Registration Dossier

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:
key study
Study period:
February 13th, 2002 - May 14th, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance dibutyl adipate (CAS 105-99-7). In accordance 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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 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: [no/yes, under following basis: ]
- Fasting period before study: no data
- Housing: single Makrolon type 1 cages with wire mesh tops on soft wood bedding.
- Diet (e.g. ad libitum): pelleted standard diet, ALTROMIN 1324, Lage, Germany.
- Water (e.g. ad libitum): tap water
- 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 (if gavage or dermal): 10 mL/kg b.w.
- Supplier: SIGMA-Aldrich Vertriebs-GmbH, Deisenhofen, Germany.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Dosing volume: 10 mL/kg b.w.
Duration of treatment / exposure:
Single administration
Frequency of treatment:
Single administration
Post exposure period:
Sampling after 24 and 48 h.
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
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide;

- Route of administration: orally
- Doses / concentrations: 40 mg/kg b.w.
- 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, 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.
Evaluation of slides with 100x oil immersion objectives.

METHOD OF ANALYSIS:
At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.
Evaluation 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)
Statistics:
nonparametric Mann-Whitney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
no clinical signs of systemic toxicity observed; no indication for cytotoxicity as the ratio of NCE/PCE was similar in test and control groups;
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 concerning: starvation period, animal strain, vehicle, route, frequency, and volume of administration.
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 no toxic reactions besides a reduction of spontaneous activity were observed. Therefore this dose was estimated 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.

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

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 mg/kg

1.200

10 - 37

2000/2235

Test substance

5/5

24

500 mg/kg

0.065

0 - 4

2000/1677

Test substance

5/5

24

1000 mg/kg

0.135

0 - 7

2000/1686

Test substance

5/5

24

2000 mg/kg

0.040

0 - 5

2000/1719

Test substance

5/5

48

2000 mg/kg

0.090

0 - 3

2000/1776

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative The test substance did not show genotoxic effects in this micronucleus assay in the mouse.