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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
07.09.2011-10.09.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Micronucleus assay in vitro in Human Lymphocytes, OECD 487, adopted July 22, 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
certificate dated 30 March 2009
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Ethyltriglycol methacrylate

Method

Target gene:
n/a (micronucleus test)
Species / strain
Species / strain / cell type:
lymphocytes: human, healthy donors not receiving medication
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a female donor (22 years old) for the first experiment, from a 34 year-old female donor for Experiment IIA and from a 32 year-old female donor for Experiment IIB. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. V. Theodor (64380 Rossdorf, Germany). The tubes were
sent to Harlan CCR to initiate cell cultures within 24 hrs after blood collection. If necessary, the blood was stored before use at 4 °C.

- Type and identity of media: Dulbecco's modified eagle medium/Ham's F12 medium, mixture 1:1, supplemented with 200 mM GlutaMax, penicillin/streptomycin (100 U/mL/100 µg/mL), phytohemagglutinin (PHA, 3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES, heparin (125 U.S.P.-U/mL)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
16.1 -2486.0 µg/ml (pre-test and main experiment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The final concentration of deionised water in the culture medium was 10 % (v/v). The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controls
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
culture medium with 10 % deionised water
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecloclin
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Exposure duration:
Experiment I:
4 hours treatment with and without metabolic activation
Experiment II A:
4 hours treatment with metabolic activation
Experiment II B:
20 hours without metabolic activation

SPINDLE INHIBITOR (cytogenetic assays): 4 mg/L cytochalasin B
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 per experiment
NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture were scored for cytogenetic damage

DETERMINATION OF CYTOTOXICITY
Method: CBPI (Cytokinesis-block proliferation index) was determined in approximately 500 cells per culture and cytotoxicity is expressed as %
cytostasis

Evaluation criteria:
The micronucleus assay is considered acceptable if it meets the following criteria:
- The number of micronuclei found in the negative and solvent controls falls within the range of the laboratory historical control data
- The positive control substances should produce significant increases in the number of cells with micronuclei.

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
-no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.
Statistics:
Chi square test

Results and discussion

Test results
Species / strain:
lymphocytes: primary human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.3 – 7.5 for solvent control as well as test item in all experiments
- Effects of osmolality: 264-289 for solvent control, 280 - 301 for test item in all experiments -> no relevant influence
- Precipitation: No precipitation of the test item was observed up to highest concentration tested

RANGE-FINDING/SCREENING STUDIES:
- Pre-test for cytotoxicity: concentrations 161, 28.3, 49.5, 86.6, 151.5, 265.1, 463.9, 811.8, 1420.6 and 2486.0 µg/ml with and without metabolic activation
Using a reduced CBPI as an indicator for toxicity in Experiment I, no cytotoxic effects were observed after 4 hrs treatment in the absence and presence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The micronucleus rates of the cells after treatment with the test item (0.05 - 0.95 % micronucleated cells) were within the range of the solvent control values (0.35 -1.05 % micronucleated cells) and within the range of the laboratory historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In Experiment I in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix no cytotoxicity was observed up to the highest applied concentration.

- In Experiment IIA in the absence of S9 mix concentrations showing cytotoxic effects were not evaluable for cytogenetic damage

- In Experiment IIB in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Exp.

Test item concentration [mg/L]

Proliferation index CBPI

Cytostasis [%]

Micronucleated cells [%]

Exposure period 4 hrs without S9 mix

I

Negative control

1.84

 

0.35

 

Solvent control

1.95

 

0.55

 

Positive control

1.80

5.1

3.85S

 

811.8

1.84

11.5

0.35

 

1420.6

1.86

9.5

0.35

 

2486.0

1.90

5.8

0.35

Exposure period 20 hrs without S9 mix

IIA

Negative control

1.97

 

0.60

 

Solvent control

1.88

 

0.60

 

Positive control

1.58

40.1

3.70S

 

265.1

1.75

14.9

0.25

 

463.9

1.71

19.2

0.55

 

811.8

1.67

24.3

0.45

Exposure period 20 hrs without S9 mix

IIB

Negative control

1.90

 

0.20

 

Solvent control

1.89

 

0.35

 

Positive control

1.38

57.6

3.25S

 

 300.0

1.83

6.2

0.15

 

 1000.0

1.65

26.5

0.20

 

 2000.0

1.45

49.3

0.15

  

* For the positive control groups, the relative values are related to the negative controls;

for the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

SThe number of micronucleated cells is statistically significantly higher than corresponding control

values

1Deionised water 10.0 % (v/v)

2MMC 2.0Mg/mL

3Demecolcin 75.0 ng/mL

4Demecolcin 125.0 ng/mL

 

 

  

Exp.

Test item concentration [mg/L]

Proliferation index CBPI

Cytostasis [%]

Micronucleated cells [%]

Exposure period 4 hrs with S9 mix

I

Negative control

1.86

 

0.40

 

Solvent control

1.96

 

0.40

 

Positive control

152

39.4

5.70S

 

811.8

1.91

5.1

0.05

 

1420.6

1.85

11.1

0.20

 

2486.0

1.93

3.2

0.60

Exposure period 4 hrs with S9 mix

IIA

Negative control

1.83

 

0.55

 

Solvent control

1.87

 

1.05

 

Positive control

1.88

n.c.

5.55S

 

811.8

1.86

0.9

0.95

 

1420.6

1.79

10.0

0.55

 

2486.0

1.77

11.7

0.70

* For the positive control groups, the relative values are related to the negative controls;

for the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

SThe number of micronucleated cells is statistically significantly higher than corresponding control

values

1Deionised water 10.0 % (v/v)

2CPA 10.0Mg/mL

3CPA 7.5Mg/mL

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Ethyltriglycol methacrylate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or to the highest
required/evaluable concentration.
Executive summary:

In a mammalian cell micronucleus assay according to OECD guideline 487 (adopted July 22, 2010), primary human lymphocyte cultures were exposed to Ethytriglycol methacrylate (98.95%) in deionised water with and without metabolic activation (S9 mix).

The following concentrations were evaluated (dose calculations adjusted to purity):

Experiment I:

4 h treatment without metabolic activation: 16.1 -2486 µg/ml

4 h treatment with metabolic activation: 16.1 -2486 µg/ml

 

Experiment IIA:

20 h treatment without metabolic activation: 49.5 – 2486 µg/mL

4 h treatment with metabolic activation: : 151.5 – 2486 µg/mL

 

Experiment IIB:

20 h treatment without metabolic activation: 75 – 2400 µg/mL

 

Ethytriglycol methacrylate was tested up to cytotoxic or the guideline limit concentration of 10 mM (corresponding to 2486.0 mg/mL). Cytotoxic effects were observed after 4 h treatment with2486 µg/ml in the absence of S9 mix; no cytotoxicity was observed in the presence of S9 mix up to 2486 µg/ml

.

In the absence and presence of S9 mix no biologically relevant increase in the number of cells carrying micronuclei was observed after treatment with the test item.

 

The micronucleus rates of the cells after treatment with the test item (0.05 - 0.95 %

micronucleated cells) were within the range of the solvent control values (0.35 -1.05 %

micronucleated cells) and within the range of the laboratory historical control data.

Positive controls induced the appropriate response. 

Ethyltriglycol methacrylate did not induce micronuclei in human lymphocytes in vitro, when

tested up to cytotoxic or the highest required/evaluable concentration. Therefore, Ethytriglycol methacrylate is considered to be non-clastogenic in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentration.

 

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