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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
February 10th to 23rd, 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Reliability of original study is 1
Justification for type of information:
Justification for Read Across is given in Section 13 of IUCLID.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Adopted July 21st, 1997
GLP compliance:
yes
Type of assay:
other: in vivo micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate
EC Number:
700-636-5
Cas Number:
5413-49-0
Molecular formula:
C10H1804
IUPAC Name:
Ethyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
This species has been routinely used as an animal model of choice for the mammalian bone marrow erythrocyte micronucleus assay. This strain was an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to the test substance.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD.
- Age at study initiation: 6 weeks old.
- Weight at study initiation: 189.9-200.0 g, 196.7-207.3 (DRF, males); 150.9-160.0 g, 156.5-162.6 g (DRF, females).
- Assigned to test groups randomly: yes. Animals were assigned to groups using a randomization procedure within Microsoft Excel.
- Housing: animals of the same sex were housed up to five per Micro-Barrier cage. Heat treated hardwood chips were used for bedding to absorb liquids.
- Diet: ad libitum, certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet).
- Water: ad libitum, tap water.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 °F.
- Humidity: 50 ± 20 %.
- Air changes: 10 changes of fresh HEPA-filtered air per hour.
- Photoperiod: 12-hour light/dark cycle.
- Other: cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- CAS No: 8001-30-7
- Lot/Batch no: M6581
- Expiration date: 15 January 2017
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dose formulations were prepared prior to dose administration as follows: appropriate amounts of test article and vehicle were added to an amber vial.

EXPOSURE TO THE SUBSTANCE
- Dose volume: 10 ml/kg/day.
- Mode of exposure: by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles).
- Animals were treated once on each of three consecutive days. The second dose occurred approximately 24 hours (24 hours ± 30 mins) after the first dose and the third dose occurred approximately 24 hours (24 hours ± 30 mins) after the second dose.

FREQUENCY OF OBSERVATIONS/PARAMETERS MEASURED
- Body weights: recorded prior to the first dose for the purpose of dose volume calculations and once on the day of sacrifice.
- Clinical observations: animals were observed prior to, approximately one and two hours after each dose administration and daily thereafter for clinical signs of toxicity.
- Euthanasia: all animals were euthanized by exposure to CO2 approximately 24 hours post the final dose.
Duration of treatment / exposure:
three days
Frequency of treatment:
single daily treatment
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3 animals/sex
Control animals:
yes, concurrent vehicle

Examinations

Details of tissue and slide preparation:
BONE MARROW COLLECTION. immediately following euthanasia, the femurs were exposed, cut just above the knee and bone marrow was aspirated into a syringe containing fetal bovine serum.

SLIDE PREPARATION: the bone marrow was transferred to a centrifuge tube containing 2 ml fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of fetal bovine serum with the pellet. Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. At least two slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of slides was stained with acridine orange for microscopic evaluation. Each slide was identified by the harvest date, study number, and animal number. Slides were coded using a random number table by an individual not involved with the scoring process.

METHOD OF ANALYSIS: bone marrow was evaluated by fluorescent microscopy. At least 1000 total erythrocytes (polychromatic (PCEs) + normochromatic erythrocytes (NCEs)) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity.
Evaluation criteria:
PCE proportions < 20 % of vehicle control value (i.e., > 80 % reduction) were considered excessively cytotoxic and would indicate bone marrow suppression.
If no toxicity was seen during the doses, the maximum dose of 2000 mg/kg/day may be used for follow-up testing.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- Mortality: no mortality occurred at any dose level during the course of the dose range finding assay.
- Clinical observations: all rats appeared normal throughout the observation period.
- Bone marrow analysis. no appreciable reductions in the PCEs/EC ratio in the test substance groups compared to the vehicle control group was observed indicating the test article did not induce cytotoxicity.

Any other information on results incl. tables

PROTOCOL DEVIATION

For the randomization and dose administration of an additional group used for vehicle control, the Group 4 (vehicle control) female animals, animals 73, 74, and 75 were assigned the incorrect body weight. Therefore, animals were dosed based on an incorrect bodyweight. Animal 73 was dosed with 2.0 ml of vehicle control but should have been dosed with 1.6 ml. Animal 74 was dosed with 2.1 ml of vehicle control but should have been dosed with 1.6 ml. Animal 75 was dosed with 2.1 ml of vehicle control but should have been dosed with 1.6 ml. Because the animals were dosed at the incorrect dose volume, this is a protocol deviation. Evaluation: even though some of the animals were dosed at a higher volume, all animals were normal and did not show any signs of toxicity. Hence, no impact is expected.

Applicant's summary and conclusion

Conclusions:
Non mutagenic under the tested conditions.
Executive summary:

The toxicity of the test substance and its ability to induce bone marrow suppression was evaluated according to the OECD Guideline 474. Test and control substance formulations were administered at a dose volume of 10 ml/kg/day by oral gavage. In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The dose levels tested were 500, 1000 or 2000 mg/kg/day in 3 animals/sex. Following scheduled euthanasia times, femoral bone marrow was collected; bone marrow slides were prepared and stained with acridine orange. Bone marrow cells were examined microscopically. The ratio of polychromatic erythrocytes (PCEs) to total erythrocytes (EC) in the test substance groups relative to the vehicle control groups was evaluated to reflect the test substance’s cytotoxicity.

No bone marrow suppression was observed in any of the groups. No appreciable reductions in the PCEs/EC ratio in the test substance groups compared to the vehicle control group was observed indicating the test article did not induce cytotoxicity. Thus, under the conditions of this study, the test article is non-toxic at and up to 2000 mg/kg in male and female rats.

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