Glycerides, C12-18 di- and tri-

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:

weight of evidence

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:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

736150-63-3

EC Number:

616-005-1

Cas Number:

736150-63-3

Molecular formula:

C25H46 O6; C27H48O8

IUPAC Name:

736150-63-3

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: RPMI medium supplemented with 10% horse serum, 200 µg/mL sodium pyruvate and 50 µg/mL gentamycin
- Properly maintained: yes (stock cultures were kept in a liquid nitrogen tank to start new stock cultures periodically in which cells were diluted daily and kept at a density of about 2E+5 to 1.5E+6)
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

First experiment:
with and without metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 3600 µg/mL
with metabolic activation: 156, 313, 625, 1250, 2500 and 3600 µg/mL

Repeat of second test:
without metabolic activation: 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitrosourea (50 µg/mL, -S9), 7,12-dimethyl-1,2-benzanthracene (3.3 µg/mL, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
First test:
- Exposure duration: +S9: 3 h, -S9: 4 h
Second main test:
- Exposure duration: +S9: 3 h, -S9: 24 h
Repeat of second main test:
- Exposure duration: -S9: 24 h
(In cell cultures with metabolic activation, the treatment period was limited to 3 h due to the cytotoxic effects induced by S9.)

- Expression time (cells in growth medium): 3 days (counting from the start of the experiment), cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates. The cell density was counted and adjusted to 3E+5 cells/mL daily.
- Selection time: approx. 10 days, cells were seeded in 2 microtitre plates with a density of 2000 cells/well in TFT selctive medium to determine the number of mutants.
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)


NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity corresponds to relative survival compared to the respective negative control values)

OTHER EXAMINATIONS:
Cloning efficiency was determined by seeding exposed cells in one microtiter plate with a density of 2 cells/well in medium without TFT.
Small and large colonies were differentiated as small colonies are capable to indicate chromosomal mutations.

Evaluation criteria:

The test item was considered as mutagenic when all of the following criteria were met:
- increases in the mutation frequency were observed in treated cultures compared to the corresponding negative control values at one or more test concentrations
- the increases showed a dose-response relationship
- the increases were reproducible between the replicates and the first and second test (when treatment conditions were the same)
- the increases were statistically significant
- the increases exceeded the historical negative control range
- the relative survival of the test groups was at least 15% at the end of the treatment period

When the above mentioned criteria were not met, the test item was considered as non-mutagenic.

Statistics:

Single values of the duplicates were determined from the examined parameters. Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA). In detail, mutation frequencies of treated samples were compared to the correpsonding negative controls with the analyis of variance test.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: starting from 3600 µg/mL (first test (4 h exposure)) and 160 µg/mL (second test (24 h exposure)), with S9: starting from 2500 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the negative and positive controls were all within the range of the historical control data despite one positive control value which was slightly higher that the historical control value. Thus, the study was considered to be valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: First Experiment - 4 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.36	1
625	91	116	57	2.04	0.86
1250	108	94	65	2.44	1.03
2500	106	83	36	2.66	1.12
3600	89	76	11	0.30*	0.12
5000	74	83	8	0.04*	0.017
ENU (50)	53	62	45	#
Table 2: First Experiment - 3 h exposure - With Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.39	1
625	75	81	65	2.59	1.08
1250	92	44	46	3.04	1.27
2500	93	30	9	0.9	0.38
3600	90	5	1	0.2	0.08
5000	84	4	1	0.47	0.2
DMBA (3.3)	24	50	29	#
Table 3: Second Experiment - 24 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	not reported	not reported
313	82	9	2	not reported	not reported
625	73	4	1	not reported	not reported
1250	68	4	1	not reported	not reported
2500	38	4	1	not reported	not reported
3600	31	1	0	not reported	not reported
ENU (50)	53	35	9	not reported	not reported
Table 4: Second Experiment - 3 h exposure - With Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2	1
156	83	95	62	1.95	0.98
313	84	101	70	1.84	0.92
625	82	105	65	1.79	0.9
1250	46	98.5	65	1.84	0.92
2500	38	88	37	1.93	0.97
3600	36	4	0	0	0
DMBA (3.3)	28	52	27	37.3	18.65
Table 5: Repeat fo Second Experiment - 24 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.17	1
`2.5	102	100	62	2.16	1
5	100	114	151	2.02	0.93
10	92	95	145	1.97	0.91
20	105	100	60	2.11	0.97
40	82	95	103	2.17	1
80	69	111	97	1.82	0.84
160	25	44	16	2.32	1.07
320	11	31	6	1.94	0.89
ENU (50)	16	31	9	51.8	23.87

 

ENU: N-ethyl-N-nitrosourea

DMBA: 7,12-dimethyl-1,2-benzanthracene

*: statistically significant with p < 0.01

#: could not be calculated as all wells contained mutant colonies

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative