Propanamide, N-[4-(2,4-dihydroxyphenyl)-2-thiazolyl]-2-methyl-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

yes

Remarks:

The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer. However, this can be seen as uncritical, because the opacity can be calculated from the extinction.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

yes

Remarks:

with acceptable restrictions: The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer. However, this can be seen as uncritical, because the opacity can be calculated from the extinction.

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

EC Number:

807-621-3

Cas Number:

1428450-95-6

Molecular formula:

C13H14N2O3S

IUPAC Name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used for classification.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Slaughterhouse Müller Fleisch GmbH, Birkenfeld, Germany
- Donor animals: between 12 and 60 months old
- Date and time of eye collection: on the day of the test
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution supplemented with 0.01% penicillin and 0.01% streptomycin

INCUBATION MEDIUM
- MEM (Minimum Essential Medium) without phenol red supplemented with 1% FCS, L-Glutamine and NaHCO3 (= complete MEM (cMEM) without phenol red); pre-warmed to 32±1 °C
- MEM (Minimum Essential Medium) with phenol red supplemented with 1% FCS and L-Glutamine (= complete MEM (cMEM) with phenol red); pre-warmed to 32±1 °C

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Equilibration time of the corneas: 1 h at 32±1 °C in cMEM without phenol red
- Quality check of the equilibrated corneas: free of macroscopic defects

DETERMINATION OF THE INITIAL OPACITY
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded.
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via a photometer at 570 nm.
- Specification of the device: spectral photometer, Specord 205, Analytik Jena, Germany

Test system

Vehicle:

other: olive oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 750 µL
- Concentration (if solution): 20% solution in olive oil

VEHICLE
- Substance: olive oil
- Amount(s) applied in the test: Example: 750 µL

POSITIVE SUBSTANCE
- Substance: 20% Imidazole solution in 0.9% NaCl
- Amount(s) applied in the test: 750 µL

Duration of treatment / exposure:

4 h at 32 ± 1 °C

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

number of eyes for the test item: 3
number of eyes for the positive control: 3
number of eyes for the negative control: 3
number of eyes for the solvent control: 3

Details on study design:

TEST CONDITIONS
- Short description of the method used: open-chamber method
The glass window from the anterior chamber was removed prior to treatment. The controls or test substance were applied directly to the epithelial surface of the cornea. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system. Corneas were exposed for 4 h with the test substance or the controls.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed from the anterior chamber and the epithelium washed at least three times.
- Medium for washing the corneas: cMEM containing phenol red
- Medium for final rinsing: cMEM without phenol red;
Thereafter both chambers of the cornea holder were filled with fresh cMEM without phenol red and final opacity was recorded.

DETERMINATION OF THE FINAL OPACITY
- Method: corneal opacity was determined via a photometer at 570 nm
- Specification of the device: Spectral photometer, Specord 205, Analytik Jena, Germany

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: The cMEM without phenol red was then removed from the anterior chamber, and 1 mL sodium fluorescein solution (concentration 5 mg/mL) was added. The chambers were then closed again and incubated for 90 ± 5 min at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density at 490 nm.
- Specification of the device: Spectral photometer, Specord 205, Analytik Jena, Germany
- Correction of Measured Absorption at 490 nm: As cuvettes with a pathlength of 0.2 cm are used in the measurement of the sodium fluorescein solution in the spectral photometer, the pathlength must be corrected to 1 cm. Coefficient: 1/0.2 = 5: all absorptions were multiplied with this coefficient.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Value:

ca. 0.908

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Value:

ca. 0.668

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

The absorption (570 nm) and opacity values which were measured before and after exposureare given in the following table:

Table 1  Absorption and Opacity Values Negative Control and Positive Control

Parameter	Negative control			Positive control
Absorption before	0.1567	0.1616	0.1597	0.1877	0.1214	0.1272
Absorption after	0.2596	0.1937	0.2709	1.9489	1.6818	2.0496
Opacity before	1.4345	1.4508	1.4444	1.5406	1.3225	1.3403
Opacity after	1.8180	1.5621	1.8659	88.8996	48.0618	112.0986
Opacity Difference	0.3835	0.1113	0.4215	87.3590	46.7393	110.7583

Mean opacity difference of the negative control is 0.3054.

 

Table 2  Absorption and Opacity Values Test Item and Solvent Control

Parameter	Solvent control			Test ItemN-[4-(2,4-dihydroxyphenyl)thiazol-2-yl]isobutyramide
Absorption before	0.1990	0.1294	0.2167	0.1510	0.1651	0.1535
Absorption after	0.3601	0.2348	0.4103	0.2368	0.2617	0.2209
Opacity before	1.5812	1.3471	1.6470	1.4158	1.4625	1.4240
Opacity	2.2914	1.7171	2.5722	1.7250	1.8268	1.6630
Opacity Difference	0.7101	0.3700	0.9251	0.3092	0.3643	0.2391

Mean opacity difference of the solvent control is 0.6684.

 

For the permeability measurement, three replicates for each treatment group were measured. The optical density values at 490 nm are given in the following tables:

Table 3  Optical density at 490 nm Negative Control and Positive Control

Repl.	Negative Control			Positive Control
Meas.	0.0090	0.0076	0.0075	0.2231	0.2604	0.2774
Corr.	0.0450	0.0380	0.0375	1.1155	1.3020	1.3870
Mean	0.0402			--

 

Table 4  Optical density at 490 nm Test Item and Solvent Control

Repl.	Solvent control			Test ItemN-[4-(2,4-dihydroxyphenyl)thiazol-2-yl]isobutyramide
Meas.	0.0116	0.0078	0.0056	0.0095	0.0074	0.0065
Corr.	0.0580	0.0390	0.0280	0.0475	0.0370	0.0325
Mean	0.0417

Note: In order to correct the path length, a factor of 5 was taken into account when calculating the IVIS

 

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Table 5  IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control 0.9 % NaCl	1.059	0.908	22.0 %
	0.681
	0.984
Solvent control Olive oil	1.580	1.293	24.4 %
	0.955
	1.345
Test Item 20% suspension in olive oil	-0.272	-0.405	37.0 %
	-0.375
	-0.567
Positive Control 20 % imidazole in 0.9% NaCl	103.183	99.733	32.9 %
	65.361
	130.655

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

CLP: not classified

Note: Guidance on the Application of Regulation (EC) No. 1272/2008
3.3.2.1.2.4 Testing methods: in-vitro methods
“The OECD has at present adopted three in vitro tests for the identification of substances inducing serious eye damage, i.e. the Isolated Chicken Eye (ICE) test (OECD TG 438; TM B.48), the Bovine Corneal Opacity and Permeability (BCOP) test (OECD TG 437; TM B.47) and the Fluorescein Leakage (FL) test (OECD TG 460). These tests are recommended for use as part of a tiered-testing strategy for regulatory classification and labelling (e.g. Top-Down Approach). A substance can be considered as causing serious eye damage (Category 1) based on positive results in the ICE test, the BCOP test, the FL test, the Isolated Rabbit Eye (IRE) test or the Hen's Egg Test on Chorio-allantoic Membrane (HET-CAM) test78. Negative results from the ICE and BCOP test methods can be used for classification purposes i.e. ‘bottom-up approach’. For other test methods the negative in vitro corrosivity responses in these tests must be followed by further testing (Guidance on IR/CSA Section R.7.2.4.1).
In addition an in vitro test method has been validated by ECVAM and is under consideration for the development of an OECD TG: the Cytosensor Microphysiometer (CM) test. This can be used for the identification of category 1-substances within a Top-Down Approach.
There are no in vitro tests with regulatory acceptance for eye irritation at present. However, two human corneal epithelium models, EpiOcular and SkinEthic, have been submitted to ECVAM for validation.”

3.3.2.3.2. In-vitro data
“A substance can be considered as causing serious eye damage (Category 1) based on positive results in the ICE test, the BCOP test, the Isolated Rabbit Eye (IRE) test or the Hen's Egg Test on Chorio-allantoic Membrane (HET-CAM) test79. Negative results from the ICE and BCOP test methods can be used for classification purposes i.e. ‘bottom-up approach’, but for other test methods the negative in vitro corrosivity responses in these tests must be followed by further testing (Guidance on IR/CSA Section R.7.2.4.1).
There are currently no validated in vitro eye irritation test methods available.”

Executive summary:

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle which were between

12 and 60 months old.

The test item N-[4-(2,4-dihydroxyphenyl)thiazol-2-yl]isobutyramide was tested as 20%

suspension in olive oil.. 750 μL of the suspension were brought onto the cornea of a bovine

eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for one

hour and whose opacity had been measured. The test item was incubated on the cornea

for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values

were measured.

Physiological sodium chloride solution was used as negative control. The negative control

showed no irritating effect on the cornea.

Olive oil was used as solvent control. The solvent control showed no irritating effect on the

cornea.

20% Imidazole solution (dissolved in 0.9% NaCl) was used as positive control. The positive

control induced serious eye damage on the cornea.

The test item N-[4-(2,4-dihydroxyphenyl)thiazol-2-yl]isobutyramide (tested as 20% suspension

in olive oil) showed no effects on the cornea of the bovine eye. The calculated

IVIS (in vitro irritancy score) is -0.405.