Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

Molecular formula:

Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

IUPAC Name:

Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

Details on test material:

- Name of test material (as cited in study report): DHW 80
- Molecular weight: approximately 333 g/mol
- Physical state: liquid at room temperature
- Colour: brown
- Analytical purity: approximately 49 - 51 % (water content 50 %)
- Lot/batch No.: 10393
- Expiration date of the lot/batch: 1994-11-04
- Stability: pure - months; in solvent - in water stable for several hours, unstable in alkaline solutions
- Storage condition of test material: at room temperature

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Mininmal Essential Medium (MEM), SEROMED, D-12247 Berlin, supplemented with 10 % fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fractions (S9 mix), prepared from livers of 8-12 weeks old male rats treated with Aroclor 1254 in olive oil 5 days before

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 3.0*, 6.0, 10.0, 30.0, 60.0, 100.0** µg/mL
with S9 mix: 16.5, 60.0, 100.0, 15.0 µg/mL

Experiment II:
without S9 mix: 6.0, 10.0, 30.0, 40.0, 50.0**, 60.0** µg/mL
with S9 mix: 16.5, 60.0, 100.0, 165.0 µg/mL

*not evaluated, culture not continued; ** not evaluable, toxic effects

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (E. MERCK, D-64293 Darmstadt, purity > 99.5%)
- Justification for choice of solvent/vehicle: chosen according to its solubility properties and its non-toxicity for the cell culture system, final concentration in cell culture medium did not exceed 1% (v/v)

Controls

Untreated negative controls:

yes

Remarks:

untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: EMS (Ethylmethanesulfonate) - 0.6 mg/mL = 4.8 mM dissolved in nutrient medium (MEM without FCS), without metabolic activation; DMBA (7,12-dimethylbenz(a)anthracene - 3.85 µg/mL = 15.0 mM dissolved in DMSO, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in serumfree medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): Cells were treated for 4 hours on day 2. After incubation cells were washed and incubated with fresh complete growth medium. On day 5 cells of experiments without S9 mix were subcultivated in 175 cm² flasks, cells out of experiments with S9 mix were subcultivated on day 6. Selection with TG6 medium was carried out from day 9 to day 17.
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): fixation was carried out on one day

SELECTION AGENT (mutation assays): Thioguanine - 11 µg/mL (SIGMA gmbH, D-82041 Deisenhofen)

NUMBER OF REPLICATIONS: duplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Test substance is classified "positive" if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response for one of the test points.
Test substance is classified "mutagenic" if it induces reproducibly a mutant frequency that is three times higher then the spontaneous mutation frequency in the experiment or if there is a reproducible concentration-related increase of the mutation frequency.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

concentration-dependent toxicity was observed, which was less pronounced in experiment I compared to experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Without S9 mix test substance reduced the cloning efficiency of V79 cells at concentrations higher than 30.0 µg/mL. With S9 mix no toxicity could be observed up to the limit of solubility of the test substance.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1: Experiment I: without metabolic activation

Concentration [µg/mL]	Cloning Efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 Surviving Cells	Mutation Factor
Negative control	100	100	7.8	1.0
Solvent control (DMSO)	100	100	9.2	1.2
Positive control (EMS)	52.3	88.9	394	50.5
6.0	111.5	107.8	11.5	1.5
10.0	92.1	114.6	13.8	1.8
30.0	63.9	97.6	6.9	0.9
60.0	2.6	79.1	10.8	13.9

 

Table2: Experiment I: with metabolic activation

Concentration [µg/mL]	Cloning Efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 Surviving Cells	Mutation Factor
Negative control	100	100	1.6	1.0
Solvent control (DMSO)	100	100	13.6	8.5
Positive control (DMBA)	55.1	75.3	451.9	282.4
16.5	136.3	101.9	4.2	2.6
60.0	136.5	102.3	4.3	2.7
100.0	45.3	75.9	2.1	1.3
165.0	20.2	95.4	8.6	5.4

Table3: Experiment II: without metabolic activation

Concentration [µg/mL]	Cloning Efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 Surviving Cells	Mutation Factor
Negative control	100	100	17.1	1.0
Solvent control (DMSO)	100	100	7.2	0.4
Positive control (EMS)	82.8	87.6	448	26.2
6.0	91.1	92.4	19.1	1.1
10.0	72.5	106.7	6.4	0.4
30.0	0.5	85.9	17.5	1.0
40.0	0.3	45.4	16.4	1.0

Table4: Experiment II: with metabolic activation

Concentration [µg/mL]	Cloning Efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 Surviving Cells	Mutation Factor
Negative control	100	100	24.5	1.00
Solvent control (DMSO)	100	100	0.0	0.0
Positive control (DMBA)	61.9	109.8	466.3	19.0
16.5	103.5	117.1	17.5	0.7
60.0	94.6	104.6	17.6	0.7
100.0	98.2	96.6	16.1	0.7
165.0	93.1	90.2	10.8	0.4

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells. Therefore, DHW 80 is considered to be non-mutagenic in this HPRT assay.