Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jan 2016 - 7 Jul 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Request to provide this particular data in a ECHA decision of Feb 2015

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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
Specific details on test material used for the study:
Identity: DIETHYL CARBONATE
CAS No.: 105-58-8
EINECS No.: 203-311-1
Composition: > 99.5 % diethyl carbonate
Purity: > 99.98 % (GC)
Appearance: clear, colourless liquid
Homogeneity: homogenous
Supplier: Novasol S.A., Mechelsesteenweg 26, BE-1970 Wezembeek-Oppem, Belgium
Batch no. PC 11879

Test animals

Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Hsd: ICR (CD-1) mice
Sex:
male
Details on test animals and environmental conditions:
Supplier of animals: Envigo RMS srl, San Pietro al Natisone (UD), Italy
Number of animals: 50, well received from supplier in good health conditions (for preliminary toxicity tests and Main Assay)
Sex of animals: 40 male, 10 female. Since the preliminary toxicity experiments did not show substantial differences between sexes, the Main Assay was performed including male animals only.

Age of animals: 5 to 6 weeks old at the time of arrival

Acclimatisation: At least five days were allowed for acclimatisation and quarantine. During this period the health status of the animals was assessed by daily observations.

Animal husbandry:
Housing up to 5 per cage, by sex, in polysulphone H-Temp solid bottomed cages with suitable nesting material. Animal room controls
were set to maintain temperature and relative humidity at 22 °C +/- 2 °C and 55% +/- 15% respectively. Actual conditions were monitored daily, recorded and the records retained. The animals were kept in a 12 hour light/dark cycle. Food and drinking water were supplied ad libitum. The animals were maintained on a commercially available laboratory rodent diet (Mucedola 4RF21 S.r.l., Settimo Milanese, Milano, Italy). Records of analysis of drinking water and diet are retained at RTC Veterinary Services.

Allocation of animals:
On the day of arrival, animals were identified by ink marking on the tail. They were weighed and 10% of males had a body weight range between 18.0-21.0 g while 2 females out of 10 had a body weight of 17.0-20.0 g respectively. On the day before treatment of toxicity tests, animals were weighed and allocated to treatment groups. For the Main Assay, animals were weighed and allocated to treatment groups approximately one week before dosing (15-Feb-2016) and were weighed also on the day before dosing (22-Feb-2016).

Administration / exposure

Route of administration:
intravenous
Vehicle:
sterile distilled water of injectable grade (batch no. 15D2902 obtained from Baxter)
Details on exposure:
Selection of administration route:
According to the topical requirements of OECD TG474, a proof of absorption needs to be given. That means evidence needs to be given that the erythrocytes were exposed to the test item (in this case diethyl carbonate).
Therefore, the intravenous injection route was chosen to ensure direct contact of erythrocytes and the test item. The oral administration route was not chosen as the contact and proof of contact of erythrocytes to diethyl carbonate
could have been uncertain.

Selection of dose levels:
The selection of dose levels for the Main Assay, was performed on the basis of two preliminary toxicity experiments. In a first experiment, four groups each of two male and two female animals were dosed at 320, 160, 80.0 and 40.0 mg/kg body weight/day. The highest dose level was selected according to the solubility test performed by the Test Site during the validation of the analytical method for formulation analysis.

Animals were dosed at a rate of administration of 0.5 mL/min (intravenous injection), dose volume was 20 mL/kg body weight. Animals were inspected immediately after dosing, approximately 1 hour after dosing, at the end of the working day and daily throughout the study for signs of reaction to treatment. Besides, they were weighed daily starting from Day 1 of treatment.

Approximately twenty-four hours after the second dosing, animals were sacrificed and bone marrow smear slides were prepared. Scoring was performed on slides prepared from the femurs of all surviving animals. Signs of toxicity were not consistent among the treated groups. Since the most reliable reason for these observations was the formation of the CO2 in the blood as degradation product of the test item depending on the rate of administration of test item solutions, it was considered appropriate to perform an additional toxicity trial where a solution of test item at the maximum concentration of 16.0 mg/mL was administered at the low flow rate of 0.2 mL/min by using a pump set (intravenous injection). Animals were inspected immediately after dosing, approximately 1 hour after dosing, few hours after dosing and daily throughout the study for signs of reaction to treatment. Besides, they were weighed daily starting from Day 1 of treatment. For these animals, it was not considered necessary to prepare bone marrow smear slides.
Duration of treatment / exposure:
All animals were dosed twice at a 24 hour interval (vehicle control & various dose levels of test item), dose volume see under "Details on exposure".
The animals of the positive control group were treated once (dose volume 10 mL/kg body weight).
Frequency of treatment:
All animals were dosed twice at a 24 hour interval (vehicle control & various dose levels of test item).
The animals of the positive control group were treated once (dose volume 10 mL/kg body weight).
Post exposure period:
Animals from the vehicle control group and animals from groups treated with
the test item were sacrificed 22-23 hours after last dosing. Animals from the
positive control group were sacrificed 24 hours after dosing.

Animals were sacrificed by dislocation of the cervical vertebrae.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle; main assay
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
test item; main assay
Dose / conc.:
160 mg/kg bw/day (nominal)
Remarks:
test item; main assay
Dose / conc.:
320 mg/kg bw/day (nominal)
Remarks:
test item; main assay
Dose / conc.:
2 mg/kg bw/day (nominal)
Remarks:
Mitomycin-C; main assay
No. of animals per sex per dose:
Preparations of the test item, positive control item or vehicle were administered
to groups of 5 male mice, with the exception of Group 4 where additional 3 reserve
animals (nos. 52 to 56) were treated at the high dose level to allow substitution in case of mortalities.
Control animals:
yes
Positive control(s):
A solution of Mitomycin-C (label name Mitomycin-C from Streptomyces subspicatus), batch no. SLBM6528V, obtained from Sigma, was prepared in distilled water of injectable grade (batch no. 14C2403, Baxter) immediately before use, and served as a positive control.

Examinations

Tissues and cell types examined:
The femurs of animals were removed and bone marrow cells obtained by flushing with foetal calf serum.
Details of tissue and slide preparation:
The bone marrow cells were centrifuged and a concentrated suspension
prepared to make smears on slides. These slides were air-dried and then
stained with May-Gruenwald and Giemsa solutions in phosphate buffer (pH
6.8). Three slides were made from each animal.
Evaluation criteria:
The slides were randomly coded by a person not involved in the subsequent
microscope scoring. They were examined under low power and one or two
slides from each animal were selected according to staining and quality of
smears. Four thousands PCEs per animal were examined for the presence of
micronuclei at high power (x 100 objective, oil immersion). At the same time,
the numbers of normal and micronucleated normochromatic erythrocytes
(NCEs) were also recorded.
Statistics:
Only counts obtained from polychromatic cells were subjected to statistical
analysis. Using the original observations (and not the micronucleus frequencies
per 1000 cells), a modified 2 calculation was employed to compare treated
and control groups. The degree of heterogeneity within each group was
first calculated and where this was significant, it was considered in the
comparison between groups. Variance ratios or 2 values were taken to show
the significance of any differences between each treated group and the control.
In addition, a test for a linear trend (Snedecor and Cochran) was performed
in order to evaluate dose effect relationship.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Acceptance criteria
The assay is considered to be valid if the following criteria are met:
– The incidence of micronucleated PCEs of the vehicle control group falls
within the historical negative control range.
– The positive control item induces a significant increase in the frequency
of micronucleated PCEs and the response falls within the historical
positive control range.
– The appropriate number of doses and cells have been analysed (at least
4000 thousand cells per animal and three doses).

Criteria for outcome of assay
The test item is considered to induce micronuclei if the following three criteria
are met:
– At least one of the treatment groups exhibits a statistically significant
increase (p<0.05) in the frequency of micronucleated PCEs compared
with the concurrent negative control.
– The incidence shall be out of the range of historical values observed in
our laboratory for negative controls.
– The increase shall be dose related when evaluated with an appropriate
trend test (Snedecor and Cochran).
The test item is not considered to induce micronuclei if none of the above
mentioned criteria is fulfilled and there is bone marrow exposure to the test
item.

Historical controls were included in the Appendix of the study report.

Any other information on results incl. tables

Results & Conclusions incl. tables from original study report, see attached pdf file under "Overall remarks, attachments".

Applicant's summary and conclusion

Conclusions:
ANALYSIS OF RESULTS

The incidence of micronucleated PCEs of the vehicle control group fell within
the historical negative control range (95% confidence limits).
A statistically significant increase in the incidence of micronucleated PCEs
over the negative control value was seen in the positive control group.
The induced response was compatible with the historical control range
demonstrating the laboratory proficiency in the conduct of the test.

Three dose levels and five animals per group were available for micronucleus slide
analysis. The appropriate number of cells (4000 PCEs) was analysed per
animal. Based on the stated criteria, the assay was therefore accepetd as valid.
Following treatment with DIETHYL CARBONATE, no statistically significant
increase in the incidence of micronucleated PCEs was observed in any
dose group. The incidences were within the range of our historical control
values and no significant dose effect relationship was found after a trend test
evaluation. Heterogeneity in response was observed within the positive control group but
this did not affect the integrity of the study. A summary of the results obtained for the Main Assay is presented in Table 4,
which shows the mean incidence of micronucleated PCEs for each group, the
standard error of the mean and the range of values observed. The percentage
over the negative control value of the PCEs/(PCEs+NCEs) ratio for each
group is also shown.

CONCLUSION
On the basis of the results obtained, it is concluded that DIETHYL CARBONATE,
administered by intravenous route, does not induce micronuclei in the
polychromatic erythrocytes of treated mice, under the reported experimental
conditions.