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Toxicological information

Genetic toxicity: in vivo

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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:
weight of evidence
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

Constituent 1
Chemical structure
Reference substance name:
Dibutyl adipate
EC Number:
203-350-4
EC Name:
Dibutyl adipate
Cas Number:
105-99-7
Molecular formula:
C14H26O4
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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested
for 24 h sampling
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

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