Dimethyl sebacate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: other: chromosome aberration and aneuploidy

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 February 2016 - 21 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26 September 2014

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl sebacate

EC Number:

203-431-4

EC Name:

Dimethyl sebacate

Cas Number:

106-79-6

Molecular formula:

C12H22O4

IUPAC Name:

1,10-dimethyl decanedioate

Test material form:

other: white solid

Details on test material:

storage at room temperature

Specific details on test material used for the study:

- Name of test material: Dimethyl Sebacate
- Synonym: DIMETHYL SEBACATE(DMS)
- CAS No.: 106-79-6
- Batch No.: 20150702
- Description: colorless solid to transparent liquid (the test item is a colorless solid below +31°C and it is a transparent liquid above +31°C)
- Analytical purity: 99.7%
- Expiry date: 12 June 2017
- Storage condition: at room temperature.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Justification: rodent species generally accepted by Regulatory Authorities for this type of study
- Source: breeder: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: approximately 6 weeks old on the day of treatment
- Mean body weight at study initiation: the mean body weight was 188 g (ranging from 173 g to 211 g) at the beginning of the main study
- Housing: the animals were housed by three to five per group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the day of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 30 to 70%
- Air changes (per hr): at least 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 16 February 2016 to 21 March 2016.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Concentration of test material in vehicle: 50, 100 and 200 mg/mL
- Justification for choice of vehicle: the test item was found soluble in the vehicle, which was compatible with the test system and the route of administration
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test item solutions were prepared according to an homogeneity and stability study. These solutions were stable up to 10 days at room temperature, based on test item dose formulation stability.

Duration of treatment / exposure:

Two treatments separated by 24 hours (+/- 2 hours)

Frequency of treatment:

One treatment per day.

Post exposure period:

Sacrifice: Animals from the test item-treated groups and from the vehicle control group were sacrificed between 18 and 24 hours after the last treatment and animals from the positive control group were sacrificed between 18 and 24 hours after the single treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

- preliminary dose range finding test: 6 male rats were used,
- main study (micronucleus test): 28 male rats were used,
- blood sampling: 3 male rats were used.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate
- Route of administration: oral
- Frequency: one treatment on Day 2
- Volume: 10 mL/kg.

Examinations

Tissues and cell types examined:

Bone marrow: polychromatic (PE) and normochromatic (NE) erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The high test item dose formulation used in the main study (micronucleus test) was based on toxic effects observed in the preliminary dose range finding test. For this, a single preliminary assay was performed on two groups of three males each (one group treated with the test item and another group treated with the vehicle).
Since no toxic effects were observed in the animals given the test item at 2000 mg/kg/day, this dose-level was selected as the top dose-level for the main study (micronucleus test) in accordance with the criteria specified in the international guidelines. The two other selected dose-levels were 1000 and 500 mg/kg/day.

SAMPLING TIMES:
At sacrifice, between 18 and 24 h after the last treatment.

DETAILS OF SLIDE PREPARATION:
After sacrifice, the femurs were removed and bone marrow was flushed and suspended in fetal calf serum. The separation of anucleated erythrocytic cells from other myeloic cells was carried using a cellulose column. This elution step enables the production of slides containing only polychromatic and normochromatic erythrocytes without any nucleated cells or mast cell granules. After centrifugation of the eluate containing the cells, the supernatant was removed and the cells in the sediment were resuspended. A drop of this cell suspension was placed and spread on a slide. At least two slides were prepared for each animal. The slides were air-dried and stained with Giemsa and then coded for "blind" scoring.

METHOD OF ANALYSIS:
For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 Polychromatic Erythrocytes; the
Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 Erythrocytes (PE + NE).

Evaluation criteria:

Evaluation of a positive response: a test item is considered clearly positive if all the following criteria are met:
- a statistically significant increase in the frequency of MPE is observed when compared to the vehicle control group,
- the increase in the frequency of MPE is dose-related,
- for at least one dose-level, the frequency of MPE is above the corresponding vehicle historical range.

Evaluation of a negative response: a test item is considered negative if none of the criteria for a positive response are met.

Statistics:

The analyses of MPE/1000PE and PE/NE ratios were performed with SAS software.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw/day
- Clinical signs of toxicity in test animals: none

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction
- Ratio of PCE/NCE (for Micronucleus assay): The mean values of the PE/NE ratio in the groups treated with the test item were not statistically significantly different from that of the respective vehicle control animals (0.40 to 0.48 for test item-treated groups vs. 0.56 for the vehicle control group). Thus no toxicity of the test item for the bone marrow was evidenced.
- Appropriateness of dose levels and route: oral route, since it is a possible route of Human exposure. Highest recommended dose level since no toxic effects were recorded in the range-finding study.
- Statistical evaluation: No statistically significant difference was noted in the mean value of MPE in the groups treated with the three dose-levels of test item and no dose-response relationship was evidenced.

Applicant's summary and conclusion

Conclusions:

The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells, after two oral administrations, 24 hours apart, at dose-levels of 500, 1000 and 2000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of rats.

 

The study was performed according to the international guidelines (OECD guideline No. 474 and Commission Directive No. B.12) and in compliance with the Principles of Good Laboratory Practice.

 

Methods

A preliminary dose range finding test was performed to define the dose-levels to be used for the main study (micronucleus test).

 

In the main study (micronucleus test), Sprague-Dawley male rats received two oral treatments of the test item at the dose-levels of 500, 1000 and 2000 mg/kg/day, at a 24-hour interval. Five rats were treated with the low and intermediate dose-levels, and eight rats were treated with the high dose-level to ensure sufficient animals were available for micronucleus analysis.

One group of five males received the vehicle (corn oil) under the same experimental conditions, and acted as vehicle control group. One group of five males received the positive control item (cyclophosphamide) once by oral route at the dose-level of 15 mg/kg/day.

Moreover, for the evaluation of plasma level of the test item, three satellite males received two oral treatments 24 hours apart, of the test item at the highest tested dose-level of 2000 mg/kg/day.

 

Animals from the test item-treated groups and from the vehicle control group were sacrificed between 18 and 24 hours after the last treatment and animals from the positive control group were sacrificed between 18 and 24 hours after the single treatment. Bone marrow smears were then prepared.

 

For each animal of each group, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 4000 Polychromatic Erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocytes ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Results

 

Mortality and clinical signs in the main study (micronucleus test)

No unscheduled mortality was observed in any groups during the main study.

A reflux at dosing was observed on Day 2 in one of the supernumerary males treated at 2000 mg/kg/day.

No other clinical signs were recorded in any other treated animals.

Body weight

No relevant changes in body weight were observed in treated groups in comparison to the vehicle group.

 

Cytogenetic test results

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data.

Moreover, cyclophosphamide induced a highly significant increase in the frequencies of MPE, indicating the sensitivity of the test system under our experimental conditions tested. The study was therefore considered to be valid.

 

The mean values of the PE/NE ratio in the groups treated with the test item were not statistically significantly different from that of the respective vehicle control animals.

The mean values of MPE in the groups administered the test item at 500, 1000 and 2000 mg/kg/day were similar to those of the vehicle control animals. No statistically significant difference was noted, no dose-response relationship was evidenced and all the frequencies obtained remained within the vehicle historical range. Therefore, these results met the criteria of a negative response.

Conclusion

The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells, after two oral administrations, 24 hours apart, at dose-levels of 500, 1000 and 2000 mg/kg/day.