (1.55 and 1.45 %). The values are in the range of the laboratory historical control data (0.05 - 1.70 % micronucleated cells) and therefore biologically irrelevant. In the presence of S9 mix one single value (1.60 %) exceeded the range of the laboratory historical control data (0.05-1.50 % micronucleated cells) after treatment with 1000.0 µg/mL. The value is not statistically significant and therefore biologically irrelevant. Mitomycin C (0.1 µg/mL), Griseofulvin (8.0 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mutation study in mammalian cells

Specific details on test material used for the study:

Yellow, clear liquid
Expiration date: November 21, 2015

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I (4 hours exposure):
without S9 mix: 0.6 - 1.3 - 2.5 - 5.0 - 10.0 µg/mL
with S9 mix: 5.0 - 10.0 - 20.0 - 40.0 - 80.0 µg/mL

Experiment II:
without S9 mix (24 hours exposure): 2.5 - 5.0 - 10.0 - 20.0 - 40.0 µg/mL
with S9 mix (4 hours exposure): 5.0 - 10.0 - 20.0 - 40.0 - 80.0 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Evaluation criteria:

- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
- A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concen-trations in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
- However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Phase separation or turbidity visible to the unaided eye, was noted at the highest evaluated concentration with metabolic activation.
- The dose range in the presence of metabolic activation was limited by the solubility of the test item in aqueous medium. Turbidity or phase separation was noted at 80.0 μg/mL in both main experiments with metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Relevant cytotoxic effects indicated by a relative cloning efficiency below 50% in both parallel cultures occurred in the first experiment at 10.0 μg/mL without metabolic activation and in experiment II at 40.0 μg/mL without metabolic activation.

CONTROL DATA
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.4 up to 15.6 mutants per 106 cells; the range of the groups treated with the test item was from 3.8 up to 29.1 mutants per 106 cells. EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The induction factor of three times the corresponding solvent control was not reached or exceeded at any test point. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. There was no significant dose dependent trend in any of the experimental parts.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005/11/24-2005/12/23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: C-SAT 050078; Sovermol 1102
- Physical state: liquid at room temperature
- Analytical purity: 100 % active ingredient
- Lot/batch No.: 1134003
- Expiration Date: 01.06.2007
- Other: solubility in vehicles: soluble in acetone, oil, DMSO

Target gene:

His

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of Phenobarbital/ß-naphtoflavone induced rats.

Test concentrations with justification for top dose:

Pre- Experiment/ Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate (with and without metabolic activation)
Experiment Il: 33; 100; 333; 1000; 2500; and 5000 µg/plate (with and without metabolic activation)

The test item precipitated at 5000 µg/plate in the overlay agar in both experiments with and without metabolic activation. The undissolved particles of the test item had no influence on the data recording.

Vehicle / solvent:

The test item was soluble in DMSO which is nontoxic to bacteria.

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See any other information on materials and methods incl. tables

Details on test system and experimental conditions:

EXPERIMENT 1
METHOD OF APPLICATION:
plate incorporation (experiment I)
DURATION:
48 hours at 37°C
NUMBER OF REPLICATIONS:
3
DETERMINATION OF CYTOTOXICITY:
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

EXPERIMENT 2
METHOD OF APPLICATION:
preincubation (experiment 2)
DURATION
- Preincubation period: 60 min. at 37°C
- Selection time: 48 hours at 37°C
NUMBER OF REPLICATIONS:
3
DETERMINATION OF CYTOTOXICITY
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

OTHER
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation of the test item the some plates were counted manually.

Evaluation criteria:

- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
At the maximal test item concentration of 5000 µg/plate a slight precipitation of the test item was observed. This precipitate did not increase toxicity or impair with the colony counting.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in ail strains used.
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation in experiment I. Minor toxic effects were observed in experiment Il in strain TA 100 without metabolic activation and in strains TA 1537 and TA 100 with metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
In experiment Il without metabolic activation, the data in the negative control of strain TA 100 were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genotoxicity data on the substance itself is available. Information is therefore derived from two in vivo micronucleus assays (OECD 474) with structurally related substances. Both substances are negative in this assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to chapter 13 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Read-across from CAS# 151661-88-0

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Read-across from Oleic methyl ester, epoxidized, reaction products with glycerol (OEG)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

An Ames test and an HPRT test are available for the substance itself. Further information is provided on reas-across substances. None of the tests gave any indication for a genotoxic potential. The studies are summarised below.

 

Ames test:

The potential of the test item to induce gene mutations was examined by means of two independent S. typhimurium reverse mutation assays (Ames test) according to OECD 471, adopted in 1997. The bacterial strains of TA 1535, TA 1537, TA 98, TA 100, and TA 102 were used. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment Il was performed as a pre-incubation assay. The test item was incubated in nominal concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/ plate in experiment I and 33; 100; 333; 1000; 2500; and 5000 µg/ plate in experiment II. The test was performed in triplicates. After an incubation period of 48 hours, revertant colonies were counted. Negative and positive controls were included. The plates incubated with the test item showed normal background growth up to 5000 µg/ plate, with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups, with and without S9 mix in experiment I. Minor toxic effects were observed in experiment Il in strain TA 100 without S9 mix and in strains TA 1537 and TA 100 with S9 mix. No increase in revertant colony numbers of any of the five tested strains was observed following treatment with the test item at any concentration and neither with or without S9 mix. In experiment Il without S9 mix, the data in the negative control of strain TA 100 were slightly above our historical control range. This slight effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies. Based on the results the test item is considered to be not mutagenic in bacteria (RCC 2006). 

 

HPRT test:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The following concentrations were applied: Experiment I (4 hours exposure): without S9 mix: 0.6 - 1.3 - 2.5 - 5.0 - 10.0 µg/mL; with S9 mix: 5.0 - 10.0 - 20.0 - 40.0 - 80.0 µg/mL Experiment II: without S9 mix (24 hours exposure): 2.5 - 5.0 - 10.0 - 20.0 - 40.0 µg/mL; with S9 mix (4 hours exposure): 5.0 - 10.0 - 20.0 - 40.0 - 80.0 µg/mL. Phase separation or turbidity visible to the unaided eye, was noted at the highest evaluated concentration with metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I below 50% in both parallel cultures occurred in the first experiment at 10.0 μg/mL without metabolic activation and in experiment II at 40.0 μg/mL without metabolic activation. The dose range in the presence of metabolic activation was limited by the solubility of the test item in aqueous medium. Turbidity or phase separation was noted at 80.0 μg/mL in both main experiments with metabolic activation. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The induction factor of three times the corresponding solvent control was not reached or exceeded at any test point. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. There was no significant dose dependent trend in any of the experimental parts. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.4 up to 15.6 mutants per 106 cells; the range of the groups treated with the test item was from 3.8 up to 29.1 mutants per 106 cells. EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells (Harlan 2013).

 

Ames test (CAS 188831-96-1):

The test item was tested for its mutagenic potential in two independent experiments according to the OECD guideline 471 at a concentration range of 8.0 - 5000 µg/plate. The following Salmonella typhimurium strains were used: TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The test substance was tested in the absence and presence of S9 -mix which was obtained from phenobarbital/ ß-naphtoflavone treated rats. In both test series the plates were incubated for 48 h at 37°C. The findings show that the test item did not induce any reverse mutations in the absence or presence of S9 mix. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, that the test article did not induce point mutations by base-pair changes or frame shifts in the genome of the strains used. Therefore, the test item is considered to be not mutagenic in this Salmonella typhimurium reverse mutation assay (Henkel 1999).

 

HPRT test (CAS 188831-96-1):

The test item was assessed for its potential to induce gene mutations at the
HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
Phase separation was observed in experiment I at 80.0 μg/mL with and at 20.0 μg/mL and above without metabolic activation. In experiment II turbidity occurred at 40.0 μg/mL with and without metabolic activation.
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 10.0 μg/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered without metabolic activation. The concentration range of the experimental parts with metabolic activation and 24 hour treatment without metabolic activation was limited by the solubility of the test item in culture medium.
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls. The induction factor of three times the corresponding solvent control was not reached or exceeded at any test point.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of experiment I without metabolic activation. However, the trend was judged as biologically irrelevant as the mutation frequency did not exceed the threshold described above.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.5 up to 26.8 mutants per 106 cells; the range of the groups treated with the test item was from 3.7 up to 43.1 mutants per 10^6 cells.

EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Ames test (CAS 85586-35-2)

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The used dose ranges were 33 μg up to 5 500 μg/plate  in the SPT and PIT. Standard plate test (SPT) and preincubation test (PIT) were both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 2 750 μg/plate. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.
Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (40M0114/12M063, 2012).

HPRT test (CAS 85586-35-2)

The study was performed to investigate the potential ofthe test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The highest concentration used in the range finding pre-experiment was 4000 μg/mL of the test item based on the solubility properties of the test item. The concentration range of the main experiments was limited by phase separation of the test item and cytotoxic effects. The test item was dissolved in DMSO.
No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Micronucleus test in vitro (CAS 85586-35-2)

The test substance was assessed for its potential to induce micronuclei in V79 cells of the Chinese hamster in vitro in three independent experiments. In each experimental group two parallel cultures were analyzed and 1000 cells per culture were scored for micronuclei. The highest applied concentration (4000 µg/ml) was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline No. 487. In the absence and presence of S9 mix no clear cytotoxicity was observed up to the highest applied concentration. No mutagenicity was observed at the concentrations evaluated. In Experiment IA and IB in the absence and presence of S9 mix two statistically significant increases were observed after treatment with 1000µg/ml (1.20 and 1.30%). The values are in the range of the laboratory historical control data and therefore not biologically relevant. In the absence of S9 mix one single value (1.60%) exceeded the range of the laboratory historical control data after treatment with 1000µg/ml. The valueis not statistically significant and therefore not biologically relevant. Appropriate mutagens were used as positive controls. They induced statistically significant increases in the percentage of micronucleated cells.

Micronucleus test in vivo (CAS 151661-88-0):

The substance was tested in vivo for its DNA damaging potential in a micronucleus test. The study was performed in albino mice of the strain CFW1 following the draft OECD 474 (initial assessment). Six male and six female mice were used per experimental group. The test item was administered by oral gavage, using arachis oil as vehicle. Based on the results of the dose range finding study a dosage of 5000 mg/kg bw was applied in the main trial. The test item was administered once, and six male and six female animals per group were sacrificed at time intervals of 24, 48 and 72 hours after the administration. Bone marrow smears from both femurs of each animal were prepared and the bone marrow preparations from the first five animals of each group were examined for micronuclei in 1000 polychromatic erythrocytes of each animal. A vehicle control and MMS as positive control were included in this study. No statistically significant enhanced mean values of micro-nucleated cells in polychromatic erythrocytes were observed at all examined time intervals compared to negative control values. Toxic effects as indicated by an enhanced mortality rate or by a reduction in the ratio of polychromatic to normochromatic erythrocytes (PCE/ NCE) were not noticed. Under the experimental conditions selected the test item did not induce chromosomal mutations in the bone marrow of mice. The test item is considered to have no DNA damaging potential in mice in vivo (Henkel 1990).

 

Micronucleus test in vivo (Oleic methyl ester, epoxidized, reaction products with glycerol):

The structural analogue substance (read across) was tested in the micronucleus test for the incidence of chromosomal aberration in male and female mice according to OECD guideline 474. Seven animals /sex were treated with 10000 mg/kg bw which was suspended in water with the aid of CMC (2 %) and cremohore (0.5 %) and was administered by oral gavage. Bone marrow smears from both femurs were prepared from each animal 24, 48 and 72 hours after the administration of the product and evaluated for micronuclei. In addition, one treatment group obtained an extra dosages of 5000 mg/kg bw and one group of 1000 mg/kg bw 24 h after the first application by oral gavage. The group treated with 10000 mg/kg bw showed a micronucleated cell count which was comparable to the current controls. Therefore the additional dosages of 5000 and 1000 mg/kg bw were not evaluated. It was concluded from the obtained results that there was no evidence of mutagenic potential of the test item, when administered orally in this in vivo micronucleus test (Henkel 1985).

 

Justification for classification or non-classification

The substance was not mutagenic in the Ames test and the HPRT assay in V79 cells, and it was, based on information from structural analogues, not clastogenic in vivo in the mouse micronucleus test. Therefore, based on the available data, classification for genetic toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.