Registration Dossier

Administrative data

Description of key information

Skin Irritation:

In the in vitro skin corrosion test according to OECD 431 guideline, the test item showed tissue viability 92.23% after 240 min exposure. Thus, methylthiouracil is non-corrosive.

In the in vitro skin irritation test according to OECD 439 guideline the determined viability of culture treated by methylthiouracil (89.22%) fulfilled the criteria for non-irritancy.

Eye Irritation:

In vitro eye irritation test according to the OECD 437 guideline no prediction on classifiction cannot be made for eye irritation/serious eye damage or as causing serious eye damage

In the eye rritation test according to OECD 492 methylthiouracil showed a relative percent viability > 60 % . According to the criteria, it is concluded that the test item is non-irritant.  

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiSkin from SkinEthic Laboratories France.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
20 mg
Duration of treatment / exposure:
3, 60 and 240 minutes
Duration of post-treatment incubation (if applicable):
3 hour incubation with MTT
Number of replicates:
Two
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
90.83
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure
Value:
96.64
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240 min exposure
Value:
92.23
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
he absolute mean OD570 of the negative control tissues was 0.9712, 0.8067 and 0.9037 for the 3 -minute, 60 -minute and 240 -minute exposures, respectively.

Mean OD values of individual epidermis units.

3 -minutes exposure

      Adsorption (OD570)
   R1  R2
Negative control   1,0449

 0,9851

 Test item

 0,9804

0,8715 

 

60 -minutes exposure

        Adsorption (OD570)
   R1  R2
 Negative control  0,8406 0,8605 
 Test item  0,8172 0,8297 

240 -minute exposure

          Adsorption (OD570)
Negative control   0,9423 0,8527 
Positive control  0,0840 0,0788 
Test item  0,8786 0,8759 
Interpretation of results:
GHS criteria not met
Conclusions:
After 240 -minute exposure in the Reconstructed Human Epidermis model the test item showed tissue viability 92.23%. Taken together, methylthiouracil is non-corrosive in the in vitro skin corrosion test performed according to OECD 431 guideline.
Executive summary:

The test item, methylthiouracil, was tested for its possible skin corrosion potential using a three dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application. The study was perfomed according to OECD 431 guideline. The absolute mean OD570 of the negative control tissues was 0.9712, 0.8067 and 0.9037 for the 3 -minute, 60 -minute and 240 -minute exposures, respectively. The positive control has a mean cell viability of 4.16 % after 240 -minute exposure. Thus, the results of the controls indicate that the test system functioned properly.

After 240 -minute exposure the test item showed tissue viability 92.23%. Taken together, the test item methylthiouracil is non-corrosive in the in vitro skin corrosion test.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE

EpiSkin Small Model kit provided by SkinEthic

- Model used: EpiDerm™ (EPI-200-SIT)
- Tissue batch number(s): 17-EKIN-044
- Delivery date: 31 October , 2017
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 mg
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 min exposure
Value:
89.22
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes

Table 1. True OD values of individual epidermis units.

               Absorption (OD570)
   R1 R2  R3  Mean  SD 
 Negative control  0,7248 0,7315  0,7210  0,7258 0,0053 
 Positive control 0,0432  0,0433  0,0453  0,0439  0,0012 
 Test item 0,6519  0,6398  0,6510  0,6476  0,0067 

OD: optical density

R1, R2, R3:triplicate exposures

Ture OD value=OD Raw-OD Blank

Table 2. Individual tissue viability of epidermis units (relative).

               % Individual viability
  R1  R2  R3  Mean  SD 
 Positive control  5,95 5,96  6,24  6,05  0,17 
 Test item  89,82 88,15  89,69  89,22  0,93 
Interpretation of results:
GHS criteria not met
Conclusions:

In the in vitro skin irritation test according to OECD 439 guideline the determined viability of culture treated by methylthiouracil (89.22%) fulfilled the criteria for non-irritancy.
Executive summary:

The test item methylthiouracil was tested for its possible skin irritation potential using a three dimestional Reconstructed Human Epidermis model, EpiSkin. The test was performed according to OECD 439 guideline. The magnitude of viability was quantified by using MTT test. Validity of the test method was ascertained by positive control 5% SDS. Three tissue replicates were used for each treatment (exposure time 15 minutes), including negative and positive control.  

The tissue viability met the acceptance criterion. Mean OD570of negative control was  0,7258. The viability of culture treated by positive control 5% SDS was 6.05%. The positive control met the acceptance criterion: mean tissue viability less than 20%. Determined viability of culture treated by methylthiouracil (89.22%) fulfilled the criteria for non-irritancy. Therefore, methylthiouracil is considered to be non-irritant to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Korde Chemicals Pvt. Ltd. / 151117
- Expiration date of the lot/batch: 14.11.2019
- Purity test date: 15.11.2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Ambient temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Final preparation of a solid:

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source:
Local abattoir (Slaughter house), Near Frazer town, Bengaluru
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight):
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples:
- Indication of any antibiotics used:
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
100 mg
Duration of treatment / exposure:
4 h
Details on study design:
SELECTION AND PREPARATION OF CORNEAS

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES

NEGATIVE CONTROL USED

SOLVENT CONTROL USED (if applicable)

POSITIVE CONTROL USED

APPLICATION DOSE AND EXPOSURE TIME

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: yes/no. If YES please specify duration

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
Irritation parameter:
in vitro irritation score
Value:
6.699
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes

Table 1. Opacity calculations of negative control.

 Cornea nro. Blank value Io (lux)

 Pretreatment cornea

reading I (lux)

 Initial opacity

Post-treatment

cornea reading I (lux)

Final opacity

Change of

opacity

  

Corrected

opacity 

 Mean

opacity

 4  502  455 450  0 na 
 5 502  449   435 na 
 6  502 497  480  na 

Table 2 . Opacity calculations of positive control.

 Cornea nro. Blank value Io (lux)

 Pretreatment cornea

reading I (lux)

 Initial opacity

Post-treatment

cornea reading I (lux)

Final opacity

Change of

opacity

  

Corrected

opacity 

 Mean

opacity

 27  502  453 123  123  118 117  162,67 
 28 502  442  80 211  205  204  162,67 
 29  502 454  94  173  168  167  162,67 

Table 3. Opacity calculations of test item, methylthiouracil.

 Cornea nro. Blank value Io (lux)

 Pretreatment cornea

reading I (lux)

 Initial opacity

Post-treatment

cornea reading I (lux)

Final opacity

Change of

opacity

  

Corrected

opacity 

 Mean

opacity

 4  502  455 450  0 na 
 5 502  449   435 na 
 6  502 497  480  na 
Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, the test item 6-Methyl-2-Thiouracil cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.
Executive summary:

The evaluation of the occular irritancy of methylthiouracil was carried out using the bovine corneal opacity and permeability assay (BCOP). 100 mg of test item was applied undiluted on the corneal surface by open chamber method. The IVIS scores were as follows: negative control (distilled water) 1.228, positive control (chlorhexidine) 163.377 and test item 6.699. The test item was classified as no prediction can be made based on UN GHS criteria.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Korde Chemicals pvt ltd RRR House, Plot 80, sector 23, Turbhe Cidco Industrial area Navi Mumbai-400705, India / 151117
- Expiration date of the lot/batch:
14.11.2019
- Purity test date: 15.11.2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Ambient (+15 to +25°C), avoid sunlight
- Stability under test conditions:
stable

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
Serious eye damage refers to the production of tissue damage in the eye, or serious physical decay of vision, following application of a test chemical to the anterior surface of the eye, which is not fully reversible within 21 days of application, as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).
The assessment of serious eye damage/eye irritation has typically involved the use of laboratory animals (OECD Test Guideline TG 405). In relation to animal welfare concerns, TG 405 recommends the use of a sequential testing strategy for the determination of the serious eye damage/eye irritation potential of chemicals. This testing strategy is described in a Supplement to the Guideline and includes the use of validated, scientifically valid and accepted in vitro test methods, thus decreasing or avoiding pain and suffering of animals. Most international regulation includes a requirement for in vitro tests for serious eye damage/eye irritation for substances manufactured or imported.
EpiOcular™ EIT is an in vitro test method able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS (1), and the test method was recommended as scientifically valid for the purpose.
One of the in vitro test method currently covered by this Test Guideline (OECD TG 492) is the EpiOcular™ Eye Irritation Test (EIT), which makes use of a commercially available RhCE tissue construct as test system.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The RhCE tissues come from Normal human epidermal keratinocytes (NHEK) derived from Neonatal-foreskin tissue. The tissues/cells are screened for: HIV, Hepatitis-B, Hepatitis-C, and mycoplasma.
Source of the Test System
MatTek Invitro Life Sciences Laboratories
Mlynske Nivy 73,
Bratislava, Slovakia.
EpiOcular tissues are produced by MatTek In vitro Life Sciences Laboratories and procured directly through world courier
Test System Cat No. Lot No Shipping Date Receipt Date
EpiOcular tissues OCL-200-EIT 27019 15 January 2018 16 January 2018
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
17 h 47 m
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used
according to OECD 492

- RhCE tissue construct used, including batch number
EpiOcular™ MatTek Invitro Life Sciences Laboratories Mlynske Nivy 73, Bratislava, Slovakia.
EpiOcular tissues are produced by MatTek In vitro Life Sciences Laboratories and procured directly through world courier.
Test System Cat No. Lot No Shipping Date Receipt Date
EpiOcular tissues OCL-200-EIT 27019 15 January 2018 16 January 2018

- Doses of test chemical and control substances used
50 μL each of negative control (sterile deionised water), positive control (methyl acetate) and 50 mg of test item

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
post soak: After rinsing, the tissues were immediately transferred to a pre-labelled 12 well plate containing 5 mL of previously warmed assay medium, immersed and kept at room temperature for 22 minutes to facilitate the removal of any residue test item.
Post incubation: At the end of the Post-Soak immersion, each tissue was removed from the assay medium, the medium was decanted off the tissue, and the tissue construct was blotted on absorbent material.
The tissues were then transferred to a pre-labeled 6 well plate containing 1 mL of warm assay medium. The tissues were incubated for 17 hours and 47 minutes at 37oC, 5% Co2.

- Description of any modifications to the test procedure
: none
- Description of the method used to quantify MTT formazan
: according to OECD 492
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model

If the test item-treated tissue viability is > 60.0% relative to negative control-treated tissue viability, the test item is labeled as non-irritant.
If the test item-treated tissue viability is ≤ 60.0% relative to negative control-treated tissue viability, the test item is labeled as irritant.

- Complete supporting information for the specific RhCE tissue construct used
Serious eye damage refers to the production of tissue damage in the eye, or serious physical decay of vision, following application of a test chemical to the anterior surface of the eye, which is not fully reversible within 21 days of application, as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).
The assessment of serious eye damage/eye irritation has typically involved the use of laboratory animals (OECD Test Guideline TG 405). In relation to animal welfare concerns, TG 405 recommends the use of a sequential testing strategy for the determination of the serious eye damage/eye irritation potential of chemicals. This testing strategy is described in a Supplement to the Guideline and includes the use of validated, scientifically valid and accepted in vitro test methods, thus decreasing or avoiding pain and suffering of animals. Most international regulation includes a requirement for in vitro tests for serious eye damage/eye irritation for substances manufactured or imported.
EpiOcular™ EIT is an in vitro test method able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS (1), and the test method was recommended as scientifically valid for the purpose.
One of the in vitro test method currently covered by this Test Guideline (OECD TG 492) is the EpiOcular™ Eye Irritation Test (EIT), which makes use of a commercially available RhCE tissue construct as test system.

- Acceptable variability between tissue replicates for positive and negative controls
- Acceptable variability between tissue replicates for the test chemical
• The negative control OD was in between > 0.8 and < 2.5.
• The mean tissue viability of the positive control was < 50% compared to the negative control.
• The difference of viability between two tissue replicates were less than 20% for both the test item and positive control treated tissue, whereas for negative control treated tissues the difference of viability between two tissue replicates was greater than 20%. Since this variability will not have any impact on the outcome of test results, data was considered for further analysis and interpretation.
Irritation parameter:
other: relative OD viability
Run / experiment:
Experiment
Value:
100.41
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:
no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
yes
- Acceptance criteria met for positive control: yes

Calculations

·        Mean OD value of the blankcontrol wells (ODBlk) were calculated for each experiment.

·        Blank corrected valueswere obtained by subtracting ODBlkvalue from each OD value of the same experiment.

·        Mean value of the two aliquotsof each tissue were calculated to get corrected test item OD.

·        Mean value of two relating tissueswere calculated to obtain the corrected mean OD.

Corrected OD value of negative control corresponds to 100% viability.

Corrected negative control OD = Negative control OD - ODBlk= 100% Viability

Calcultaion for viablity

Percentage viability was calculated for two relating tissues of controls and test item relative to the negative control (100 %).

 

Viability % =

corrected test item OD

x 100

corrected mean negative control OD

Mean test item viability was calculated and the test item was classified according to the prediction model, described in section 8.5.

Results

Code Viability Tissue 1 Viability Tissue 2 Mean Diff Classifi-
cation
Ali 1 Ali 2 Mean 1 Ali 1 Ali 2 Mean 2 1 & 2 1 & 2    
NC 100.76 96.53 98.64 102.45 100.26 101.36 100 2.71 NI
PC 38.34 38.37 38.36 38.06 38.32 38.19 38.28 0.16 I
Test item 96.92 102.64 99.78 100.96 101.13 101.05 100.41 1.27 NI

NC: Negative Control (deionized water), PC: Positive control (methyl acetate), I: Irritant, NI: Non-irritant, Ali: Aliquot, Diff: difference of viability between 2 tissues.

OD Values of individual epiocular tissues

Treatment OD570nm Values
Tissue 1 Tissue 2
R1 R2 R1 R2
Negative Control 1.736 1.6649 1.7644 1.7277
Positive Control 0.6859 0.6864 0.6812 0.6856
Test item (6-Methyl-2-Thiouracil) 1.6715 1.7676 1.7394 1.7423
Interpretation of results:
GHS criteria not met
Conclusions:
Under the test method and test conditions employed the tissues treated with test item 6-Methyl-2-Thiouracil showed a relative percent viability > 60 % hence, it is concluded that the test item 6-Methyl-2-Thiouracil was Non-Irritant (NI).
Executive summary:

In vitro eye irritation test was carried out using Reconstructed Human Cornea-like Epithelium with an objective to evaluate Eye Irritation potential of the test item 6-Methyl-2-Thiouracil. Pre-checks were performed on 6-Methyl-2-Thiouracil to identify if the test item was a direct MTT reducers and/or colour interfering substance. Test item was found to be non- reducer of MTT and did not react with either water or isopropanol to result in colour formation.  

EpiOcularTMtissues were procured from MatTek In Vitro Life Science Laboratories. Upon receipt, the tissues were equilibrated to room temperature for 15 minutes. Tissues were inspected for any air bubbles between the agarose gel and insert. Then the tissues were carefully removed from agarose, blotted to remove agarose sticking to the inserts and transferred into 6 well plate containing 1 mL of assay medium and incubated at 37°C, 5% Co2for 1 hour. After the incubation period assay medium was replaced with fresh assay medium and incubated at 37°C, 5% Co2overnight for 16 hours.

Following overnight incubation, tissues were pre-wetted with 20 μL of Ca++Mg++Free-DPBS and incubated at 37°C, 5% Co2for 30 minutes. After prewetting, 50 µL each of negative control (sterile deionized water) and positive control (Methyl Acetate), 50 mg of test item 6-Methyl-2-Thiouracil was added on to the tissue surface. All the treatments were carried in duplicates (2 EpiOcular tissues/treatment) and incubated at 37oC, 5% Co2for 6 hours.

Post treatment, all the tissues were made free of negative control, positive control and test item by rinsing with Ca++Mg++-free DPBS. Post rinsing, the tissues were dried by blotting on to the tissue paper and soaked in 5 mL of assay medium filled in 12 well plate and incubated further for 22 minutes at room temperature.

Post soaking in media, tissues were transferred to pre labelled 6 well plate containing 1 mL of assay medium and incubated at 37oC, 5% Co2for 17 hours 47 minutes.

After the recovery period, MTT assay was carried out by transferring inserts into a 24 well plate containing 0.3 mL of MTT solution and incubated at 37°C, 5% Co2for 180 minutes. The inserts were transferred to a pre labelled 6 well plate containing 1 mL of isopropanol.The plate was sealed and stored overnight at 2-8°C in the dark and on the next day, the plates were kept on orbital shaker for 2 hours 50 minutes to extract the MTT. After extraction, the tissue inserts were removed and the liquid within each insert was decanted in to the same well from where it is removed and 1 mL of isopropanol was added to the extract mixed and was quantified by optical density (OD) measurement at 570 nm. OD values were analyzed to calculate the relative percent viability of the tissues.

The relative percent viability of the tissues treated with the test item 6-Methyl-2-Thiouracil was 100.41 % considering the mean negative control as 100 % viability. Under the same conditions the positive control Methyl Acetate showed only 38.28 % viability confirming the sensitivity of the test system.

Classification of test items as irritants and non-irritants was carried according to prediction model described in study plan. Under the test method and test conditions employed the tissues treated with test item showed a relative percent viability > 60 % hence, it is concluded that the test item 6 -Methyl-2-Thiouracil was Non-Irritant (NI). 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item, methylthiouracil, was tested for its possible skin corrosion potential using a three dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application. The study was perfomed according to OECD 431 guideline. The absolute mean OD570of the negative control tissues was 0.9712, 0.8067 and 0.9037 for the 3 -minute, 60 -minute and 240 -minute exposures, respectively. The positive control has a mean cell viability of 4.16 % after 240 -minute exposure. Thus, the results of the controls indicate that the test system functioned properly.

After 240 -minute exposure the test item showed tissue viability 92.23%. Taken together, the test item methylthiouracil is non-corrosive in the in vitro skin corrosion test.

The test item methylthiouracil was tested for its possible skin irritation potential using a three dimestional Reconstructed Human Epidermis model, EpiSkin. The test was performed according to OECD 439 guideline. The magnitude of viability was quantified by using MTT test. Validity of the test method was ascertained by positive control 5% SDS. Three tissue replicates were used for each treatment (exposure time 15 minutes), including negative and positive control.  

The tissue viability met the acceptance criterion. Mean OD570of negative control was  0,7258. The viability of culture treated by positive control 5% SDS was 6.05%. The positive control met the acceptance criterion: mean tissue viability less than 20%. Determined viability of culture treated by methylthiouracil (89.22%) fulfilled the criteria for non-irritancy. Therefore, methylthiouracil is considered to be non-irritant to the skin.

The evaluation of the occular irritancy of methylthiouracil was carried out using the bovine corneal opacity and permeability assay (BCOP). 100 mg of test item was applied undiluted on the corneal surface by open chamber method. The IVIS scores were as follows: negative control (distilled water) 1.228, positive control (chlorhexidine) 163.377 and test item 6.699. The test item was classified as no prediction can be made based on UN GHS criteria.

The second test for eye irritation was performed according to OECD 492 guideline with Reconstructed Human Cornea-like epithelium (RhCE). The relative percent viability of the tissues treated with the test item 6-Methyl-2-Thiouracil was 100.41 % considering the mean negative control as 100 % viability. Under the same conditions the positive control Methyl Acetate showed only 38.28 % viability confirming the sensitivity of the test system.Classification of test items as irritants and non-irritants was carried according to prediction model described in study plan.Under the test method and test conditions employed the tissues treated with test item showed a relative percent viability > 60 % hence, it is concluded that the test item 6 -Methyl-2-Thiouracil was Non-Irritant (NI). 

Justification for classification or non-classification

Skin Irritation:

The results of two key studies indicate that no classification is warranted for skin irritation according to CLP Regulation 1272/2008.

Eye Irritation

The results of two key studies do not indicate the test substance to be classified for eye irritant according to CLP Regulation 1272/2008.