Fatty acids, tall-oil, reaction products with acrylic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

not relevant

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Diacid 1550

IUPAC Name:

Diacid 1550

Details on test material:

- Name of test material (as cited in study report): Diacid 1550
- Physical state: viscous liquid
- Lot/batch No.: Confidential
- Storage condition of test material: At room temperature

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12 nutrient medium supplemented with 10% heat-inactivated fetal bovine serum (HIFBS) and 2 mM L-glutamine (stock medium) and medium containing 10% HIFBS, 2 mM L-glutamine, 50 units/mL of penicillin and 50 ug/mL of streptomycin (test medium).
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction and "CORE" (1:4)."CORE" = 24 mg NADP and 45 mg DL-isocitric acid per mL in deionized, distilled water

Test concentrations with justification for top dose:

Without metabolic activation: 150, 100, 75, 50, 25 and 13 ug/mL
With metabolic activation: 100, 75, 50, 25, 13 and 6.3 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: easily miscible

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Without activation: 16 hr
With activation: 2 hr
- Fixation time (start of exposure up to fixation or harvest of cells): 15.25 hours until harvested

SPINDLE INHIBITOR (cytogenetic assays): Colcmid (0.1 µg/mL), last 2 hr of incubation
STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: 4 replicate cultures (2 for cytoxicity determination in the Parallel Toxicity Test, and 2 for the evaluation of induced chromosome aberration)

NUMBER OF CELLS EVALUATED: 100 metaphases per dose

DETERMINATION OF CYTOTOXICITY
- Method: A range-finding test at the maximum concentration of 5000 µg/mL and nine lower concentrations was performed to determine the doses for the main test. Cytotoxicity was evaluated on the basis of Relative Cell Growth (RCG). Furthermore, the Average Generation Time (AGT) was determined to fix the harvest time for the main test.

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: yes

OTHER: Validity: the assay was considered valid based on:
1 - Negative control: the number of cells with aberrations (excl. gaps and endoreplications) should not exceed 6%
2 - At least 25% of scored cells in the positive control should show one or more chomosome aberrations
3 - At least one of the scored test doses should show more than 25% reduction in the RCG

Evaluation criteria:

A result was considered a positive response if:
1) The test article showed a positive dose-response trend and a statistically significant increase over that of the solvent controls in the number of cells with aberrations at one or more dose levels.
2) In the event there is no positive dose-response trend, at least two consecutive test doses should show a statistically significant increase in the number of cells with chromosome aberrations.

The test substance was considered to have caused an equivocal response if:
1) One of the test doses shows a statistically significant increase in the number of cells with aberrations without an accompanying positive dose-response trend.
2) The test article shows a statistically significant dose-response trend, but none of dose levels shows a significant increase in the number of cells with aberrations.

The test article was considered to have caused a negative reponse if no indication of a positive dose response was observed and none of the test doses showed a statistically significant increase in the percentage aberrant cells.

Statistics:

A statistical analysis was not performed for the test doses since the percentage of cells with aberrations in the test dose was lower than in the solvent control both with and without metabolic activation.

The data for the positive controls were analyzed using a Chi-square test (P ≤ 0.05 was considered significant).

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Cytotoxic from a dose of 500 ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: Test doses showed an osmolality value below 427
- Water solubility: formed a turbid suspension with water
- Precipitation: Precepitation was only observed at concentrations of 5000, 1000 and 500 ug/mL, not in the test doses.
- Other confounding effects: not relevant

RANGE-FINDING/SCREENING STUDIES: Calls did not survive at concentrations >500 µg/mL both with and without metabolic activation. The highest dose at which cells did not survive was 100 µg/mL, in which RCG was reduced to 54% without metabolic activation and 13% with metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: no information available

ADDITIONAL INFORMATION ON CYTOTOXICITY: no information available

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Percentage of cells with Aberrations without metabolic activation:

	Solvent control	Positive control	Negative control	Tested concentrations
				100µg/mL	75µg/mL	50µg/mL	25µg/ml
% of cells with aberrations	4.0	58	4.0	3.0	3.0	4.0	1.0
P-value	-	0.000	-	-	-	-	-

 

Percentage of cells with Aberrations with metabolic activation:

	Solvent control	Positive control	Negative control	Tested concentrations
				75µg/mL	50µg/mL	25µg/mL	13µg/ml
% of cells with aberrations	3.0	29	3.0	2.0	1.0	2.0	1.0
P-value	-	0.000	-	-	-	-	-

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In an in vitro Chromosome Aberration assay, Diacid 1550 did not cause a significant increase in the number of cells with aberrations or a positive dose response trend for concentrations of 6.3 to 100 ug/mL in Chinese Hamster Ovary Cells with and without metabolic activitation under the conditions of this test.

Executive summary:

In this study the ability of Diacid 1550 to induce chromosome aberrations in Chinese Hamster Ovary Cells was investigated. The study procedures used in this study were according to OECD Guideline 473 and the study was performed under GLP conditions..

Prior to the Chromosome Aberration assay, a miscibility test, osmolality test and a range finding test were performed in order to determine suitable doses for the main test.

In the Chromosome Aberration assay, concentrations of 150, 100, 75, 50 25, and 13 ug/mL were used without metabolic activation, whereas concentrations of 100, 75, 50, 25, 13 and 6.3 ug/mL were used with metabolic activation. Cells were exposed to the test substance for 16 hours without metabolic activation and for 2 hours with metabolic activation. After harvesting, cells were air dried and stained in Giemsa stain. A total of 100 metaphases were scored per dose for chromosome aberrations.

Diacid 1550 did not cause a significant increase in the number of cells with aberrations or a positive dose response trend for concentrations of 6.3 to 100 ug/mL in Chinese Hamster Ovary Cells with and without metabolic activitation under the conditions of this test.