Didocosyl sebacate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Apr - 10 Jun 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Version / remarks:

(2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)

Version / remarks:

(1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

Didocosyl sebacate

EC Number:

255-730-4

EC Name:

Didocosyl sebacate

Cas Number:

42233-75-0

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Decanedioic acid, diesters with Fatty alcohols C20-22 (even numbered)

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Physical state: white solid
- Storage condition of test material: room temperature in the dark
- Expiration date of the lot/batch: 30 Jun 2013

Test animals

Species:

rat

Strain:

other: RCCHan(TM)WIST

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK, Ltd., Oxon, UK
- Age at study initiation: 7 - 12 weeks
- Weight at study initiation: 186 - 218 g
- Housing: Animals were housed in groups of five in solid-floor polypropylene cages with woodflake bedding (Datesand Ltd., Cheshire, UK)
- Diet: 2014C Teklad Global Certified Rodent diet (Harlan Laboratories UK, Ltd., Oxon, UK), ad libitum
- Water: (tap/filtered) water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 – 25
- Humidity (%): 30 – 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: Arachis oil was selected as the solvent based on data from Harlan Laboratories Ltd. (28 Day Repeated Dose Oral Toxicity Study in the rat).
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was freshly prepared as required as a suspension at the appropriate concentration in arachis oil. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Duration of treatment / exposure:

Not applicable

Frequency of treatment:

single treatment

Post exposure period:

24 and 48 h after treatment

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

7 males (test group and negative control)
5 males (positive control)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): Due to the known properties of inducing chromosomal aberrations cyclophosphamide was selected as the appropriate positive control.
- Route of administration: intraperitoneal
- Doses / concentrations: 25 mg/kg bw / 2.5 mg/mL

Examinations

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow cells

Details of tissue and slide preparation:

DETAILS OF TISSUE AND SLIDE PREPARATION:
Two to four hours prior to sample collection, animals are injected intraperitoneally with 4 mg/kg Colchicine, and samples are collected 2-4 hours thereafter. Cells are harvested from the bone marrow, swollen, fixed and stained, and analyzed for chromosomal aberrations.

CRITERIA FOR DOSE SELECTION:
A range finding study was performed to find the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
One group of rats from each dose level was killed by cervical dislocation approximately 24 hours following treatment and a second group dosed with 2000 mg/kg bw was killed at approximately 48 hours.

DETAILS OF SLIDE PREPARATION:
Slides were fixed and stained with 5% Giemsa-solution for 10 minutes.

METHOD OF ANALYSIS:
100 metaphase cells of adequate quality were scored per animal for both numerical and structural chromosome aberrations.

OTHER:
A mitotic index (MI) value was also obtained for each animal by recording the number of metaphase cells that were associated with 1000 cells.

Evaluation criteria:

Experiments with rat bone marrow cells have established a range of aberration frequencies acceptable for vehicle control animals; these are commonly in the range of 0 to 3% cells with structural aberrations. A positive response was recorded for a particular treatment if the % cells with aberrations markedly exceeded that seen in the vehicle control. For modest increases in aberration frequency, appropriate statistical tests may be applied in order to record a positive response.

Statistics:

Comparisons were made between the vehicle control group and each treatment dose group, with a chi-squared test, using observed numbers of cells with aberrations. Analysis of mitotic index data was performed using a Student´s t-Test.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: In animals dosed with the test item at 2000 mg/kg bw via the oral and intraperitoneal routes there were no clinical signs or premature deaths observed.
- Evidence of cytotoxicity in tissue analyzed: The test item showed no difference in its toxicity to male or female rats; therefore the main test was performed using male rats only.
- Rationale for exposure: The maximum recommended dose of 2000 mg/kg bw was selected as the top dose.

RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: No marked decreases in the mitotic index mean value were observed in any of the test item dose groups when compared to the vehicle control group. There was no evidence of a statistical significant increase in the incidence of cells with chromosome aberrations excluding gaps in animals dosed with the test item, when the dose groups were compared to the vehicle control group. Two of the test item dose groups, 24-hour 2000 mg/kg bw and 24-hour 1000 mg/kg bw, both had animals which did not have 100 metaphases suitable for scoring, but since there was no marked response in these dose groups this was considered to be acceptable. The test item did not induce a significant increase in the numbers of polyploid cells in any of the treatment groups. The positive control group animals showed highly significant increases in the frequency of aberrations indicating that the test method itself was operating as expected. It should be noted that due to the toxic response seen with cyclophosphamide in the bone marrow there were insufficient metaphases for scoring in one of the animals and this animal was excluded from scoring. Since there was an adequate response seen in the remaining animals this was considered to be acceptable.

Any other information on results incl. tables

MORTALITY DATA:

There were no premature deaths seen in any of the test item dose groups, except in the 24 -hour maximum dose level group (2000 mg/kg bw) where one animal was lost shortly after dosing but this was considered due to a technical error during dosing rather than the effects of the test item.

Table 1: Bone marrow chromosome analysis - dose dependence

	Dose (mg/kg bw)
	0	500	1000	2000	CPA
Post-exposure period [h]	24	24	24	24	24
Total no. of animals	7	7	7	7	4
Analyzed metaphases	700	700	655	573	250
Aberrant metaphases (%)
Including gaps	0.4	0.6	0.9	0.7	29.2
Excluding gaps	0.4	0.4	0.6	0.5	26.8***
Polyploidy	0	0	0	0	0
Mitotic Index (MI)	1.41	2.74	2.00	1.67	0.60

CPA: Cyclophosphamide

***= P< 0.001

Table 2: Bone marrow chromosome analysis - time dependence

	Dose (mg/kg bw)
	0	2000	2000	CPA
Post-exposure period [h]	24	24	48	24
Total no. of animals	7	7	7	4
Analyzed metaphases	700	573	700	250
Aberrant metaphases (%)
Including gaps	0.4	0.7	0.7	29.2
Excluding gaps	0.4	0.5	0.7	26.8***
Polyploidy	0	0	0	0
Mitotic Index (MI)	1.41	1.67	1.91	0.60

CPA: Cyclophosphamide

***= P< 0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test item did not induce any significant or dose-related increases in the frequency of chromosome aberrations. The test item was considered to be non-clastogenic to rat bone marrow cells in vivo.