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

Endpoint summary

Description of key information

Three in vitro tests were perormed to determine skin irritation / corrosion and eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted in 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test system:

human skin model

Remarks:

EpiDerm™; reconstructed three-dimensional human epidermis (EPI-200)

Cell type:

other: Human-derived epidermal keratinocytes

Cell source:

other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Vehicle:

water

Details on test system:

REMOVAL OF TEST SUBSTANCE
- Washing: The test substance was washed from the skin surface with phosphate buffered saline.
- Time after start of exposure: 3 and 60 min

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, tissues were incubated with 300 µL prewarmed MTT solution for 3 h at 37 ± 1°C, 5 ± 1 % CO2 and 80-95 % RH. After aspiration of the MTT solution, tissues were washed several times in phosphate buffered saline followed by tissue drying. Extraction of the formazan product was carried out in 2 mL isopropanol. The optical density was measured at 570 nm wave length in a plate spectrophotometer.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg ± 2.5 mg moistened with 25 µL demineralized water

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: 8 M KOH

Duration of treatment / exposure:

3 min at room temperature and 60 min at 37 °C in an incubator

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

ca. 86.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

ca. 105.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Table 1: MTT assay after 3 minutes exposure

	OD Tissue 1	OD Tissue 2	OD Mean	SD	Viability %	RSD %
Negative Control	1.856 1.840 1.819	1.817 1.802 1.812	1.824	0.02	100	---
Test Substance	1.501 1.645 1.580	1.588 1.588 1.578	1.580	0.05	86.6	0.4
Positive Control	0.409 0.424 0.438	0.431 0.404 0.400	0.418	0.02	22.9	2.0

Table 2: MTT assay after 60 minutes exposure

	OD Tissue 1	OD Tissue 2	OD Mean	SD	Viability %	RSD %
Negative Control	1.515 1.659 1.584	1.867 1.865 1.847	1.723	0.16	100	---
Test Substance	1.871 1.894 1.867	1.754 1.757 1.747	1.815	0.07	105.3	4.9
Positive Control	0.141 0.141 0.141	0.163 0.161 0.161	0.151	0.01	8.8	9.7

Table 3: Historical Control Data

Parameter	OD Negative Control	OD Negative Control	Viability % Positive Control	Viability % Positive Control
Incubation Time	3 min	60 min	3 min	60 min
Mean	1.936	1.920	27.2	14.1
SD	0.157	0.168	5.6	4.4
Range	1.614 – 2.355	1.598 – 2.326	20.2 – 43.6	8.5 – 24.2

Interpretation of results:

GHS criteria not met

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat. 1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.

Executive summary:

One valid experiment was performed.

Two tissues of the human skin model EpiDerm TM were treated with N-[4-(2,4-dihydroxyphenyl)

thiazol-2-yl]isobutyramide for three minutes and one hour, respectively. In average,

24. 7 mg of the solid test item were applied to each tissue and spread to match the tissue

size.

Demineralised water was used as negative Qontrol, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of

the tissues was evaluated by addition of MTT which can be reduced to a blue formazan.

Formazan production was measured by measuring the optical density (OD) of the resulting

solution.

After treatment with the negative control, the absorbance values were within the required

acceptability criterion of mean OD ~ 0.8 and !5 2.8 for both treatment intervals thus showing

the quality of the tissues. The positive control showed clear corrosive effects for both

treatment intervals.

After three minutes treatment with the test item, the relative absorbance values were reduced

to 86.6 %. This value is well above the threshold for corrosion potential (50 %). After

one hour treatment, relative absorbance values were increased to 105.3 %. This value,

too, is well above the threshold for corrosion potential (15 %). In the guideline, values

greater or equal to the threshold are considered as ''non-corrosive to skin".

Therefore, N-[4-(2,4-dihydroxyphenyl)thiazol-2-yl]isobutyramide is considered as

not corrosive in the Human Skin Model Test.

Reference 2

Endpoint:

skin irritation: in vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 3

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted in 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test system:

human skin model

Remarks:

EpiDerm™; reconstructed three-dimensional human epidermis (EPI-200)

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: MatTek In Vitro Life Science Laboratories, Bratislava

Vehicle:

other: Dulbecco’s Phosphate Buffered Saline

Remarks:

CaCl2 and without MgCl2

Details on test system:

REMOVAL OF TEST SUBSTANCE
- Washing: The test substance was washed from the skin surface with phosphate buffered saline.
- Time after start of exposure: 60 min
- Post-treatment incubation period: 42 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed 42 h after the incubation period. Therefore, tissues were incubated in 300 µL prewarmed MTT solution for 3 h ± 5 min at 37 °C, 5 % CO2 and 80 - 95 % humidity. After aspiration of the MTT solution, tissues were washed 3 times in PBS followed by tissue drying. Extraction of the formazan product was carried out in 2 mL isopropanol. The optical density was measured at 570 nm wave length in a plate spectrophotometer.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25.4, 25.1 and 24.2 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: SDS, 5 % (v/v), 30 µL

Duration of treatment / exposure:

35 min at 37 °C and 25 min at RT

Duration of post-treatment incubation (if applicable):

Not applicable. Post-treatment incubation period: 42 h

Number of replicates:

3

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

100

Remarks on result:

other:

Remarks:

Basis: mean value of negative controls (DPBS). Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

2.1

Remarks on result:

other:

Remarks:

Basis: mean value of positive controls (5 % SDS). Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

92.7

Remarks on result:

other:

Remarks:

Basis: mean value of test substance. Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Table 1: MTT assay after 60 minutes exposure

	OD Tissue 1	OD Tissue 2	OD Tissue 3	OD Mean (blank corrected)	SD	Viability %	RSD %
Negative Control	2.668 2.617	2.395 2.358	2.505 2.500	2.471	0.133	100	---
Test Substance	2.209 2.177	2.417 2.404	2.434 2.320	2.290	0.117	92.7	5.1
Positive Control	0.092 0.093	0.086 0.087	0.090 0.087	0.053	0.003	2.1	5.8

Absorption value blank isopropanol (OD at 570 nm): 0.037

Table 2: Historical Control Data

Parameter	OD Negative Control	Viability % Positive Control
Mean	1.823	7.2
SD	0.333	3.9
Range	1.024 – 2.254	2.0 – 17.1

Interpretation of results:

GHS criteria not met

Conclusions:

CLP: not classified

Executive summary:

Three tissues of the human skin model EpiDermTM were treated with N-[4-(2,4-

dihydroxyphenyl)thiazol-2-yl]isobutyramide for 60 minutes.

In average, 25 mg of the solid test item (wetted with 25 μL DPBS-buffer) were applied to

each tissue and spread to match the tissue size (0.63 cm2; as indicated by supplier).

DPBS-buffer was used as negative control, 5% SDS-solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required

acceptability criterion of ≥ 0.8 mean OD ≤ 2.8. The positive control showed clear irritating

effects. Variation within tissues was acceptable (< 18 %).

After the treatment with the test item, the relative absorbance values were reduced to

92.7 %. This value is well above the threshold for irritation potential (≤ 50%).

Therefore, N-[4-(2,4-dihydroxyphenyl)thiazol-2-yl]isobutyramide is considered as

not irritant in the Human Skin Model Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

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

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.

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

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

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.

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

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

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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

After three minutes treatment with the test item, the relative absorbance values were reduced

to 86.6 %. This value is well above the threshold for corrosion potential (50 %). After

one hour treatment, relative absorbance values were increased to 105.3 %. This value,

too, is well above the threshold for corrosion potential (15 %). In the guideline, values

greater or equal to the threshold are considered as ''non-corrosive to skin".

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 does not

require classification for eye irritation or serious eye damage. Based on the outcome of the

study, the test item is therefore not classified for eye irritation or serious eye damage, as

defined by the UN GHS of classification and labelling of chemicals and the Regulation on

classification, labelling and packaging of substances and mixtures (EU CLP).