Registration Dossier

Administrative data

Description of key information

Two studies are available.

GLP studies.

The test Skin Irritation:

The test was performed according to the OECD Test GuidelineNo.439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method(2015) and Protocol for: In Vitro EpiDermTMSkin Irritation Test

BCOP test:

The test was performed according to the OECD Test Guideline No. 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, Adopted 26thJuly 2013

Klimish score 1.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26.07.-29.07.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
One tissue was rejected from evaluation due to having much higher OD570 than the other two. As absorbances of these two tissues were close together and not close to cut-off value, this deviation had no impact on the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
other: normal human-derived epidermal keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: model EpiDermTM
- Tissue: The reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 23347 kitL)
- Date of initiation of testing: 26.7.2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C


Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 g

NEGATIVE CONTROL
PBS (phosphate buffered saline) prepared 25/02/2016, exp. 25/08/2016 and prepared 23/03/2016 exp. 23/09/2016
- Amount(s) applied: 25 µL


POSITIVE CONTROL
5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 012616TMB, exp. 26/01/2017
Duration of treatment / exposure:
60 min.
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid

Table No. 1: OD570values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

 

Treatment

OD570

Avg

SD

Average viability

 

1

2

3

 

 

(% NC)

NC

 

PBS

1.926

1.806

1.646

1.793

0.115

100.0 

viability (%)

107.4

100.7

91.8

100.0

6.4

C4

 

322/16

1.763

1.822

2.942

1.793

0.030

100.0 

viability (%)

98.3

101.6

164.1

100.0

1.6

PC

 

5% SDS

0.052

0.052

0.046

0.050

0.003

2.8

viability (%)

2.9

2.9

2.6

2.8

0.16

Interpretation of results:
GHS criteria not met
Conclusions:
Under the above-described experimental design, average viability of tissues treated by the test substance Methylcentralit was 100.0 % of negative control average value. No correction was done.
The test item is considered to have no category:
(ii) if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
Even if the value obtained lay close cut-off value for the test, the effect of the test item was negative in EpiDermTM model.
According to the classification criteria given in chapter 4.5. of this report, the test item, Methylcentralit, is considered to have no category in accordance with UN GHS 1.


Executive summary:

The test item, Methylcentralit, was assayed for the in vitro skin irritation
in human epidermal model EpiDermTM.The test was performed according tothe OECD Test Guideline No.439:In VitroSkin Irritation: Reconstructed Human Epidermis Test Method (2015) and Protocol for: In Vitro EpiDermTMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4).

After pre-incubation of tissues, 25 mg of the test substance was placed directly a top to the previously moistened tissue and it was spread on the entire tissue surface. Length of exposition was 60 minutes. Three tissues were used for the test substance and every control.

After removal of the test substance, tissues were post-incubated for approximately 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Isopropyl alcohol extract of one of three tissues was much higher than those of the other two. The outlying tissue was rejected from evaluation.

Average viability of the two evaluated treated tissues was 100.0% i.e. viability was >50 %.

The effect of the test item was negative in EpiDermTMmodel (tissues were not damaged).

According to the classification criteria given in chapter 4.5.,the test item, Methylcentralit, is considered to have no categoryin regard to skin irritation.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03.08.-04.08.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: Bovine eye
Details on test animals or tissues and environmental conditions:
Bovine eyes source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic
Eyes were collected by slaughterhouse employees The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
Vehicle:
Hank's balanced salt solution
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test substance was tested as suspension prepared from test substance at 20% concentration in a 0.9% sodium chloride solution. 2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.
Duration of treatment / exposure:
4hr
Duration of post- treatment incubation (in vitro):
1,5 hr
Number of animals or in vitro replicates:
Exposed group (test substance) - 3 corneas (No. 1, 2, 4)
Positive control group (20% Imidazole) – 3 corneas (No. 12, 14, 16)
Negative control group (0.9% NaCl) – 3 corneas (No. 5, 7, 13)
Details on study design:
PROCEDURE SCHEME
Selection of corneas, mounting in holders → incubation with EMEM 1hour (32 ± 1°C) → removed EMEM, measurement of baseline opacity → treatment by positive and negative control substances and test substance (incubation 4 hours) → washing of epithelium, measurement of opacity after application → application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C) → measurement of absorbance (490 nm).


SELECTION AND PREPARATION OF CORNEAS
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

From 21 eyes the 6 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 1, 2, 4, 5, 7, 12, 13, 14 and 16,), 3 eyes was superfluous and remaining 3 were used for the testing of another substance.

QUALITY CHECK OF THE ISOLATED CORNEAS
The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

NEGATIVE CONTROL USED: YES
Name: 0.9% NaCl
Lot: 162748131
Expiration: 6/2019
Supplier: B.Braun Melsungen, Germany


POSITIVE CONTROL USED: YES
Name: Imidazole
Lot no: WXBC1234V
Expiration: 01/2024
Supplier: Sigma-Aldrich


Irritation parameter:
in vitro irritation score
Value:
0.32
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Table No. 3: Appearance of corneas  

Group

Cornea No.

Appearance after exposure

Negative control

5

Without macroscopic damage

 

7

Without macroscopic damage

 

13

Without macroscopic damage

Positive control

12

Corneal opacity

 

14

Corneal opacity

 

16

Corneal opacity

Test substance

1

Without macroscopic damage

 

2

Without macroscopic damage

 

4

Without macroscopic damage

Interpretation of results:
GHS criteria not met
Conclusions:
The In Vitro Irritancy Score (IVIS) for Methylcentralit was 0.32.
This value of IVIS is ≤ 3 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: No category.
Executive summary:

The test substance, Methylcentralit, was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the OECD Test Guideline No. 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, Adopted 26thJuly 2013

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured.The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

 

The In Vitro Irritancy Score (IVIS) forMethylcentralit,was 0.32.

This value of IVIS is≤ 3 therefore the classification according to UN GHS criteria foreye irritation or serious eye damage is: No category.

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

Additional information

Justification for classification or non-classification

Based on the available results of the test substance Methylcentralit is not classified