Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable data from a publication with solid information on materials/methodology and results
Principles of method if other than guideline:
In this publication of Mortelmans et al. is referred to various publications including the essential publication of Ames et al. (1975) to describe and justifiy materials and methodology that were applied.
GLP compliance:
not specified
Type of assay:
other: Salmonella/mammalian microsome assay
Species / strain / cell type:
other: Salmonella TA1535, TA1537, TA98, TA100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix, rat and hamster liver
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 6666 µg/plate
Vehicle / solvent:
Solvent dimethylsulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA1535, TA100), 4-nitro-o-phenylenediamine (TA98), 9-aminoacridine (TA97, TA1537)
Species / strain:
other: Salmonella TA1535, TA1537, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test substance diethyl carbonate showed no mutagenicity on the different bacterial strains in the dose-level range from 0-6666 µg/plate.
Endpoint:
genetic toxicity in vitro
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: BG Chemie review gives a summary of an extended half-page abstract published in a renowned scientific journal (Mutation research)
GLP compliance:
not specified
Species / strain / cell type:
yeast, other: Saccharomyces cerevisiae D4

Citation from BG Chemie review:

7.6 Genotoxicity

7.6.1 In vitro

Cells of the Saccharomyces cerevisiae strain D4, which is used to determine gene conversion at the try5 and ade2 loci, contain ten

times more cytochrome P-450 when they are grown with glucose as the sole carbon source than when they are cultured with galactose. Dose-dependent induction of gene conversion was found in a 4-hour incubation (at 37 °C) of cells from glucose cultures with carbonic acid diethyl ester (dose not specified). In contrast, when the cells were from galactose cultures, no induction of gene conversion was observed after incubation with carbonic acid diethyl ester. The authors concluded that this suggested that the mixed-function oxidase system of the yeast enabled the substance to be metabolized.

-------------

Cytochrome P-450 and the activation of aflatoxin B1, in Saccharomyces cerevisiae (by Callen et al. 1978):

In Saccharomyces cerevisiae the cellular content of cytochrome P-450 was investigated and shown to be at a maximum when cells were harvested from log phase cultures growing with glucose as sole carbon source. Log cells of the strain D4 harvested from cultures growing on medium containing glucose possessed about 10x more cytochrome P·450 than log cells from cultures growing

on medium containing galactose. The diploid strain D4 can be used to monitor gene conversion at the try5 and ade2 loci. When log phase cells from glucose cultures were incubated for four hours at 37° C with dimethylnitrosamine, ß-naphthylamine, ethyl carbonate and dimethylsulphoxide a dose response could be established for the induction of convertants. Treatment of cells harvested

from cultures growing on medium containing galactose did not result in any significant induction of convertants. This suggests that the mixed-function oxydase system of yeast is capable of metabolizing these compounds. However, aflatoxin B, induced convertants when incubated with cells harvested from cultures growing on medium containing either glucose or galactose as carbon source. Since aflatoxin B, is thought to be metabolized to an epoxide by the mixed-function oxydase system, these results suggest that this cornpound is metabolized to an active form in yeast cells grown on medium containing galactose by either an efficient and apparently specific cytochrorne P-450 or by alternative enzymes.

Conclusions:
Interpretation of results (migrated information):
other: mutagenic activity depended on the growth medium (on glucose medium but not galactose medium mutagenic activity), dose dependency
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study plan: 2011-11-09, Performance of study: 2012-04-04, Draft report: 2012-04-12, Final report: 2012-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Official testing guideline according to OECD 487, GLP compliant with certificate, detailed description of methods conducted.
Qualifier:
according to
Guideline:
other: OECD 487 "In Vitro Mammalian Cell Micronucleus Test"
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
other: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
Blood lymphocytes were obtained from adequate donors (Experiment I, one female, 34 years old; Experiment II, one female, 33 years old) which were healthy, non-smoking, no known recent exposures to genotoxic chemicals or radiation. Blood samples were drawn by venous puncture and collected in heparinized tubes. Blood cultures were set up within 24 hours after sample collection.
Metabolic activation:
with and without
Metabolic activation system:
S9 liver enzyme mixture from the livers of male Sprague-Dawley rats treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
Test concentrations with justification for top dose:
Experiment I: 1203, 601.5, 300.8, 150.4, 75.2, 37.6, 18.8, 9.4 µg/mL (with and without S9)
Experiment II: 1003, 752.3, 401.2 µg/mL (with and without S9)
Negative solvent / vehicle controls:
yes
Remarks:
Culture medium without fetal calf serum
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: dissolved in Aqua demin, final concentrations 0.15 and 0.3 µg/mL, without exogenous metabolic activation
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: dissolved in 0.9 % NaCl, final concentrations 7.5 and 15 µg/mL, with exogenous metabolic activation (S9 mix).
Evaluation criteria:
Evaluation of the slides was performed using Zeiss microscopes with 100 x oil immersion objectives. In all replicates, the cytokinesis-block proliferation index (CBPI) was determined to assess cell proliferation using at least 500 cells per culture. From these determinations, the test item concentrations, which were evaluated for micronuclei, were defined.

CBPI = ((MONC*1) + (BNC*2) + (MUNC*3))/n

n: total number of cells
MONC: mononucleate cells
BNC: binucleate cells
MUNC: Multinucleate cells

Cytotoxicity was calculated as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
Cytostasis % = 100 -100 [(CBPI(T) - 1) / (CBPI(C) - 1)]

CBPI(T): Cytokinesis-block proliferation index of test item resp. positive control
CBPI(T): Cytokinesis-block proliferation index of solvent control

The number of binucleated cells with and without micronuclei in each treatment group was compared with the solvent control value.

Acceptability:
The genotoxicity assay is considered acceptable if it meets the following criteria:
- the micronucleus induction in human lymphocytes of the solvent control is within the range of the historical control data or within literature data
- the positive controls induce a detectable increase over the background, which demonstrates the sensitivity of the test system
Statistics:
Statistical significance was tested using Fisher's exact Test with the following equation:

φ(a) = [(a+b)!(c+d)!(a+c)!(b+d)!]/[n!a!b!c!d!]

a: number of binucleated cells with micronuclei of the solvent control
b: number of binucleated cells with micronuclei of the test item of the respective concentration
c: number of binucleated cells without micronuclei of the solvent control
d: number of binucleated cells without micronuclei of the test item of the respective concentration

This equation represents a hypergeometric distribution. The resulting probability of the respective distribution was halved and the probabilities of the more extreme distributions (down to a value of 0 in the controls) were added to give cumulated p-value of the tail of the distribution.
Species / strain:
other: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Classification:
The test item was considered to have no genotoxic effects if:
- the number of micronucleated cells in all evaluated dose groups was in the range of the laboratory's historical control data of the solvent control
- no statistically significant or concentration-related increase in the number of micronucleated cells was observed

The test item was considered to have genotoxic effects if:
- the number of micronucleated cells in all evaluated dose groups was above the range of the historical laboratory control data
- either a concentration-related increase of micronucleated cells or a statistically significant increase in the number of cells containing micronuclei was observed

Cytotoxicity Test (experiment I): in absence of S9

Treatment   Precipitation  Haemolysis  Average CBPI  % Cytostasis
 Solvent controlculture medium  no  no  1.94  --
 Positive control MMC (0.3 µg/mL)  no  no  1.79  16.0
 Diethyl carbonate        
 1203 µg/mL  no  no  1.87  7.4
 601.5 µg/mL  no  no  1.95  -1.3
 300.8 µg/mL  no  no  1.88  6.0
 150.4 µg/mL  no  no  1.86  7.9
 75.2 µg/mL  no  no  1.95  -1.6
 37.6 µg/mL  no no  1.96  -2.1
 18.8 µg/mL  no  no  2.00  -7.0
 9.4 µg/mL  no  no  1.96  -2.6

Cytotoxicity Test (experiment I): in presence of S9

Treatment   Precipitation  Haemolysis  Average CBPI  % Cytostasis
 Solvent control culture mediumSolvent control 0.9 % NaCl nono  no no  1.90 1.83  -- --
 Positive control CPA (15 µg/mL)  no  no  1.40  51.5
 Diethyl carbonate        
 1203 µg/mL  no  no  1.88  2.6
 601.5 µg/mL  no  no  1.95  -5.6
 300.8 µg/mL  no  no  1.91  -1.2
 150.4 µg/mL  no  no  1.86  4.6
 75.2 µg/mL  no  no  1.94  -3.6
 37.6 µg/mL  no no  1.95  -5.5
 18.8 µg/mL  no  no  1.92  -2.1
 9.4 µg/mL  no  no  1.89  1.3

 

On the basis of the results of the cytotoxicity tests, the following concentrations were selected for evaluation: 1203, 601.5 and 300.8 µg/mL. After evaluation of the upper three concentrations, further concentrations were selected for evaluation, in order to confirm the concentration-effect-relationship which was inconclusive when observing the upper three concentrations only: without S9 mix / 4 +/-1 hours exposure: 150.4, 75.2 and 37.6 µg/mL; with S9 mix / 4 +/-1 hours exposure: 150.4 and 75.2 µg/mL.

Genotoxicity Results Experiment I:

 Treatment  Average CBPI  Cytostasis (%)  Total No. of BNC examined  Total No. of MBNC  % MBNC
                Experiment I: exposure period 4 hours without S9
 Solvent control culture medium  1.94  --  2037  3  0.15
 Positive control MMC 0.3 µg/mL  1.79  16.0  2103  70  3.33
 Diethyl carbonate 1203 µg/mL  1.87  7.4  2073  37  1.78
 Diethyl carbonate 601.5 µg/mL  1.95  -1.3  2064  25  1.21
 Diethyl carbonate 300.8 µg/mL  1.88  6.0  2119  37  1.75
 Diethyl carbonate 150.4 µg/mL  1.86  7.9  2097  31  1.48
 Diethyl carbonate 75.2 µg/mL  1.95  -1.6  2074  16  0.77
 Diethyl carbonate 37.6 µg/mL  1.95  -2.1  2031  9  0.44
          Experiment I: exposure period 4 hours with S9      
 Solvent control cutlure medium  1.90  --  2092  4  0.19
 Solvent control NaCl 0.9 %  1.83  --  2069  1  0.05
 Positive control CPA 15 µg/mL  1.40  51.5  2360  45  1.91
 Diethyl carbonate 1203 µg/mL  1.88  2.6  2074  17  0.82
 Diethyl carbonate 601.5 µg/mL  1.95  -5.6  2162  22  1.02
 Diehtyl carbonate 300.8 µg/mL  1.91  -1.2  2103  38  1.81
 Diethyl carbonate 150.4 µg/mL  1.86  4.6  2077  36  1.73
 Diethyl carbonate 75.2 µg/mL  1.94  -3.6  2162  21  0.97

In experiment I with and without metabolic activation, no cytotoxicity, precipitation or haemolysis was observed in all concentrations. A relevant increase of the number of binucleated cells with micronuclei and a concentration-effect relationship was determined at the evaluated upper three concentrations without S9. The concentration-effect relationship was inconclusive. Therefore in addition three concentrations were evaluated and the concentration-effect relationship was confirmed.

A relevant increase of the number of binucleated cells with micronuclei was determined at the evaluated three concentrations with S9. But no concentration-effect relationship was determined. In order to check a possible concentration-effect relationship, two additional, lower concentrations were evaluated. A concentration-effect relationship was found for 300.8, 150.4 and 75.2 µg/mL. In order to verify these results and to confirm the increase of the number of binucleated cells with micronuclei, a second experiment was performed using three concentrations with and without S9.

Cytotoxicity Test (experiment II): in absence of S9

Treatment   Precipitation  Haemolysis  Average CBPI  % Cytostasis
 Solvent controlculture medium  no  no  1.91  --
 Positive control MMC (0.3 µg/mL)  no  no  1.81  10.6
 Diethyl carbonate        
 1003 µg/mL  no  no  1.60  33.7
 752.3 µg/mL  no  no  1.77  15.2
 401.2 µg/mL  no  no  1.91  0.1

Cytotoxicity Test (experiment II): in presence of S9

Treatment   Precipitation  Haemolysis  Average CBPI  % Cytostasis
 Solvent control culture medium Solvent control 0.9 % NaCl nono  no no  1.89 1.96  -- --
 Positive control CPA (15 µg/mL)  no  no  1.46  52.2
 Diethyl carbonate        
 1003 µg/mL  no  no  1.93  -4.6
 752.3 µg/mL  no  no  1.89  0.2
 401.2 µg/mL  no  no  1.87  1.9

Genotoxicity Results Experiment II:

 Treatment  Average CBPI  Cytostasis (%)  Total No. of BNC examined  Total No. of MBNC  % MBNC
                Experiment I: exposure period 18 hours without S9
 Solvent control culture medium  1.91  --  2050  3  0.15
 Positive control MMC 0.3 µg/mL  1.81  10.6  2191  82  3.74
 Diethyl carbonate 1003 µg/mL  1.60  33.7  2094  22  1.05
 Diethyl carbonate 752.3 µg/mL  1.77  15.2  2082  26  1.25
 Diethyl carbonate 401.2 µg/mL  1.91  0.1  2142  40  1.87
          Experiment II: exposure period 4 hours with S9      
 Solvent control cutlure medium  1.89  --  2169  4  0.18
 Solvent control NaCl 0.9 %  1.96  --  2045  8  0.39
 Positive control CPA 15 µg/mL  1.46  47.8  2128  71  3.34
 Diethyl carbonate 1003 µg/mL  1.93  -4.5  2124  24  1.13
 Diethyl carbonate 752.3 µg/mL  1.89  0.2  2080  19  0.91
 Diehtyl carbonate 401.2 µg/mL  1.87  1.9  2123  20  0.94

In experiment II with metabolic activation, no cytotoxicity, precipitation or haemolysis was observed in all concentrations. In the treatment without metabolic activation, no precipitation or haemolysis was observed, but a medium toxicity of 33.7 % in the highest concentration was detected. A relevant increase of the number of binucleated cells with micronuclei was determined at the evaluated three concentrations without S9. No concentration-effect relations was determined. Significance was given, though. A relevant increase of the number of binucleated cells with micronuclei was determined at the evaluated three concentrations with S9. A weak concentration-effect relationship was determined.

Conclusions:
Interpretation of results (migrated information):
positive with and without metabolic activation (S9 mix)

In this study, conducted according to OECD testing guideline 487 "In Vitro Mammalian Cell Micronucleus Test", a statistically significant and biologically relevant increase in binucleated cells with micronuclei was observed (in the presence and absence of S9 mix) after treatment with the test substance diethyl carbonate. The range of binucleated cells with micronuclei after treatment with the test substance was significantly higher than the range of the solvent control values. A concentration-effect relationship was observed only for treatments below 400 µg/mL. In conclusion of the presented experimental results of this study, the test substance diethyl carbonate showed clear evidence of genotoxic activity in this in vitro test for the induction of micronuclei.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
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
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).

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.
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.
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.
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.

Results & Conclusions incl. tables from original study report, see attached pdf file under "Overall remarks, attachments".
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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test substance diethyl carbonate showed no mutagenicity on different bacterial strains in the dose-level range from 0-6666 µg/plate (see endpoint record 7.6.1.001, Mortelmans, K. et al. 1986, publication, key data for in-vitro gene mutation in bacteria).

In a study conducted according to OECD testing guideline 487 "In Vitro Mammalian Cell Micronucleus Test", a statistically significant and biologically relevant increase in binucleated cells with micronuclei was observed (in the presence and absence of S9 mix) after treatment with the test substance diethyl carbonate. The range of binucleated cells with micronuclei after treatment with the test substance was significantly higher than the range of the solvent control values. A concentration-effect relationship was observed only for treatments below 400 µg/mL. In conclusion of the presented experimental results of this study, the test substance diethyl carbonate showed clear evidence of genotoxic activity in this in vitro test for the induction of micronuclei (see endpoint record 7.6.1.003, study report, Novasol, 2012, key data for in-vitro micronucleus test in mammalian cells)

Based on Guidance on information requirements and chemical safety assessment Chapter R.7a: Endpoint specific guidance, R.7.7.6.3 Testing strategy for mutagenicity (ECHA, May 2008) a test proposal will be made as one in vitro mutagenicity test showed a positive result (in particular the in vitro mammalian cells test); see also IUCLID endpoint record 7.6.2.001.


Justification for selection of genetic toxicity endpoint in vitro:
Official testing guideline according to OECD 487, GLP compliant with certificate, detailed description of methods conducted.

Addition information from genetic toxicity in vivo:

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.

 

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.

Justification for classification or non-classification

According to the Guidance document on CLP (Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, Version 2.0 April 2012, page 293), a positive result in genotoxicity from in vitro testing in mammalian cells (see endpoint record 7.6.1.003) is not sufficient for classification of the test substance diethyl carbonate as mutagenic (category 2). However, the generation of in vivo genotoxicity data is proposed by the author of this IUCLID dossier (Testing proposal, see IUCLID endpoint record 7.6.2.001).

Subsequently, an in vivo genotoxicity study according to OECD TG 474 (Mammalian Erythrocyte Micronucleus Test) has been performed which delivered the result "negative" (no genetic toxicity).

The substance is not classified based on the negative result of the in vivo study.