Fatty acids, C8-18 and C18-unsatd., esters with neopentyl glycol

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 based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

27 Aug - 24 Oct 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", 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:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2 (R1): 'Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (EMA/CHMP/ICH/126642/2008)'

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde Soziales, Familie, Gesundheit und Verbraucherschutz, Hamburg, Germany

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2-dimethyl-1,3-propanediyl dioleate

EC Number:

255-713-1

EC Name:

2,2-dimethyl-1,3-propanediyl dioleate

Cas Number:

42222-50-4

IUPAC Name:

2,2-dimethylpropane-1,3-diyl bisoctadec-9-enoate

Details on test material:

- Name of test material (as cited in study report): 2,2-dimethyl-1,3-propanediyl dioleate
- Analytical purity: 100%
- Physical state: light yellowish, clear liquid
- Batch No.: OE10124A
- Storage condition of test material: at room temperature, in a tightly closed container; kept away from heat, sparks, flames and direct sunlight

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: growth medium: RPMI 1640 medium supplemented with 0.05% Pluronic® F68, 2 mM L-glutamine, 220 µg/mL sodium pyruvate, 100 µg/mL gentamycin, 2.5 µg/mL fungizone and fetal bovine serum (10% v/v);
treatment medium: growth medium without sodium pyruvate, gentamycin and fungizone;
cleaning medium: growth medium supplemented with approx. 4.0E-05 M thymidine, 1.2E-04 M hypoxanthine, 3.3E-05 M glycine and 7.2E-07 M methotrexate;
recovery medium: growth medium supplemented with approx. 4.0E-05 M thymidine, 1.2E-04 M hypoxanthine and 3.3E-05 M glycine;
selection medium: growth medium containing 3 µg/mL trifluorothymidine (TFT)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary experiment: 25, 100, 250, 1000, 2500 and 5000 µg/mL
- 24 h treatment: without metabolic activation
- 4 h treatment: with metabolic activation

Main assay: 312.5, 625, 1250, 2500 and 5000 µg/mL
- Experiment I - 3 h treatment: with and without metabolic activation
- Experiment II – 24 h treatment: without metabolic activation
- Experiment II – 3 h treatment: with metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test substance was completely dissolved in acetone, but not soluble in water or DMSO.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: methylmethanesulfonate, 10 and 15 g/mL; +S9: 3-methylcholanthrene, 2.5 and 4 µg/mL

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 3 h (± S9); Experiment II: 24 h (-S9) and 3 h (+S9)
- Expression time (cells in growth medium): after the end of treatment, cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture was placed in 2 different 96 well microtiter plates (192 wells, averaging 1.6 cells/well) and incubated for 1 week, whereas the rest of the cells was incubated for 2 days for the expression period (plating efficiency step 1). The cells for the plating of survival were counted after 1 week and the number of viable clones was recorded. The cells incubated for the expression period were maintained below 10E+6 cells per mL and a minimum of 4 concentration levels plus positive and negative control were selected for 5-trifluoro-thymidine (TFT) resistance. At the end of the second expression period the selected cultures were diluted to 1E+4 cells/mL and plated for survival (plating efficiency step 2) and TFT resistance in parallel (plating efficiency step 2). The plating for survival was identical to the above described method (plating efficiency step 1 in 192 wells with average 1.6 cells/well). For the plating for TFT resistance 3 µg/mL TFT (final concentration) was added to the cultures and 0.2 mL of each suspensions placed into four 96-well microtiter plates (384 wells, averaging 2E+3 cells/well). The plates were incubated for 11-14 days.
- Selection time (if incubation with a selection agent): 11-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 11-15 days

SELECTION AGENT (mutation assays): 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: single treated cultures and duplicate solvent control cultures each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: suspension growth, relative suspension growth, plating efficiency and relative survival

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

OTHER:
- Concentration selection for the main experiment: the lowest separation factor of 2 as recommended by the guidelines was used and 5 concentrations were selected. Since no increase in the mutant frequency was observed in the preliminary experiment, it was considered acceptable not to add any further lower concentrations, as these additional lower concentrations would provide no further information.

Evaluation criteria:

The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment was a mutant frequency ≥ 2 compared to the concurrent background mutant frequency. The observation of a mutant frequency that met the minimum criterion for a single treated culture within a range of assayed concentrations was not considdered as sufficient to evaluate a test item as a mutagen.
The test results were positive, if the following criteria were met:
- a concentration-related or toxicity-related increase in mutant frequency preferably for at least 3 concentrations (depending on the concentration steps chosen and toxicity at which mutagenic activity appeared).
- the mutant frequency for a single concentration at or near the highest testable toxicity was ≥ 4 times the concurrent background mutant frequency. Smaller increases at a single concentration near the highest testable toxicity needed to be confirmed by a repeat assay.
- either parameter (concentration or toxicity (percent relative growth)) was used to establish whether the increase in mutant frequency was related to an increase in effective treatment.
- treatments inducing less than 10% relative growth were included in the assay, but not used as primary evidence for mutagenicity.
The test item was considered as negative in this assay if neither of the above criteria were met. A test item was evaluated as non-mutagenic in a single assay if the minimum increase in mutant frequency was not observed for a range of applied concentrations that extended toxicity causing 10% to 20% relative growth or a range of applied concentrations extending to at least twice the solubility limit in culture media.

Statistics:

The significance of increases in mutant frequencies (total wells with clones), by comparison with concurrent controls and the global evaluation factor (GEF), was assessed according to the recommendations of the Mouse Lymphoma Workgroup, Aberdeen, 2003. For microwell assays, the GEF is defined as 126 mutants per 1E+6 viable cells. For valid data, the test item was considered to be mutagenic in this assay if the mutant frequency (MF) of any test concentration exceeded the sum of the mean control mutant frequency plus GEF and the linear trend test was positive. This would indicate a positive, biologically relevant response.

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:

RANGE-FINDING/SCREENING STUDIES: in a preliminary cytotoxicity test with concentrations ranging from 25 to 5000 µg/mL, no signs of cytotoxicity were noted in the experiment without and with metabolic activation (24-h or 4-h exposure, respectively) at any of the concentrations tested. Hence, the highest concentration of the test substance selected for treatment in the main study was 5000 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: mutant frequencies of the positive and negative controls were within the respective historical ranges. The mutant frequencies in treated colonies were within the range of the negative control 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. Experiment I - 3 h exposure - With Metabolic Activation

Concentration [µg/mL]	Plating efficiency 2	Relative survival [%]	Mutants per 1E+06 surviving cells	% large colonies of total colonies	% small colonies of total colonies	ratio small/large colonies
SC*	1.1193	100	57.93	45.7	54.3	1.19
312.5	0.8796	79	79.30	48.0	52.0	1.08
625	0.7270	65	81.71	51.2	48.8	0.95
1250	0.9208	82	62.88	42.9	57.1	1.33
2500	1.0124	90	57.19	50.0	50.0	1.00
5000	0.9208	82	69.29	52.2	47.8	0.92
3-MCA, 2.5	0.3096	28	693.15	43.3	56.7	1.31
3-MCA, 4.0	0.3596	32	653.50	52.1	47.9	0.92

3-MCA = 3-methylcholanthrene; SC = solvent control (acetone)

* mean value of 2 parallel cultures

Table 2. Experiment I - 3 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Plating efficiency 2	Relative survival [%]	Mutants per 1E+06 surviving cells	% large colonies of total colonies	% small colonies of total colonies	ratio small/large colonies
SC*	0.8866	100	72.27	54.3	45.7	0.84
312.5	0.9649	109	66.12	45.7	54.3	1.19
625	0.9499	107	71.85	46.9	53.1	1.13
1250	0.9804	111	65.08	45.7	54.3	1.19
2500	1.2033	136	59.12	58.8	41.2	0.70
5000	1.1602	131	60.12	52.0	48.0	0.92
MMS, 10	0.1627	18	1782.42	27.8	72.2	2.60
MMS, 15	0.2248	25	1919.48	37.8	62.2	1.64

MMS = methylmethanesulfonate; SC = solvent control (acetone)

* mean value of 2 parallel cultures

Table 3. Experiment II - 3 h exposure - With Metabolic Activation

Concentration [µg/mL]	Plating efficiency 2	Relative survival [%]	Mutants per 1E+06 surviving cells	% large colonies of total colonies	% small colonies of total colonies	ratio small/large colonies
SC*	0.9070	100	53.48	47.9	52.1	1.09
312.5	0.8664	96	50.21	43.8	56.3	1.29
625	0.9208	102	55.01	51.4	48.6	0.95
1250	0.8164	90	67.37	45.0	55.0	1.22
2500	0.6769	75	62.20	41.9	58.1	1.38
5000	0.8286	91	66.38	45.0	55.0	1.22
3-MCA, 2.5	0.4203	46	894.72	48.3	51.7	1.07
3-MCA, 4.0	0.4598	51	776.64	52.0	48.0	0.92

3-MCA = 3-methylcholanthrene; SC = solvent control (acetone)

* mean value of 2 parallel cultures

Table 4. Experiment II - 24 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Plating efficiency 2	Relative survival [%]	Mutants per 1E+06 surviving cells	% large colonies of total colonies	% small colonies of total colonies	ratio small/large colonies
SC*	0.7274	100	55.94	48.3	51.7	1.07
312.5	0.6674	92	54.61	51.9	48.1	0.93
625	0.7166	99	74.73	51.3	48.7	0.95
1250	0.6866	94	61.32	51.6	48.4	0.94
2500	0.8045	111	61.16	47.2	52.8	1.12
5000	0.7065	97	55.56	55.2	44.8	0.81
MMS, 10	0.3654	50	1139.30	39.2	60.8	1.55
MMS, 15	0.2537	35	2005.13	37.6	62.4	1.66

MMS = methylmethanesulfonate; SC = solvent control (acetone)

* mean value of 2 parallel cultures

RATIO OF SMALL TO LARGE COLONIES

No change was observed in the ratio of small to large mutant colonies between treated and solvent control cultures.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative