2-Butenedioic acid (E)-, di-C16-18-alkyl esters

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Publication with summary of results only; comparable to guideline study with acceptable restrictions (no data on test substance purity, limited documentation)

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Evaluation of a Three-Exposure Mouse Bone Marrow Micronucleus Protocol

Author:

Shelby, M.D. et al.

Year:

1993

Bibliographic source:

Environmental and Molecular Mutagenesis 21: 160-179

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 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(no data on test substance purity, no samples for the peripheral blood between 36 and 48 h following the final treatment were collected))

GLP compliance:

not specified

Type of assay:

micronucleus assay

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

Molecular formula:

C22H42O4

IUPAC Name:

bis(2-ethylhexyl) adipate

Details on test material:

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

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Toxicology Program production facility at Taconic Farms
- Age at study initiation: 9 - 14 weeks
- Weight at study initiation: 25 - 33 g

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

corn oil

Duration of treatment / exposure:

3 days

Frequency of treatment:

one injection (0.4 mL) per day (3 treatments)

Post exposure period:

24 h after the last treatment

Doses / concentrations

Remarks:

Doses / Concentrations:
375, 750, 1500 and 2000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

5 males (dose groups and negative control)
40 males (positive control)
70 males (vehicle control)

Control animals:

yes, concurrent vehicle

Positive control(s):

7,12-dimethylbenzanthracene (DMBA) in corn oil

The following substances were used to develop the test protocol:
7,12-dimethylbenzanthracene; mitomycin C; benzidine
- Justification for choice of positive control(s): 7,12-dimethylbenzanthracene is slowly absorbed and metabolized; benzidine is an in vivo micronucleus inducer that is difficult to detect; mitomycin C is water soluble, direct acting and has an in vivo half live < 1h.

A protocol of 3 daily treatments (intraperitoneal injections) and a single sample time at 24 h following the final treatment was found to be the most effective for detecting all 3 postive control substances.

Examinations

Tissues and cell types examined:

Tissue: bone marrow
Cell type: erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Dose determination study: The selection of the maximum doses for the MN induction was based on either mortality, administration characteristics, depression in the percentage of bone marrow PCE (no less than 15% of the erythrocytes), or on the arbitrary maximum dose of 2000 mg/kg bw/day. Groups of 5 mice were administered the test stubstances by ip injection on three consecutive days at 200, 1000 and 2000 mg/kg bw. Animals were monitored twice daily, and 48 h after the last treatment, the surviving mice wer killed and bone marrow and peripheral blood smears (2 slides/tissue/mouse) were prepared. Based on the percentage of PCE among 200 erythrocytes, the maximum adminstration dose was estimated. As no evidence of toxicity at the daily dose of 2000 mg/kg bw/day was observed, this was the highest dose used in the main MN test.

TREATMENT AND SAMPLING TIMES: Groups of 5 mice were injected ip on 3 consecutive days with the test substance or the corresponding controls. Mice were euthanized with CO2 24 h after the third treatment.

DETAILS OF SLIDE PREPARATION: Bone marrow smears (2 slides per mouse) were prepared, fixed in absolute methanol, and stained with acridine orange.

METHOD OF ANALYSIS: Slides were evaluated at 1000x magnification for the number of MN-PCE among 2000 PCE and for the percentage of PCE among 200 erythrocytes.

Evaluation criteria:

Conclusions are based on the statistical analysis of trend and of pairwise comparisons of the solvent control with individual doses and the absolute increase in MN-PCE frequency.

Statistics:

The data were analyzed using the Micronucleus Assay Data Management and Statistical software package. The level of signifcance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE , the number of MN-PCE were pooled within each dose group and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variablity. The %PCE data were analyzed by an anylsis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each group and the concurrent solvent control group was by an unadjusted one-tailed Pearson chi-squared test which incorporated the calculated variance inflation factor for the study.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Result of the mouse bone marrow micronucleus test

Dose (mg/kg bw)	MN-PCE/1000^a)(No. of animals)	Survival	% PCE^b)
Corn oil (vehicle control)	2.12 ± 0.70	(14 groups)	Range: 1.1-3.7
0 (negative control)	2.5 ± 0.41 (4)	4/5	64.4
375	3.4 ± 0.81 (5)	5/5	39.5
750	2.3 ± 0.30 (5)	5/5	59.0
1500	2.4 ± 0.51 (5)	5/5	60.2
2000	2.6 ± 0.58 (5)	5/5	47.2
DMBA	7.93 ± 1.69	(8 groups)	Range: 4.4-10.6

^a)Micronucleated PCEs per 1000 PCE scored

^b)Percentage of erythrocytes that were polychromatic

DMBA = dimethylbenzanthracene (12.5 mg/kg bw/day)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative