1,3-dimethyl-1,3-diphenylurea

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2016

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-dimethyl-1,3-diphenylurea

EC Number:

210-283-4

EC Name:

1,3-dimethyl-1,3-diphenylurea

Cas Number:

611-92-7

Molecular formula:

C15H16N2O

IUPAC Name:

1,3-dimethyl-1,3-diphenylurea

Test material form:

solid: particulate/powder

Test animals / tissue source

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).

Test system

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

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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 26^thJuly 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.