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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of the test substance as stated in the study report: diethyl carbonate
Purity: 99,5 % min. (GC)
Production date: 2011-03-07
Expiry date: 2012-03-06
Storage: in closed vessel at room temperature

Method

Species / strain
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)
Controlsopen allclose all
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.

Results and discussion

Test results
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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.