Fatty acids, sunflower-oil, conjugated, maleated,reation products with diethanolamine, maleated tall-oil fatty acids and triethanolamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, sunflower-oil, conjugated, maleated,reation products with diethanolamine, maleated tall-oil fatty acids and triethanolamine

EC Number:

800-003-4

Cas Number:

1415316-96-9

IUPAC Name:

Fatty acids, sunflower-oil, conjugated, maleated,reation products with diethanolamine, maleated tall-oil fatty acids and triethanolamine

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0012652167 (BASF SE)
- Expiration date of the lot/batch: September 24, 2017
- Purity test date: 2016-09-13


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: guaranteed by sponsor
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle used

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 12-16h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Ham's F12 supplemented with 10% FCS except during treatment. All media were supplemented with 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine B. CO2 concentration was 5%
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Starting at 285.94µg/mL test substance concentration (corrected for purity) and emulsion was prepared.
After 4 hours treatment in the absence of S9 mix, cytotoxicity was observed as indicated by a reduced RS of about or below 20% of control at 1143.75 μg/mL and above. In addition, in the presence of S9 mix, a clearly reduced relative RS (to 14.2%) was observed after treatment with 571.88 μg/mL. No viable cells were detected at higher concentrations.
Consequently, the following doses were tested in the main experiments:
1st and 2nd experiment:
w/S9: 7.81 (first exp. only), 15.63, 31.25, 62.5, 125, 250, 500, 1000µg/mL
w/out S9: 15.63, 31.25, 62.5, 125, 250, 500, 1000, 1500µg/mL
3rd experiment:
w/out S9: 25, 50, 100, 200, 300, 500, 600, 800, 1000µg/mL

Vehicle / solvent:

Culture medium

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 20x10^6 cells /40mL

DURATION
- Preincubation period: 1 day
- Exposure duration: 4h
- Expression time (cells in growth medium):7-9 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS:

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Acceptance criteria
The HPRT assay is considered valid if:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95% control limit).
• The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range

Assessment criteria
A test substance is considered to be clearly positive if:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
And
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit)
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit)

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No change in pH value was observed up to 5000µg/mL of test compound (corrected for purity).
In the absence and presence of S9 mix, test substance precipitation was observed macroscopically in culture medium at the end of treatment at 125.00 μg/mL onward in the 1st Experiment, at 1000.00 μg/mL in the 2nd Experiment and at 600μg/mL in the 3rd Experiment.

Any other information on results incl. tables

All provided concentrations have been adjusted to the purity of the test substance.

		Exp. 1		Exp. 2		Exp.3
Conc.	Met. Act.	Mutant Frequ. (%)	RS(%)	Mutant Frequ. (%)	RS(%)	Mutant Frequ. (%)	RS(%)
0	-	1	100	5.3	100	2	100
15.63	-	3	81	1	82
31.25	-	7.2	91	3.9	85
62.5	-	7.1	81	0.3	80
125	-	2.7	76	4.6	80
200	-					3.8	103
250	-	6	79	1	87
300	-					1.1	108
500	-	13.3	76	0.4	77	0.6	103
600	-					0	103
750	-			2.1	75
1000	-	not scored	5	not scored	0	not scored	2
0	+	8.9	100	6.4	100
7.81	+	9.2	114
15.63	+	8.1	108	3.5	107
31.25	+	5.8	89	1.5	102
62.5	+	5.4	102	4	98
125	+	3.5	94	1.5	93
250	+	2.3	123	0.3	102
500	+	4.2	53	0.6	47
1000	+	not scored	0	not scored	0

Bold values indicate significant differences