4,5-bis(hydroxymethyl)-2-phenyl-1H-imidazole

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 20 August 2021 to April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to the OECD TG 438 with minor deviations not affecting the reliability of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

During preparation, eye were dissected from the skull before the assessment of corneal integrity. This deviation has no impact on the study reliability as it has been validated by there CRO and is part of their standard SOP.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,5-bis(hydroxymethyl)-2-phenyl-1H-imidazole

EC Number:

262-911-1

EC Name:

4,5-bis(hydroxymethyl)-2-phenyl-1H-imidazole

Cas Number:

61698-32-6

Molecular formula:

C11H12N2O2

IUPAC Name:

[4-(hydroxymethyl)-2-phenyl-1H-imidazol-5-yl]methanol

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: white to light-pink powder
- Storage: Keep away from heat and sources of ignition. Store in tightly closed container. Store in a well-ventilated place. Store in a cool, dark place.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep away from heat and sources of ignition. Store in tightly closed container. Store in a well-ventilated place. Store in a cool, dark place at room temperature (20°C ± 10°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: NA
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: NA
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: NA
- Reactivity of the test material with the incubation material used (e.g. plastic ware): NA

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): NA
- Preliminary purification step (if any): NA
- Final concentration of a dissolved solid, stock liquid or gel: NA
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): NA

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: NA

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Etablissement Brun, 33820 Etauliers, France
- Number of animals: Not stated
- Characteristics of donor animals (e.g. age, sex, weight):Spring chickens, 7 weeks old, 1.5 - 2.5kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. Heads collected from source at 8:45am and eyes enucleated at ICARE at 10.53am.
- Time interval prior to initiating testing: 45 minutes
- Indication of any existing defects or lesions in ocular tissue samples: Eyes where ocular lesions were present were discarded prior to testing.
- Indication of any antibiotics used: Not stated
- Selection and preparation of corneas: Not stated
- Quality check of the isolated corneas: Examination under slit-lamp microscope (Haag-Streit BP900 with a slit width of 0.095mm)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied (weight): 30 mg
- Concentration (if solution): NA

VEHICLE
- Amount(s) applied (volume or weight with unit): NA
- Concentration (if solution): NA
- Lot/batch no. (if required): NA
- Purity: NA

Duration of treatment / exposure:

10 second exposure

Duration of post- treatment incubation (in vitro):

30, 75, 120, 180 and 240 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES: The selection of eyes was based on the cornea integrity following excision.

NUMBER OF REPLICATES: 3 replicates

NEGATIVE CONTROL USED: Physiological saline – Dutscher Batch No. C0543A01 – CAS : 7647-14-5 – EC: 231-598-3 – SMILES: [Na+].[Cl-] – Storage : room temperature

POSITIVE CONTROL USED: sodium hydroxide – Fisher Scientific, Batch No. 0000080257 - CAS : 1310-73-2 – EC: 215-185-5 – SMILES: [OH-].[Na+] – Storage : room temperature

APPLICATION DOSE AND EXPOSURE TIME: 30 mg of test material was used with 10s exposure time. XX was used for both control substances with 10 s exposure time

OBSERVATION PERIOD: NA

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Two rinses with 10mL physiological saline followed by the use of a cotton bud soaked in saline to remove residual test material
- Indicate any deviation from test procedure in the Guideline: Use of cotton bud soaked with saline. However justification in the form of validated test methods does not deem this a deviation that affects the integrity of the test.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm
- Damage to epithelium based on fluorescein retention: NA
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm
- Macroscopic morphological damage to the surface: Not stated
- Others (e.g, histopathology): NA

SCORING SYSTEM: as per OECD TG 438

DECISION CRITERIA: as per OECD TG 438

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

45 minutes pretreatment, 30, 75, 120, 180 and 240 minutes post treatment

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE class I

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

45 minutes pre-test and 30 minutes post treatment

Value:

>= 0.5 - <= 2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

ICE Class III

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

45 minutes pre test, 30, 76, 120, 180 and 240 minutes post treatment

Value:

>= 0.5 - <= 0.62

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

ICE Class II

Results 4

Irritation parameter:

percent corneal swelling

Run / experiment:

45 minutes pre test, 30, 76, 120, 180 and 240 minutes post treatment

Value:

>= 3 - <= 9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

ICE Class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: cf 'Attachment"

ACCEPTANCE OF RESULTS: cf. "'Attachments'
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, as expected the negative control is classified as “No Category”, as expected.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, Sodium Hydroxide 5%, was
3 x IV. Therefore, as predicted the positive control is classified as “Corrosive/Severe Irritant.
- Range of historical values if different from the ones specified in the test guideline: as per OECD TG 438. Cf. 'Attachment'

Any other information on results incl. tables

Table 2: Negative control - Assessment of the eye corrosion/severe irritation individual and average values for evaluation of corneal lesions after treatment

Endpoint measured	Eye no.	Time (min)
		-45	30	75	120	180	240
Corneal opacity	16	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class			I	-	-	-	-
Fluorescein retention	16	0.5	0.5	-	-	-	-
Mean		0.5	0.5
ICE class		I
Corneal thickness	16	0.51	0.51	0.51	0.52	0.52	0.52
Corneal swelling (%)	16	-	0	0	2	2	2
Mean		-	0	0	2	2	2
ICE class		I
Combination of the 3 endpoints		3 x I
Classification		No Category

Table 3: Positive control - Assessment of the eye corrosion/severe irritation individual and average values for evaluation of corneal lesions after treatment

Endpoint measured	Eye no.	Time (min)
		-45	30	75	120	180	240
Corneal opacity	1	0	4	4	4	4	4
	2	0	4	4	4	4	4
	3	0	4	4	4	4	4
Mean		0.0	4.0	4.0	4.0	4.0	4.0
ICE class		IV
Fluorescein retention	1	0.5	3	-	-	-	-
	2	0.5	3	-	-	-	-
	3	0.5	3	-	-	-	-
Mean		0.5	3	-	-	-	-
ICE class		IV
Corneal thickness	1	0.54	0.84	0.98	1.05	1.10	1.11
	2	0.53	0.81	0.86	0.91	1.05	1.05
	3	0.56	0.82	0.87	0.89	1.03	1.12
Corneal swelling (%)	1	-	56	81	94	104	106
	2	-	53	62	72	96	98
	3	-	46	55	59	84	100
Mean		-	52	66	75	95	101
ICE class		IV
Combination of the 3 endpoints		3 x IV
Classification		Category I: ‘Corrosive/Severe Irritant’

Table 4: Test item Curezol 2PHZ-PW - Assessment of the eye corrosion/severe irritation individual and average values for evaluation of corneal lesions after treatment

Endpoint measured	Eye no.	Time (min)
		-45	30	75	120	180	240
Corneal opacity	7	0	4	0	0	0	0
	8	0	4	0	0	0	0
	9	0	4	0	0	0	0
Mean		0.0	0.0	4.0	0.0	0.0	0.0
ICE class		I
Fluorescein retention	7	0.5	2	-	-	-	-
	8	0.5	2	-	-	-	-
	9	0.5	2	-	-	-	-
Mean		0.5	2	-	-	-	-
ICE class		III
Corneal thickness	7	0.58	0.59	0.59	0.59	0.62	0.62
	8	0.50	0.53	0.53	0.53	0.57	0.59
	9	0.54	0.54	0.54	0.54	0.54	0.55
Corneal swelling (%)	7	-	2	2	2	7	7
	8	-	6	6	6	14	18
	9	-	0	0	0	0	2
Mean		-	3	3	3	7	9
ICE class		III
Combination of the 3 endpoints		1 x I 1 x II 1 x III
Classification		No prediction can be made

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The combination of the three endpoints for the test item CUREZOL 2PHZ-PW was 1 x I, 1 x II, 1 x III. No prediction can be made.

Executive summary:

In an in vitro eye irritation / corrosion study (Isolated Chicken Eye) performed according to the OECD TG No. 438 and in compliance with GLP, 30 mg of undiluted test item was applied to three enucleated chicken eyes for 10 seconds. The eyes were then rinsed twice with 10 mL of physiological saline. Following two rinses remnants of the product was still evident on the entire cornea of all three eyes. The remaining test substance was removed using a cotton bud soaked with physiological saline soaked. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention 45 minutes prior the treatment and at 30, 75, 120, 180 and 240 minutes  (± 5) minutes post treatment rinse.

 

The ocular reactions observed in eyes treated with the test item were:

• maximal mean score of corneal opacity: 0.0, corresponding to ICE class I;


• mean score of fluorescein retention: 2.0, corresponding to ICE class III;


• maximal mean corneal swelling: 9 %, corresponding to ICE class II.

 

The combination of the three endpoints for the test item CUREZOL 2PHZ-PW was 1 x I, 1 x II, 1 x III.

The combination of the three endpoints for the positive control, Sodium Hydroxide 5%, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

 

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, on prediction can be made on the test item CUREZOL 2PHZ-PW and additional testing is required to establish a definitive classification.