Fatty acids, C18-unsatd, dimers, hydrogenated, diisopropyl esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

27 Apr - 16 Jun 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Fatty acids, C18-unsatd., dimers, hydrogenated, 2-ethylhexyl esters (CAS 68440-06-2). In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsatd., dimers, hydrogenated, 2-ethylhexyl esters

EC Number:

500-214-9

EC Name:

Fatty acids, C18-unsatd., dimers, hydrogenated, 2-ethylhexyl esters

Cas Number:

68440-06-2

Molecular formula:

C44H84O4

IUPAC Name:

Fatty acids, C18-unsatd., dimers, hydrogenated, 2-ethylhexyl esters

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Storage condition of test material: room temperature in the dark
- Expiration date of the lot/batch: 05 May 2016

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media:
- Basic medium: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with penicillin/streptomycin (100 U/mL and 100 μg/mL, respectively), sodium pyruvate (1 mM), amphotericin (2.5 µg/mL) and 10% donor horse serum (R10 medium)
- Exposure medium: RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the study course.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of Sprague Dawley rats treated with a suspension of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).

Test concentrations with justification for top dose:

Preliminary Cytotoxicity Test:
- 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 µg/mL (with and without metabolic activation)
Experiment 1:
- 4.88, 9.77, 19.53, 39.06, 78.13, 156.25 µg/mL (with and without metabolic activation)
Experiment 2:
- 9.77, 19.53, 39.06, 78.13, 156.25, 312.5 µg/mL (without metabolic activation)
- 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, µg/mL (with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Following solubility checks performed in-house for previous studies with the same test item, the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared. The test item was considered to be a complex mixture (UVCB), therefore the maximum proposed dose level in the solubility test was set at 5000 µg/mL. However, acetone is toxic to LY5178Y cells at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5% to give a maximum achievable dose level of 2500 µg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

-S9-mix: Ethylmethanosulphonate (EMS): 400 µg/mL (experiment I), 150 µg/mL (experiment II); +S9-mix: Cyclophosphamide (CP): 1.5 µg/mL (experiment I and II)

Details on test system and experimental conditions:

METHOD OF APPLICATION
- in suspension

DURATION
- Exposure duration: Experiment I: 4 h (with and without metabolic activation); Experiment II: 24 h (without metabolic activation), 4 h (with metabolic activation)
- Expression time (cells in growth medium): On Day 2 of the experiment, the cells were plated for determination of the mutant efficiency in 96-well microtitre plates containing TFT selective medium. The microtitre plates were incubated for 10 or 14 days.
- Selection time: 10 – 14 days

SELECTION AGENT
- 4 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS
- each experiment was performed in duplicates (with and without metabolic activation).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS
- Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.

Evaluation criteria:

For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the global evaluation factor (GEF) of 126*10-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequencies, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard
deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dosedependent
manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made
after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency
of MF(controls) + 126.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test item was dosed into media.
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm.
- Precipitation: A cloudy precipitate of the test item was observed in both experiments at and above 39.06 µg/mL which turned to a greasy/oily precipitate at 156.25 µg/mL at the end of exposure.

RANGE-FINDING/SCREENING STUDIES
The dose range of the test item used in the preliminary toxicity test was 9.77 to 2500 µg/mL. In all three exposure groups there was no evidence of marked reductions in %RSG values of cells treated with test item. In the 4-hour exposure groups a cloudy precipitate of the test item was observed at and above 39.06 µg/mL which turned to greasy/oily precipitate at and above 156.25 µg/mL at the end of exposure. In the 24-hour exposure group a cloudy precipitate of the test item was observed at and above 39.06 µg/mL which turned to greasy/oily precipitate at and above 312.5 µg/mL. In the subsequent mutagenicity experiments the maximum dose level was limited to 156.25 µg/mL in the 4-hour exposures, and 312.5 µg/mL in the 24-hour exposure due to the incidence of greasy/oily precipitate (with no marked toxicity apparent).

COMPARISON WITH HISTORICAL CONTROL DATA
The results of both mutagenicity experiments were within the historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY
No evidence of marked toxicity following exposure to the test item in either the absence or presence of metabolic activation was recorded in both experiments, as indicated by the %RSG and RTG values.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of Preliminary Cytotoxicity Test

Dose (µg/mL)	%RSG (-S9) 4-hour exposure	%RSG (+S9) 4-hour exposure	%RSG (-S9) 24-hour exposure
0	100	100	100
9.77	101	98	105
19.53	117	102	103
39.06	97	103	104
78.13	93	111	102
156.25	93	97	101
312.5	105	98	104
625	92	93	104
1250	98	98	111
2500	116	99	117

%RSG: Relative suspension growth

Table 2: Results of experiment I

Dose (µg/ml)	RSG (%)	RS (day2)(%)	RTG	MF
				total	small	large
4-hour exposure –S9-mix
0	100	75.69	1.00	147.86	63.1	76.5
4.88	99	80.34	1.06	149.50	73.9	66.7
9.77	95	85.11	1.06	137.25	66.3	62.9
19.53	90	97.17	1.16	120.21	49.2	64.1
39.06	94	83.01	1.03	113.60	50.7	57.6
78.13	93	88.81	1.09	124.16	53.8	63.6
156.25	90	75.40	0.89	135.57	48.3	80.7
400 (EMS)	73	58.16	0.55	802.44	253.3	372.4
4-hour exposure +S9-mix
0	100	90.38	1.00	118.43	68.1	44.2
4.88	105	91.99	1.06	118.11	63.0	48.8
9.77	102	107.20	1.21	101.35	55.4	40.6
19.53	95	98.08	1.03	102.59	59.0	38.6
39.06	96	105.02	1.11	105.00	49.6	49.6
78.13	97	96.26	1.03	109.52	55.6	48.2
156.25	98	94.51	1.03	114.96	49.0	59.7
1.5 (CP)	76	65.31	0.56	584.31	395.7	106.8

%RSG: Relative suspension growth

RS: Relative suspension in % (day 2)

RTG: Relative total growth

MF: Mutant frequency (5-TFT resistant mutants/10⁶ viable cells 2 days after treatment

EMS: Ethylmethanosulphonate

CP: Cyclophosphamide

Table 3: Results of experiment II

Dose (µg/ml)	RSG (%)	RS (day 2) (%)	RTG	MF
				total	small	large
24-hour exposure –S9-mix
0	100	91.58	1.00	94.45	42.1	48.3
9.77	87	88.81	0.89	113.30	52.2	55.4
19.53	90	83.70	0.88	108.92	46.9	57.1
39.06	79	83.01	0.81	107.94	49.0	54.1
78.13	87	84.40	0.87	93.38	35.0	54.9
156.25	93	79.70	0.86	96.98	45.7	47.5
312.5	90	91.18	0.94	84.77	41.5	40.0
150 (EMS)	57	56.92	0.41	1304.16	398.3	461.8
4-hour exposure +S9-mix
0	100	82.33	1.00	150.94	69.5	72.1
4.88	100	87.30	1.06	122.61	54.7	61.3
9.77	104	91.18	1.16	126.31	58.7	60.3
19.53	96	91.18	1.07	131.73	61.9	61.9
39.06	86	97.17	1.02	103.56	46.2	52.1
78.13	98	82.33	0.99	90.20	46.0	40.9
156.25	82	92.81	0.92	111.86	45.3	60.8
1.5 (CP)	75	56.92	0.52	752.68	485.3	143.8

%RSG: Relative suspension growth

RS: Relative suspension in % (day 2)

RTG: Relative total growth

MF: Mutant frequency (5-TFT resistant mutants/10⁶ viable cells 2 days after treatment

EMS: Ethylmethanosulphonate

CP: Cyclophosphamide

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative