Diisopropyl adipate

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 based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

13 Feb 2002 - 14 May 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(adopted in 1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl adipate

EC Number:

203-350-4

EC Name:

Dibutyl adipate

Cas Number:

105-99-7

IUPAC Name:

dibutyl adipate

Details on test material:

- Name of test material (as cited in study report): Hexanedioic acid, Dibutyl ester; Adipinsäuredibutylester.
- Batch No: 13290016
- Physical state: colourless liquid
- Purity: 100 % active substance
- Stability in vehicle: not indicated by the sponsor
- Storage: at room temperature, light protected
- Expiration date: November, 2002

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Füllinsdorf, Switzerland.
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males: 35.1 ± 2.6 g, females: 26.8 ± 2.9 g
- Assigned to test groups randomly: yes
- Fasting period before study: animals were fasted 18 h prior to treatment
- Housing: single Makrolon type 1 cages with wire mesh tops on soft wood bedding.
- Diet: pelleted standard diet, ALTROMIN 1324, Lage, Germany, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 30 - 70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: relative non-toxicity for the animal.
- Amount of vehicle: 10 mL/kg bw
- Supplier: SIGMA-Aldrich Vertriebs-GmbH, Deisenhofen, Germany.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Dosing volume: 10 mL/kg bw
The test substance was formulated in olive oil on day of experiment

Duration of treatment / exposure:

Single administration

Frequency of treatment:

Single administration

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested
for 24 h sampling

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
for 48 h sampling

No. of animals per sex per dose:

5
(6 animals per sex, group and sampling time were treated, and 5 were finally used for evaluation)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;

- Route of administration: orally
- Doses / concentrations: 40 mg/kg bw
- Supplier: SIGMA-Aldrich Vertriebs-GmbH, Deisenhofen, Germany.

Examinations

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The highest dose (2000 mg/kg bw, maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
24 h and 48 h after administration bone marrow cells were collected for micronuclei analysis.

DETAILS OF SLIDE PREPARATION:
The femora of sacrified animals were removed, epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The centrifuged cell suspension was spread on a slide, air-dried and stained with May-Grünwald/Giemsa. At least one slide was made from each bone marrow sample.
Microscopical evaluation of slides with 100x oil immersion objectives.

METHOD OF ANALYSIS:
At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment the ratio between poly- and normochromatic erythrocytes was determined in the same sample and reported as the number of NCEs per 2000 PCEs.

Evaluation criteria:

Acceptance criteria:
- at least 80 % of animals evaluable
- dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
- comparison with historical control data (negative controls mean 0.086 +/- 0.032 PCEs with micronuclei; positive controls mean 1.678 +/- 0.479 PCEs with micronuclei)
Evaluation criteria:
A test substance is judged positive in the mouse micronucleus assay if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group

Statistics:

nonparametric Mann-Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2000 mg/kg bw group: 1 h post treatment reduction of spontaneous activity in 12/12, abdominal position in 3/12, eyelid closure in 4/12 and ruffled fur in 8/12 were evident the animals fully recovered within 24 h

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study.
The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of around 1, 2-4, 6, 24, 30 and 48 hours after administration of the test item.
At 2000 mg/kg bw no signs of toxicity were seen, except for reduction in spontaneous activity affecting all 4 animals after 1 h. After 24 hours, no more effects were seen. Therefore this dose was deemed to be suitable.


RESULTS OF DEFINITIVE STUDY:
- Induction of micronuclei (for Micronucleus assay):
In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval and with any dose level.
- Ratio of PCE/NCE (for Micronucleus assay):
The ratio was not substantially different compared to the mean values of the vehicle control, indicating that the test item had no cytotoxic effects in the bone marrow.
- Toxicity: At 2000 mg/kg bw following transient signs of toxicity were observed: 1 h post treatment reduction of spontaneous activity in 12/12 mice, abdominal position in 3/12 mice, eyelid closure in 4/12 mice and ruffled fur in 8/12 mice. Within 24 h the animals gradually recovered.

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay in NMRI mice (average values)

Experimental group	Number of animals (m/f)	Sampling time (hours after administration)	Dose (mg/kg bw)	PCEs with micronuclei (%)	Range of micronucleated cells per 2000 PCEs	PCE/NCE
Vehicle control (olive oil)	5/5	24	0	0.085	0 - 3	2000/1650
Positive control (Cyclophosphamide)	5/5	24	40	1.200	10 - 37	2000/2235
Test substance	5/5	24	500	0.065	0 - 4	2000/1677
Test substance	5/5	24	1000	0.135	0 - 7	2000/1686
Test substance	5/5	24	2000	0.040	0 - 5	2000/1719
Test substance	5/5	48	2000	0.090	0 - 3	2000/1776

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative