Oxidised Crude Tall Oil

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Sep 2014 to 25 Feb 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guidelines study under GLP, positive and vehicle controls do not fall clearly within historical control ranges in part of the experiments

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Oxidised Crude Tall Oil

EC Number:

938-639-0

Molecular formula:

C17-20H29-35CO2H. O6-10

IUPAC Name:

Oxidised Crude Tall Oil

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): EnvaMul™200
- Substance type: UVCB

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source: Genetic Toxicology Laboratory at Pfizer, Inc., Groton, CT, USA
- Type and identity of media: McCoy’s complete media
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, 12-14 hour cycling time, average 21 chromosomes

Each culture was prepared by seeding with 1.4 × 10E6 CHO cells per 75 cm2 flask in McCoy’s complete media and incubated at 37 ± 1°C in vented flasks in a humidified atmosphere of 5 ± 1% CO2 for approximately 24 hours prior to the initiation of treatment.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor™ 1254-induced rat liver S9 fraction

Test concentrations with justification for top dose:

Concentrations are based on the results of 3 pre-tests

Exp 1 (3 h without metabolic activation) : stock solution of 150 mg/mL further diluted with DMSO/ethanol(1/1) to:
6.25, 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 225, 300, and 500 µg/mL
Exp 2 (20 h without metabolic activation) : stock solution of 50 mg/mL further diluted with DMSO/ethanol(1/1) to:
6.25, 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 225, 300, and 500 µg/mL
Exp 3 (3 h with metabolic activation) : stock solution of 50 mg/L further diluted with DMSO/ethanol(1/1) to:
6.25, 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 225, 300, and 500 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO/Ethanol (1/1)
- Justification for choice of solvent/vehicle: pre-test at 500 mg/L

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO/Ethanol (1/1)

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

tests without metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO/Ethanol (1/1)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

test with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: exp 1 3 hours without metabolic activation, exp 2 20 hours without metabolic activaton, exp3 3 hours with metabolic activation
- Expression time: until 20 hours

SPINDLE INHIBITOR: Colcemid (0.1 µg/mL) 2 hours before harvesting
FIXATIVE: 3:1 methanol:glacial acetic acid

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases per replicate (+ 15 aberrant cells per replicate)

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling (with Coulter counter) + mitotic index (1000 cells)+ visible signs of cytotoxicity

OTHER EXAMINATIONS:
- Determination of polyploidy: 200 cells/replicate
- Determination of endoreplication: 200 cells/replicate

Evaluation criteria:

POSITIVE RESPONSE:
The test item would be considered positive for inducing chromosomal aberrations if a significant increase (p ≤0.01) in the mean percent of cells with chromosomal aberrations is observed at one or more dose levels, or if a significant increase (p ≤0.05) in the mean percent of cells with chromosomal aberrations is observed at two or more dose levels. If a significant increase is seen at one or more dose levels, a dose-response should be observed.
NEGATIVE RESPONSE
The test item would be considered negative for inducing chromosomal aberrations if no statistically significant increase (p ≤0.05) is observed in the mean percent of cells with chromosomal aberrations at any of the test concentrations.

Statistics:

One-tailed Fisher’s Exact test on cell numbers with aberrations
A response would be considered statistically significant for dose-response trend in the Cochran-Armitage test if p ≤0.05.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

no dose response

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 150 ug/mL and above

Vehicle controls validity:

other: within historical control ranges

Positive controls validity:

other: MMC (0.75 ug/mL) within historica control values

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 225 ug/mL and above; precipitate at 125 ug/mL and above

Vehicle controls validity:

other: outside historical control ranges

Positive controls validity:

other: MMC (0.10 ug/mL) within historical control ranges

Test results 3

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

dose response

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 75 ug/mL and above

Vehicle controls validity:

other: at the upper range of historical control values

Positive controls validity:

other: CP (0.75 ug/mL) at the lower range of historical control values

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Precipitation: see above

RANGE-FINDING/SCREENING STUDIES: performed, but only cytotoxicity part reported

COMPARISON WITH HISTORICAL CONTROL DATA: see above

Remarks on result:

other: other: 3 hours exposure

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Exp 1Cell Count Reduction and Aberration Summary: 3-Hour Incubation without Metabolic Activation

Treatment	% Mitotic Reduction	% Cytotoxicity RPD	% Cells w/Abs	% Cells w/>1 Abs	% Endo Cells	% Polyploid Cells
Vehicle (1%)	0.00	0.00	0.0	0.0	0.0	4.8
MMC 0.75 µg/mL	35.39	10.69	66.7*	46.7*	0.0	4.5
37.5 µg/mL	0.00	0.33	2.5**	0.0	0.0	2.8
75.0 µg/mL	0.00	0.67	2.5**	0.0	0.3	3.3
150 µg/mL^a	0.00	7.17	2.5**	0.0	15.0*	3.3

 

Exp 2 Cell Count Reduction and Aberration Summary: 20-Hour Incubation without Metabolic Activation

Treatment	% Cytotoxicity RPD	% Cells w/Abs	% Cells w/>1 Abs	% Endo Cells	% Polyploid Cells
Vehicle (1%)	0.00	4.0	0.0	0.0	5.0
MMC 0.10 µg/mL	15.21	50.8*	27.1*	0.0	4.8
75.0 µg/mL	13.14	1.0	0.0	0.0	5.3
100 µg/mL	23.02	2.0	0.0	0.0	6.5
125µg/mL^a	33.15	3.5	0.0	0.0	3.3

 

Exp 3 Cell Count Reduction and Aberration Summary: 3-Hour Incubation with Metabolic Activation

Treatment	% Cytotoxicity RPD	% Cells w/Abs	% Cells w/>1 Abs	% Endo Cells	% Polyploid Cells
Vehicle (1%)	0.00	2.0	0.0	8.5	4.8
CP 7.5 µg/mL	19.40	15.5*	3.5*	5.3	4.3
12.5 µg/mL	6.71	1.5	0.0	9.8	4.5
25.0 µg/mL	30.33	6.0**	1.0	9.8	5.0
75.0 µg/mL^a	54.04	12.5*	3.0**	9.0	3.0

Endo -Endoreduplicated cells

Abs -Aberrations

MMC -Mitomycin C

CP -CycloPhosphamide

RPD- Relative Population Doubling

Vehicle- 50% Dimethylsulfoxide 50% Ethanol

Percent Aberrant cells: *p=0.01, **p=0.05 using Fisher's Exact Test

aPrecipitates observed at the end of treatment

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
ambiguous

The substance was considered positive for inducing structural aberrations in CHO-WBL cells with metabolic activation and equivocal for inducing structural aberrations in CHO-WBL cells in the short-treatment without metabolic activation under the conditions of this test system. A significant test item-related increase in numerical aberrations (endoreduplication) was observed in the short-treatment without metabolic activation

Executive summary:

In a chromosome aberration test in CHO-WBL cells, structural (with metabolic activation) and numerical (without metabolic activation) aberrations were reported after 3-hour treament at concentrations upto cytotoxicity. Prolonged testing without metabolic activation did not induce any effects. Therefore it cannot be excluded that the substance exhibits clastogenic effects.