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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-11-18 to 2021-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Methacrylic acid
EC Number:
201-204-4
EC Name:
Methacrylic acid
Cas Number:
79-41-4
Molecular formula:
C4H6O2
IUPAC Name:
methacrylic acid
Test material form:
liquid
Specific details on test material used for the study:
Supplier: Röhm GmbH, Darmstadt, Gemany
Batch: R0004-66
Purity: 99.86%
Expiry Date: 12 February 2021
Storage Conditions: Room temperature

Method

Target gene:
not applicable (micronucleus test)
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this
study, blood was collected from a male donor (23 years old) for Experiment I and from a male don
or (30 years old) for Experiment II.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation used for this study was 31.7 mg/mL (Lot no. 200220).
Test concentrations with justification for top dose:
With regard to the molecular weight of the test item, 861 µg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 5.6 to 861 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
No cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 861 µg/mL were chosen as top treatment concentration for Experiment II.
Experiment I - 4 h treatment without S9 mix
5.6, 9.8, 17.1, 30.0, 52.5, 91.8, 161, 281, 492, 861 µg/ml
Evaluated concentrations: 281, 492, 861 µg/ml
Experiment II - 20 h treatment without S9 mix
52.5, 91.8, 161, 281, 492, 861 µg/ml
Evaluated concentrations: 281, 492, 861 µg/ml
Experiment I - 4 h treatment with S9 mix
Evaluated concentrations: 281, 492, 861 µg/ml

Vehicle / solvent:
Culture medium with 10.0 % deionised water
Justification for choice of solvent/vehicle:
The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Purity: 97.0 – 103.0 %
Dissolved in: Saline (0.9 % NaCl [w/v])
Concentration: 17.5 µg/mL
Untreated negative controls:
no
True negative controls:
yes
Remarks:
Culture medium with 10 % deinonised water
Positive controls:
yes
Positive control substance:
mitomycin C
other:
Remarks:
Without metabolic activation
Name: MMC; mitomycin C (pulse treatment)
Purity: 98 %
Dissolved in: Deionised water
Concentration: 1.0 µg/mL
Name: Demecolcine (continuous treatment)
Purity: ≥ 98 %
Dissolved in: Deionised water
Concentration: 100 ng/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
Experiment I: 4 h with and without metabolic activation followed by 16 h recovery period
Experiment II: 4 h with metabolic activation followed by 16 h recovery period, 20 h without metabolic
activation
- Expression time (cells in growth medium): 20 h (+ cytochalasin B)
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h
SPINDLE INHIBITOR (cytogenetic assays): 4 mg/L cytochalasin B
STAIN (for cytogenetic assays): Giemsa
3
NUMBER OF REPLICATIONS: 2 per experiment
NUMBER OF CELLS EVALUATED: at least 1000 binucleate cells per culture 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
Rationale for test conditions:
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline 487 for In Vitro Mammalian Cell Micronucleus Test requesting for the top concentration clear toxicity with cytostasis of 55 ± 5 %, and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 μL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible.
The highest applied concentration in this study (861 μg/mL of the test item, approx. 10 mM) was
chosen with regard to the molecular weight of the test item and with respect to the current OECD
Guideline 487.
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 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:
The Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for
statistical computing and graphics.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Additional information on results
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: solvent control – 7.37 (exp. I, ); test item 861 μ/ml – 6.58 (without S9 mix)
- Effects of osmolality: solvent control – 293; test item 861 μ/ml – 297 -> no relevant influence
- Precipitation: no precipitation of the test item in the culture medium observed.
RANGE-FINDING/SCREENING STUDIES:
- pre-experiment with 10 concentrations: 5.6, 9.8, 17.1, 30.0, 52.5, 91.8, 161, 281, 492, 861 µg/ml exposure duration 4 h with and without metabolic activation
- no toxicity observed
HISTORICAL CONTROL DATA:
For the solvent controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The calculated 95% control limit of the solvent controls (realized as 95% confidence interval) was applied for the evaluation of acceptability and interpretation of the data. Control charts of the corresponding experiments are added as quality control method. For the positive controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The min-max range of the positive controls was applied for the evaluation of acceptability
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I and II in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
OTHER:
In Experiment I and II in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.
Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Any other information on results incl. tables









































































































































































Exp.

Preparation interval


Test item
concentration
in µg/mL
Proliferation
index
CBPI
Cytostasis
in %*
Micronucleated
cells
in %**
95% Ctrl limit
in %
Exposure period 4 h without S9 mix
I40 hSolvent control11.74 0.400.00 – 1.04
  Positive control21.3454.4 12.60S 
  2811.687.60.55 
  4921.688.10.35 
  8611.6610.70.30 
Trend test: p-value 0.417
Exposure period 20 h without S9 mix
II40 hSolvent control12.05 0.40 
  Positive control31.5944.3 3.95S0.00 – 0.86
  2811.986.70.35 
  4921.9212.90.10 
  8611.8221.60.20 
Trend test: p-value 0.272
Exposure period 4 h with S9 mix
I40 h

Solvent control1



1.54



 


0.450.00 – 1.03
  

Positive control4


1.2455.9 3.30S 
  

281


1.532.80.50 
  

492


1.58n.c.0.40 
  

861


1.55n.c.0.40 
Trend test: p-value 0.363

*       For the positive control groups and 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


S       The number of micronucleated cells is statistically significantly higher than corresponding control values


T           Trend analysis via linear regression is significant (p ˂ 0.05)


1       DMSO            0.5 % (v/v)
2            MMC               0.8 µg/mL
3            Demecolcine     75 ng/mL
4            CPA               15.0 µg/mL


Applicant's summary and conclusion

Conclusions:
In this micronucleus test methacrylic acid did not induce micronuclei in human lymphocytes in vit
ro when tested was tested up to and including the limit concentrations (ca. 10 mM).
Executive summary:

In a mammalian cell micronucleus assay according to OECD guideline 487, adopted 29 July 2016, primary human lymphocyte cultures were exposed to methacrylic acid in culture medium with 10.0 % deionised water at concentrations of 0 (control), 281, 492, 861 µg/ml with and without metabolic activation (S9 mix). Methacrylic acid was tested up to and including the limit concentration (ca. 10 mM).


No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis was observed up to and including the highest applied concentration. Positive controls induced the appropriate response.


There was no evidence of micronucleated cells induced over background. Therefore, methacrylic acid is considered to be non-clastogenic in this micronucleus test, when tested up to and including the limit concentration.

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