Fatty acids, C16-18 and C18-unsatd., iso-Bu 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 based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

05 Jan - 05 Mar 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions (analytical purity of test substance not specified).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

analytical purity of test substance not specified

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:

Reference substance 002

Cas Number:

163961-32-8

Details on test material:

- Physical state: Amber liquid
- Analytical purity: no data
- Storage condition of test material: Room temperature in the dark
The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Method

Target gene:

thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- details on cell strain: L5178Y +/- 3.7.2c mouse lymphoma cells
- Type and identity of media: RPMI 1640 medium with Glutamax-l and HEPES buffer
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Experiment I: 4 hour treatment with and without S9 mix - 156.25, 312.5, 625, 1250, 2500, 5000 µg/mL
Experiment II: 24 hour treatment without and 4 hour treatment with S9 mix - 156.25, 312.5, 625, 1250, 2500, 5000 µg/mL
Experiment III: 4 hour treatment with S9 mix - 39.06, 78.13, 117.2, 156.25, 234.38, 312.5, 468.75, 625, 937.5, 1250 µg/mL

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate (EMS, 150 + 400 µg/mL without S9); Cyclophosphamide (CP, 2 µg/mL with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h with and without metabolic activation increased to 24 h in the second experiment (without metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): 5-fluorothymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Cell counts, viability after 2 days: the cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the % relative suspension growth that gives an indication of post treatment toxicity

OTHER:
The numbers of small and large colonies seen in the TFT mutation plates were also recorded

Evaluation criteria:

- The vehicle control values have to be in range with historical data.
- Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
- For a test material 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. The IMF must exceed the Global Evaluation Factor (GEF).
- when a test material 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.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

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:

TEST-SPECIFIC CONFOUNDING FACTORS
A cloudy precipitate of the test material was observed at and above 78.13 µg/mL forming a greasy/oily precipitate at and above 1250 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there appeared to be variable exposure of the test material to the cells as indicated by the reductions in the Relative Suspension Growth (%RSG) at certain dose levels. This was considered to be due to the nature of the test material formulations. In the 24-hour exposure in the absence of S9 there was a greater reduction in %RSG values forming a plateau effect down the dose range. A cloudy precipitate of the test material was observed at and above 78.13 pglml forming a greasy/oily precipitate at and above 1250 pglml. Due to the variable levels of toxicity observed the maximum recommended dose of 5000 pgiml was investigated in Experiments I and II.

COMPARISON WITH HISTORICAL CONTROL DATA:
All results were within the ranges of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The dose range used in the preliminary toxicity test was 19.53 to 5000 µg/mL.
Due to the variable levels of toxicity observed the maximum recommended dose of 5000 µg/mL was investigated in experiments 1 and 2.

Any other information on results incl. tables

Table 1: Results for the Relative Suspension Growth (%RSG) in the preliminary toxicity test were as follows:

Dose (µg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
19.53	88	84	61
39.06	83	92	61
78.13	74	92	56
156.25	91	78	58
312.5	85	78	53
625	75	64	55
1250	84	84	50
2500	85	77	44
5000	65	91	64

 

Table 2: Results from Experiment 1

Treatment (µg/mL)	4-Hours without S9			Treatment (µg/mL)	4-Hours with S9
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	100.35	0	100	1.00	122.76
156.25	130	1.34	96.34	156.25	98	1.05	88.56
312.5	112	1.28	98.69	312.5	92	1.00	106.06
625	136	1.57	91.80	625	82	0.86	137.37
1250	129	1.20	138.71	1250	94	0.92	122.97
2500	127	1.47	82.69	2500	102	0.87	110.73
5000	152	1.43	119.09	5000	109	0.95	112.66
Linear Trend                                      NS				Linear Trend                                      NS
EMS 400	124	0.83	748.75	CP 2	94	0.51	951.61

 

Table 3: Results from Experiment 2

Treatment (µg/mL)	24-Hours without S9			Treatment (µg/mL)	4-Hours with S9
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	88.69	0	100	1.00	131.27
156.25	63	0.69	95.98	156.25	47	0.41	210.65
312.5	66	0.62	57.47	312.5	58	0.46	229.00
625	72	0.77	99.53	625	35	0.33	213.23
1250	63	0.61	64.44	1250	88	0.82	150.73
2500	76	0.81	82.48	2500	113	1.03	128.99
5000	99	1.l2	90.36	5000	127	1.09	133.50
Linear Trend                                      NS				Linear Trend                                      NS
EMS 400	103	0.73	1021.46	CP 2	67	0.527	1305.55

 

 

Table 4: Results from Experiment 3

Treatment (µg/mL)	4-Hours withS9
	%RSG	RTG	MF§
0	100	1.00	165.58
39.06	110	0.98	151.66
78.13	101	1.03	185.56
117.2	61	0.41	167.55
156.25	50	0.39	170.51
234.38	138	1.32	174.72
312.5	124	1.32	128.11
468.75	64	0.63	176.42
625	60	0.41	228.91
937.5	63	0.46	190.85
1250	65	0.46	156.75
Linear Trend                                      NS
CP 2	62	0.22	856.01

%RSG: Relative Suspension Growth

RTG : Relative Total Growth

MF§ : 5-TFT resistant mutants/10^6 viable cells 2 days after treatment

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative not mutagenic in mouse lymphoma cells

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5l78Y cells and is therefore considered to be non-mutagenic under the conditions of the test.