Didodecyl fumarate

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 from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report which meets basic scientific principles (NTP study type).

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Chromosome Aberrations and Sister Chromatid Exchanges in Chinese Hamster Ovary Cells: Evaluations of 108 Chemicals

Author:

Galloway, S.M. et al.

Year:

1987

Bibliographic source:

Environmental and Molecular Mutagenesis 10(10):1-175

Reference 2

Reference Type:

secondary source

Title:

Diesters Category of the Aliphatic Esters Chemicals (Test Plan and Robust Summaries for Substances in the HPV Test Plan)

Author:

US-EPA (American Chemistry Council's Aliphatic Esters Panel)

Year:

2010

Bibliographic source:

High Production Volume (HPV) Chemical Challenge Program (201-16837A and 201-16837B)

Reference 3

Reference Type:

secondary source

Title:

Bis(2-ethylhexyl)adipate (DEHA) CAS N°: 103-23-1

Author:

OECD

Year:

2000

Bibliographic source:

SIDS Initial Assessment Report for SIAM 10; Tokyo, Japan, 15-17 March 2000

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

(no duplicate culture; no data on cytotoxicity; differing tretament times; no continuous treatment; sampling time not equivalent to about 1.5 normal cell cycles; no data on gaps and gaps are not included in analysis)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) adipate

EC Number:

203-090-1

EC Name:

Bis(2-ethylhexyl) adipate

Cas Number:

103-23-1

IUPAC Name:

bis(2-ethylhexyl) adipate

Details on test material:

- Name of test material (as cited in study report): Di(2-ethylhexyl)adipate, DEHA
- Analytical purity: no data

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

other: cloned Chinese hamster ovary cells (CHO-W-B1)

Details on mammalian cell type (if applicable):

- Type and identity of media:
Mc-Coy's 5a medium with 10% fetal calf serum, L-glutamine and antibiotics

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

40, 130, 400 µg/mL with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water, DMSO, ethanol or acetone in that order of preference
- Justification for choice of solvent/vehicle: depending on solubility

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: triethylenemelamine (TEM, 0.15 µg/mL) (without S9) and cyclophosphamide (CP, 15 µg/mL) (with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 14 hours without metabolic activation, 2 hours with metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 14 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Results of one experiment are reported.

NUMBER OF CELLS EVALUATED: 100 cells per dose from each of the three highest dose groups having sufficient metaphases for analysis

DETERMINATION OF CYTOTOXICITY
- Method: cell monolayer confluence and mitotic activity, cell cycle delay

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The analysis examined the evidence for a dose relation and the absolute increase over the solvent control at each dose. All types of aberrations were recorded seperately, but for data analysis they were grouped into categories of "simple" (breaks and terminal deletions), "complex" (exchanges and rearrangements), "other" (includes puleverized chromosomes) and "total". Gaps and endoreplications were recorded but were not included in the totals. Aberrations and polyploid cells were not scored but metaphases with 19-23 chromosomes were used.
A decision scheme was used to combine the results of the trend test with the evaluation at individual doses for aberration analyses. A test with one dose that is positive but without a positive trend test is considered “?”. The designation “weak-positive’’ (“ + w”) was used in cases for which there was weak evidence for a positive response and is not an indication of potency. However, if there was a very strong trend as a result of a large increase in aberrations at a single dose, we called the result ‘‘w + *” to denote the fact that the level of aberrations was high (see decision table under Any other information on material and methods including tables).

Statistics:

Linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binominal sampling assumption (as opposed to Poison) was used, and the test was that described by Margolin et al. (1983). The p values were adjusted by Dunnett's method to take into account the multiple dose comparisons. For data analysis, the "total" aberration category was used, and the criterion for a positive response was that the adjusted P value be < 0.05.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Initially, dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hours after treatment. The top doses selected for the cytogenetic assay were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analyses of aberration. The aim was to obtain results at the highest dose at which sufficient metaphase cells would be available for analysis. Observations on cell growth and cell cycle kinetics from the SCE test were also used to select the doses and fixation times for the chromosome aberration tests.

COMPARISON WITH HISTORICAL CONTROL DATA: not included in statistical analysis due to variability from day to day and from reader to reader, control data were however valuable in forming the decision scheme for data evaluation and are useful in deciding wether to repeat an assay

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table2a: CA results - Short Overview

	-S9	+S9	Summary
Result	+W	-	+W
Range	40 - 400	40 - 400
LEC	400	-

Least Effect Concentration tested (LEC) is 400 µg/mL which is the highest concentration tested. This concentration showed a high trend but due primarily to a statistically significant increase at this single dose.

Table2b: CA results - Detailed overview

without S9 mix
DOSE	Cells	Percent cells with aberrations
(µg/mL)		Total	Simple	Complex
0.0	100	1	1	0
40.0	100	3	2	1
130.0	100	4	2	2
400.0	100	7*	5	2
positive control - TEM
0.15	100	28	18	15
with S9
0.0	100	2	2	0
40.0	100	5	5	0
130.0	100	5	5	0
400.0	100	6	5	1
positive control CP
15.0	100	35	18	22

*In the tables, any value significantly greater than the concurrent control (Dunnett's adjusted P < 0.05) is marked with an asterisk.

Simple: breaks and terminal deletions

Complex: exchanges and rearrangements

Total: sum of all types of aberrations (simple + complex + other); Gaps and endoreduplications were recorded but were not included in the totals.

Applicant's summary and conclusion

Conclusions:

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