1-Hexyl 4,5-diamino pyrazole hemisulfate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Mar. 27, 2013 to Apr. 29, 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

the corneal thickness was measured using the Depth Measuring Attachment # II for the Haag-Streit slit-lamp microscope. However, according to the OECD 438 guideline, the corneal swelling scores shown in Table 3 to be used for the determination of the ICE classes, are only applicable if the thickness is measured with the depth-measuring device n° 1 and a slit-width setting at 9 1/2 equalling 0.095 mm. This has an impact on result analysis

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to OECD principles of GLP

Test material

Test material information

Constituent 1

Reference substance name:

2-hexylpyrazole-3,4-diamine;sulfuric acid

EC Number:

696-231-5

Cas Number:

1361000-03-4

Molecular formula:

C9H18N4 x 0.5 H2SO4

IUPAC Name:

2-hexylpyrazole-3,4-diamine;sulfuric acid

Constituent 2

Reference substance name:

C6 pyrazole hemisulfate

IUPAC Name:

C6 pyrazole hemisulfate

Constituent 3

Reference substance name:

4,5-diamino-1-hexyl-1H-pyrazole hemisulfate

IUPAC Name:

4,5-diamino-1-hexyl-1H-pyrazole hemisulfate

Test material form:

solid: crystalline

Details on test material:

- Name of test material: 1-hexyl-1H-pyrazole-4,5 diamine sulphate (2:1) (Code: A021277)- TSIN: WR804146- Substance type: Pure active substance- Physical state: Colourless crystals- Storage condition of test material: Ambient conditions (protected from light)

Test animals / tissue source

Species:

other: Chicken

Strain:

not specified

Test system

Vehicle:

other: Water (containing NaoH and Ascorbic acid (0.1%))

Amount / concentration applied:

TEST MATERIAL- Test concentration: 1.5%, 5% and 100% w/w - Amount applied: 30 μL of 1.5% and 5% w/w solutions; 30 mg of 100% (neat) test substance NEGATIVE CONTROL: Physiological saline- Amount applied: 30 µL POSITIVE CONTROL: NaoH- Amount applied: 30 mg

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment. Fluorescein retention was only scored at approximately 30 minutes after treatment.

Details on study design:

DETAILS OF TEST SYSTEM:- Test system used: Isolated chicken eye (ICE) - Source: The eyes were isolated from either male or female spring chicken heads (ROSS) obtained from poultry slaughter house van Miert, Breukelen, The Netherlands. The eyes were dissected in the test facility for experiment.- Age of animal: Approximately 7 week old- Weight of animal: Approximately 1.5 - 2.5 kg- Collection and transportation of chicken heads: Animal heads were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline and maintained at ambient temperature.EXPERIMENTAL PROCEDURE:- Preparation of the eyes: Within 2 h after kill of chickens, eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium BP 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. The fluorescein treated cornea was examined with a slit-lamp microscope to ensure that the cornea was not damaged. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (TNO, Zeist, The Netherlands).- Conditions of superfusion apparatus: The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a rate of approximately 0.10 - 0.15 mL/min (peristaltic pump, Watson-Marlow 205CA, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32°C (water pump set at 36.4°C; Lauda 103,Germany).- Selection Criteria for eyes used in the ICE: Eyes with a corneal thickness (measured using the Depth Measuring Attachment # II for the Haag-Streit slit-lamp microscope) deviating more than 10% of the average corneal thickness of the eyes, eyes that showed opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced, if required.- Number of test eyes: 3 per dosing volume- Number of control eyes: 3 per dosing volume for positive control and 2 eyes for negative control.- Application of test substance: Once all eyes have been examined and approved, the eyes were incubated for 45 to 60 min to equilibrate them to the test system (as each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention) prior to treatment. Following the equilibration period, a zero reference value was recorded for corneal swelling calculations. At time = 0 (time immediately after the zero reference measurement) the clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards and the eyes (corneas) were treated with the test substance/saline.REMOVAL OF TEST SUBSTANCE- Washing: After exposure, eyes were rinsed thoroughly with 20 mL of isotonic saline (ambient temperature).EVALUATION1. SCORING SYSTEM: After treatment, eyes were scored as follows:A. Corneal swelling: Corneal swelling (observed at 30, 75, 120, 180 and 240 min), expressed as a percentage, was calculated according to the following formula: Corneal swelling = ((Corneal thickness at time t - Corneal thickness at time t = 0)/ Corneal thickness at time t = 0) × 100- A negative swelling up to -5% (not unusual for control eyes) was presented as 0% swelling. The mean percentage of swelling for the three test eyes was calculated for each of the observation time points. The maximum mean percentage was used for classification into one of four categories. B. Corneal opacity: Opacity degree (observed at 30, 75, 120, 180 and 240 minutes) of density (area most dense taken for scoring) No opacity………………………………………………………………………..................................................................0Very faint opacity…………………………………………………............................................................................0.5Scattered or diffuse areas, details of iris clearly visible……………………………………………….……….…...……….1Easily discernible translucent area, details of iris slightly obscured…………..…….........................…………2Severe corneal opacity, no specific details of iris visible, size of pupil barely discernible.....……………….3Complete corneal opacity, iris invisible……………………………………………………………………….……………….………4The mean corneal opacity value for all test eyes was calculated for the observation time points of 30, 75, 120, 180, and 240 min. Note: In case of score 4, thickness assessment will not be possibleC. Fluorescein retention (observed at 30 min)No fluorescein retention………………………………………………………………………………………………………..0Very minor single cell staining………………………………………..……………………………………………………0.5Single cell staining scattered throughout the treated area of the cornea……………….........……..1Focal or confluent dense single cell staining…………………………………………………………….……………..2Confluent large areas of the cornea retaining fluorescein………………………………………..…………….3- The mean fluorescein retention value for all test eyes was calculated for the observation time point of 30 min only. If desired or in case of test substances that have adhered to the cornea, fluorescein retention can be determined at t=240 min or whenever the test compound is removed. 2. MORPHOLOGICAL EVALUATION: These include "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings was subjective to the interpretation of the investigator.3. MICROSCOPIC EVALUATION: Corneal lesions are determined by microscopical examination. The effects include but are not limited to erosion, necrosis and vacuolation of the epithelium, disorder of stromal fibers, pyknotic nuclei in the stroma and necrosis of the endothelium. The classification of these findings is subject to the interpretation of the investigator.TOOL USED TO ASSESS SCORE: All observations were carried out with Slit lamp microscope (Slit-lamp 900 CN, Haag-Streit AG, Liebefeld-Bern, Switzerland).

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Corneal swelling and opacity observed at 30, 75, 120, 180 and 240 min. Fluorescein retention observed at 30 min.

Value:

ca. 31

Remarks on result:

other: 30 mg of neat test substance

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Corneal swelling and opacity observed at 30, 75, 120, 180 and 240 min. Fluorescein r etention observed at 30 min.

Value:

ca. 13

Remarks on result:

other: 1.5% (w/w) concentration of test substance in Ascorbic acid

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Corneal swelling and opacity observed at 30, 75, 120, 180 and 240 min. Fluorescein r etention observed at 30 min.

Value:

ca. 65

Remarks on result:

other: 5% (w/w) concentration of test substance in Ascorbic acid

Other effects / acceptance of results:

Irritant / corrosive response data
- Neat test substance (30 mg): Caused very slight swelling (1%), slight opacity (1.0), and very slight fluorescein retention (0.5). Microscopic examination of the corneas revealed no abnormalities of epithelium, stroma or endothelium. The calculated Irritation Index was 31.- 1.5% w/w test substance: Caused very slight swelling (3%), very slight opacity (0.5), and no fluorescein retention (0.0).
Microscopic examination of the corneas revealed no abnormalities of epithelium, stroma or endothelium. The calculated Irritation Index was 13.- 5% w/w test substance: Caused slight swelling (15%), slight opacity (1.0), and slight to moderate fluorescein retention (1.5). Microscopic examination of the corneas revealed very slight or slight erosion and very slight vacuolation of the epithelium. No abnormalities of the stroma and endothelium were observed. The calculated Irritation Index was 65.

Other effects
HISTOPATHOLOGICAL EFFECTS: Microscopical examination of the corneas generally confirmed the effects observed by slit-lamp examination.

Any other information on results incl. tables

Table 1 - Summary results of the slit-lamp examination after 30 second treatment with 4,5-diamino-1-hexyl-1H-pyrazole hemisulfate (study # 78056)

Test material	Maximum mean score			Irritation categories	Irritation Index	Classification
	Corneal swelling (%)	Corneal opacity	Fluorescein retention
Neat test substance (30 mg)	1	1	0.5	I;II;I	31	NC
1.5% (w/w) test concentration	3	0.5	0	I;I;I	13	NC
5.0% (w/w) test concentration	15	1	1.5	II;II;II	65	2B
Negative control (Saline)	0	0	0	Not applicable; two eyes tested
Positive control (NaOH)	49	4*	3	IV;IV;IV	189	Category 1

Irritation categories: I = no effect; II = slight effect; III = moderate effect; IV = severe effect

*Immediate iris constriction

'NC' denotes Not classified

Applicant's summary and conclusion

Interpretation of results:

other: not classified

Conclusions:

Under the condition of this isolated chicken eye (ICE) test, 1-hexyl-1H-pyrazole-4,5 diamine sulphate can be assigned to following irritation categories (as per UN-GHS system of classification): Neat test substance or 1.5% w/w solution : Not classified.
5% w/w solution: Category 2B (mildly irritating to eyes)

Executive summary:

Thein- vitro eye irritation of 1-hexyl-1H-pyrazole-4,5 diamine sulphate (2:1) (Hexylpyrazole) was determined by following the OECD guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants).

The eyes were isolated from approximately 7 week old male or female spring chickens (ROSS) obtained from poultry slaughterhouse van Miert, Breukelen, The Netherlands. The isolated chicken eyes were exposed to a single application of 30 mg neat test substance or 30 μL of 1.5% and 5% w/w aqueous solutions of test substance for 10 seconds followed by a 20 mL saline rinse.

Sodium hydroxide and physiological saline (0.9%) served as positive and negative controls respectively.

The test included three test substance (or positive control) treated eyes and two negative control treated eyes.

After treatment three main parameters, corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells, were observed. Corneal thickness and corneal opacity were scored at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment using a slit microscope. Fluorescein retention was only scored at approximately 30 minutes after treatment. In addition, histopathology was performed on the cornea to assess the nature and depth of injury.

Neat test substance (30 mg) caused very slight swelling (1%), slight opacity (1.0), and very slight fluorescein retention (0.5). Microscopic examination of the corneas revealed no abnormalities of epithelium, stroma or endothelium. The calculated Irritation index was 31.

1.5% w/w test substance caused very slight swelling (3%), very slight opacity (0.5), and no fluorescein retention (0.0). Microscopic examination of the corneas revealed no abnormalities of epithelium, stroma or endothelium. The calculated Irritation index was 13.

5% w/w test substance caused slight swelling (15%), slight opacity (1.0), and slight to moderate fluorescein retention (1.5). Microscopic examination of the corneas revealed very slight or slight erosion and very slight vacuolation of the epithelium. No abnormalities of the stroma and endothelium were observed. The calculated Irritation index was 65.

The negative control eye did not show any corneal effects and demonstrated that the general conditions during the tests were adequate.

The positive control eyes showed severe corneal effects and demonstrated that the ICE test met the acceptance criteria to be considered a valid study.

Under the condition of this isolated chicken eye (ICE) test, 1-hexyl-1H-pyrazole-4,5 diamine sulphate can be assigned to following irritation categories (as per UN-GHS system of classification):

Neat test substance or 1.5% w/w solution : Not classified

5% w/w solution: Category 2B (mildly irritating to eyes) 

Thisin- vitro acute eye irritation test is classified as acceptable, and satisfies the guideline requirements of the OECD 438 method.