Fatty acids, C8-10, tetraesters with pentaerythritol

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:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (shorter exposure period. Lack of data on test substance, no positive controls for 40 h time point)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

(in both experiments, cultures without metabolic activation were exposed to the test substance for about 16 h, no positive control for the 40 h time point)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

189200-42-8

Cas Number:

189200-42-8

IUPAC Name:

189200-42-8

Details on test material:

- Name of test material: only trade name given
- Physical state: pale yellow liquid
- Analytical purity: no data
- 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: McCoy's 5A Medium containing 10% (v/v) fetal bovine serum and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

other: WBL clone

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague-Dawley rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

40, 80 and 160 µg/mL with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on results of a solubility test, acetone was selected as the vehicle. The test substance was not soluble in water or dimethyl sulfoxide at any of the concentrations (10, 25, 50% (v/v)) tested. The test substance was soluble as a 50% mixture in acetone.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - S9: N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 µg/mL (v/v) in acetone; + S9: 7,12-Dimethylbenz[a]anthracene (DMBA), 10 µg/mL (v/v) in acetone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
+S9: ca. 3 h (± 0.5 h)
- S9: ca. 16 h (± 0.5 h)
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 16 h (± 0.5 h); second experiment - ca. 16 and 40 h (± 0.5 h)

SPINDLE INHIBITOR (cytogenetic assays): 0.2 mL Colcemid® (10 mg/mL (v/v) in cell culture medium)
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2 replications (16 h) and 1 replication (40 h), respectively

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

Evaluation criteria:

A test substance was considered positive in the chromosome aberration test if:
1. A statistically significant dose-related increase in the percentage of aberrant cells and in at least one of the treatment groups, the percentage of aberrant cells exceeds 5%. OR
2. A reproducible and statistically significant response for at least one of the treatment groups is observed. In addition, the mean percentage of aberrant cells exceeds 5%.
A positive result indicates that under the test conditions the test substance induces chromosomal aberrations in cultured mammalian somatic cells.
If neither of the above conditions exist, the test substance is considered nonmutagenic or negative for inducing chromosomal aberrations in this system.

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance is not soluble in water, therefore it was dissolved in acetone.
- Precipitation: Final concentrations of the test substance in medium of 10, 20, 39, 78, 156, 313, 625, 1250 and 2500 µg/mL were tested by visual and microscopic methods for precipitation immediately, 30 minutes and 3 h after dosing. Traces of the test substance were observed microscopically at all test concentrations equal to or greater than 78 µg/mL. Therefore, the upper limit of the culture medium solubility of the test substance was considered to be between 39 and 78 µg/mL. Based on these results, the study director selected the following concentrations for the toxicity pretest: 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL.
In the main experiments, slight precipitation was observed in the second experiment after 16 h at 160 µg/mL without metabolic activation. Precipitation was not noted at any other harvest of a 160 µg/mL culture.

RANGE-FINDING/SCREENING STUDIES: To determine a concentration selection for the aberration assay, a toxicity pretest was conducted with concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL of the test substance with and without metabolic activation. The concentrations tested were based on the results of a culture medium solubility test. The cultures with metabolic activation were treated for 3 h (± 0.5 h). The cultures without metabolic activation were treated until 2-3 h prior to harvest. All cultures were harvested about 16 h from the beginning of treatment. After harvest, the number of cells that survived treatment were counted using a hemacytometer to evaluate cytotoxicity and the mitotic indices (number of mitotic cells per 1000 total cells) were determined to evaluate cell cycle suppression. The selected concentrations for the aberration assay were based on the results of the cell count data and mitotic index data. The highest reduction in cell survival was observed at 160 µg/mL without metabolic acvtivation, where reduction in viability of 37% was noted. Other less notable reductions in cell survival were noted (see table 3), but were not indicative of a concentration-related trend. Based on these results, the concentrations selected for the aberration assay were 40, 80 and 160 µg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1

Test item	Concentration	Mitotic Index	Aberrant cells	Aberration frequency
	in µg/mL	in %	in %	in %
Exposure period 16 h, fixation time 16 h, without S9 mix
vehicle	0.5% (v/v)	6.8	0.5	0.5
MNNG	0.6	6.2	22.5**	24.5
Test substance	40	5.6	0.5	0.5
	80	6.5	0.5	0.5
	160	7.2	1.0	1.0
Exposure period 3 h, fixation time 16 h, with S9 mix
Acetone	0.5% (v/v)	5.5	1.0	1.0
DMBA	10	2.6	33.5**	42.0
Test substance	40	5.3	1.5	1.5
	80	6.3	0.5	0.5
	160	4.6	2.0	2.0

 

**statistically significantly higher than vehicle control (p<0.001)

MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)

 

Table 2. Test results of experiment 2

Test item	Concentration	Mitotic Index	Aberrant cells	Aberration frequency
	in µg/mL	in %	in %	in %
Exposure period 16 h, fixation time 16 h, without S9 mix
vehicle	0.5% (v/v)	7.2	1.0	1.0
MNNG	0.6	5.4	17.5**	17.5
Test substance	40	7.1	1.0	1.0
	80	5.4	0.5	0.5
	160	7.1	2.5	2.5
Exposure period 3 h, fixation time 16 h, with S9 mix
Acetone	0.5% (v/v)	2.2	0.0	0.0
DMBA	10	4.5	33.0**	43.0
Test substance	40	2.2	1.0	1.0
	80	2.4	2.0	2.0
	160	2.0	1.5	1.5
Exposure period 16 h, fixation time 40 h, without S9 mix
Acetone	0.5% (v/v)	3.4	2.5	2.0
MNNG #	0.6	---	---	---
Test substance	40	3.0	4.0	4.5
	80	2.2	3.0	3.0
	160	3.4	2.0	2.0
Exposure period 3 h, fixation time 40 h, with S9 mix
Acetone	0.5% (v/v)	4.8	2.5	2.5
DMBA #	10	---	---	---
Test substance	40	5.4	2.0	2.0
	80	4.8	2.0	2.0
	160	5.0	0.5	0.5

 

**statistically significantly higher than vehicle control (p<0.001)

MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)

# According to the study report, positive controls were not required for the 40 h harvest.

Table 3. Toxicity pretest results

Treatment Group in µg/mL	Cell Survival in %*
	+ S9	- S9
non-treated	107	102
vehicle	100	100
0.625	122	114
1.25	95	72
2.5	121	88
5	108	68
10	111	108
20	89	102
40	99	93
80	78	93
160	120	63

  * % cell survival as compared to vehicle

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative