1-Hexyl 4,5-diamino pyrazole hemisulfate

Endpoint summary

Description of key information

1-Hexyl 4,5-diamine pyrazole sulfate tested as neat substance in state-of-the-art in vitro test methods for skin corrosion (TER assay) and skin irritation (EpiSkinTM RHE method), showed that 1-hexyl 4,5-diamine pyrazole sulfate is non-corrosive and non-irritant.

On the basis of the results obtained in the ICE study, it can be concluded that 1-hexyl 4,5 -diamine pyrazole sulfate as neat substance and at 1.5% (w/w) in aqueous solution is not a strong eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Apr. 04, 2013 to Apr. 05, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to UK GLP in accordance with OECD, EU, US EPA, US FDA and Japan principles of GLP

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: One (RccHan™:WIST) rat was obtained from Harlan Laboratories U.K. Ltd., Oxon, UK- Age at study initiation: 23 days old (the animal was in telogen phase of hair growth and little or no hair growth was visible)- Sex: Female- Weight at study initiation: Not reported- Housing: During the acclimatization period the animal was housed in a suspended plastic cage furnished with softwood wood flakes.- Diet: Rodent 2014C Teklad Global Certified Diet (ad libitum)- Water: Tap water (ad libitum)- Acclimation period: 2 daysExperiment Initiation Date: Apr. 04, 2013Experiment Completion Date: Apr. 05, 2013

Type of coverage:

open

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL: Sufficient neat test material to cover the epidermal surface was used in this study.

Duration of treatment / exposure:

Contact period of 24 h

Observation period:

24 h

Number of animals:

One

Details on study design:

PRE-TEST PROCEDURES: - Skin disk preparation: Post acclimation period, the animal was shaved to remove hair from the dorsal surface. The shaved area was washed using an antibiotic wash and a second antibiotic wash was performed after 3 days. Three days later, the animal was killed by inhalation of carbon dioxide followed by cervical dislocation. The dorsal skin of animal was removed as a single pelt and after removal of excess fat the pelt was mounted epidermal side uppermost, onto a polytetrafluoroethylene (PTFE) tube. The tissue was secured in place using a rubber "O" ring. Excess tissue was trimmed away and the "O" ring/PTFE interface was sealed with soft paraffin wax. The tube was supported by a clamp inside a labeled 30 mL glass receptacle containing 10 mL electrolyte solution (154 mM MgSO4).- Skin disk quality control: Two skin discs of approx. 0.79 cm2 were taken from the pelt and the TER (Transcutaneous Electrical Resistance) was measured as a quality control procedure. Each disc had to give a resistance value of greater than 10 kΩ in order for the remainder of the pelt to be used in the assay. If either disc fell below the 10 kΩ threshold, the pelt was discarded. The quality control discs were then discarded and new discs from the acceptable pelt were mounted on the PTFE tubes.TEST PROCEDURE:- Number of test replicates: 3 skin discs- Controls: 3 positive (approximately 36% hydrochloric acid) and 3 negative control (sterile distilled water) discs were assayed.- Test material or negative /positive control administration: Test material/negative control/positive control was applied for 24 hours to cover the epidermal surface of skin disk. 150 µL of distilled water was applied with test material to ensure good contact with the skin. - Removal of test substance: After 24 hours of exposure, the test material was removed by washing the skin disc with a jet of warm tap water until no further test material could be removed. - Determination of Transcutaneous Electrical Resistance (TER): The TER was measured using a Wheatstone Bridge with a low voltage alternating current. Prior to measurement of the resistance, the surface tension of the skin disc was reduced by adding a sufficient volume of 70% ethanol to cover the epidermis. The ethanol was removed by inverting the tube after approximately 3 seconds. The measurement was taken and a value in Ω/kΩ per skin disc was displayed on the databridge display. The mean TER for the skin discs was calculated. Details are provided in the study report.

Irritation parameter:

other: Transcutaneous Electrical Resistance (TER)

Basis:

mean

Time point:

other: 24h

Remarks on result:

other: Mean TER value of test material: 28.9 ± 5.6 kΩ (Above the Cut off TER value of 5 kΩ)

SKIN DISK QUALITY CONTROL RESULT: Two skin discs were evaluated for quality control. Each disc had to give a resistance value of greater than 10kΩ in order for the remainder of the pelt to be used in the assay. If either disc fell below the 10 kΩ threshold, the pelt was discarded. The quality control discs were then discarded and new discs from the acceptable pelt were mounted on the PTFE tubes.

TRANSCUTANEOUS ELECTRICAL RESISTANCE ASSAY RESULTS: The individual and mean value of three TER measurement for test material, positive control and negative control after a 24 hours contact period are provided in below table:

Table 1: Individual and Mean TER (Transcutaneous Electrical Resistance) measurements (Study # 78119

Contact Period	Tissue Number	TER	Mean TER	Standard Deviation
4,5-Diamino-1-hexyl-1H-pyrazole, hemisulfate
24 hours	1	24.6 kΩ	28.9 kΩ	± 5.6
	2	26.8 kΩ
	3	35.2 kΩ
Positive Control (approximately 36% Hydrochloric acid)
24 hours	4	x	873.5 Ω*	± 71.4
	5	823 Ω
	6	924 Ω
Negative Control (Sterile distilled water)
24 hours	7	19.0 kΩ	17.4 kΩ	± 1.4
	8	16.5 kΩ
	9	16.7 kΩ

TER: Transcutaneous Electrical Resistance

X: Reading unobtainable due to perforated disc, which was considered due to the corrosive nature of the positive control.

*: Based on 2 skin discs due to perforation of 1 skin disc

Interpretation of results:

other: non corrosive

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

4,5-Diamino-1-hexyl-1H-pyrazole, hemisulfate (2:1) was considered non corrosive in the in-vitro skin corrosion (Transcutaneous Electrical Resistance (TER)) assay.

Executive summary:

The in-vitro skin corrosion test of 4,5-Diamino-1-hexyl-1H-pyrazole, hemisulfate(2:1) was determined following the OECD 430 guideline (In-Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER)).

Dorsal skin disks were removed as a single pelt from one female Wistar strain rat (RccHan™: WIST) obtained from Harlan Laboratories U.K. Ltd., Oxon, UK.

The skin discs were evaluated for TER using a Wheatstone Bridge with a low voltage alternating current. The measurements were taken in Ω/kΩ per skin disc and the mean TER for the skin discs were calculated. The undiluted test material (neat) was used in this study. Undiluted test material, the positive control (36% HCl) and the negative control (sterile distilled water) were tested in triplicate.

The mean TER value recorded for 1-hexyl-1H-pyrazole-4,5 diamine sulfate (2:1) after 24 hours contact period was 28.9 kΩ (TER > 5 kΩ: Non corrosive test material).

1-hexyl-1H-pyrazole-4,5 diamine sulfate (2:1) was considered non corrosive in thein-vitro skin corrosion (Transcutaneous Electrical Resistance (TER)) assay.

This in-vitro skin corrosion test is classified as acceptable, and satisfies the guideline requirements of the OECD 430 method.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Apr. 23, 2013 To May. 29, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Qualifier:

according to guideline

Guideline:

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

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:

according to UK, OECD and EC principles of GLP

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied: 10 mg for pre-test; 10 ± 2 mg solid for main testNEGATIVE CONTROL (Sterile water)- Amount(s) applied: 10 µL- Concentration: Applied as such (100%)POSITIVE CONTROL (Sodium Dodecyl Sulphate): - Concentration: 5.0% w/v aqueous solution

Duration of treatment / exposure:

15 minutes

Details on study design:

TEST DESIGN:- Test method: Colourimetric MTT reduction assay TEST SYSTEM: RHE (Reconstituted Human Epidermal) model obtained from SkinEthic Laboratories, Lyon, France consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. TEST PROCEDURE:- Preparation of MTT stock solution: A 3 mg/mL MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required. - PRE-TEST (Assessment of direct test material reduction of MTT): A pre-test was performed to assess the ability of test material to directly reduce MTT to formazan thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. 10 mg of the solid test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay/maintenance medium and incubated in the dark at 37°C, 5% CO2 for 3 hours. Untreated MTT solution was used as a control. If the MTT solution color turned blue relative to the control, the test material was considered to have the ability to reduce the MTT.- FUNCTIONAL CHECK WITH WATER-KILLED TISSUES: This was conducted to avoid false negative results. If the test substance was able to reduce MTT directly and not totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, a skin irritation test with water-killed tissues (possess no metabolic activity but absorb and bind the test substance like viable tissues) was performed in parallel. In addition to the normal test procedure, each MTT reducing test substance was applied to a water-killed tissue. In addition, one water-killed tissue remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue. Details on the preparation of water-killed tissues were provided in the study report.- Pre-Incubation (Day 0: tissue arrival): Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert. 2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of pre-labelled 12-well plates. Each epidermis tissue was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test material and each control. The tissues were incubated at 37°C, 5% CO2 in air for at least 24 hours.MAIN TEST: Application of test material and rinsing (Day 1): - Number of tissues treated with test material: 9 (3 for relative tissue viability analysis, 3 for color correction procedure and 3 for histology)- Number of tissues treated with negative control: 9 (3 for relative tissue viability analysis, 3 for color correction procedure and 3 for histology) - Number of tissues treated with positive control: 6 (3 for relative tissue viability analysis and 3 for histology)- Application of test material: Approximately 10 mg of the solid test material was applied to the epidermis surface. Tissues were exposed for 15 minutes in the biological safety cabinet at room temperature.- Rinsing: After 15 minutes of exposure, each tissue was removed from the well and rinsed with Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each tissue was then totally submerged in 150 mL of PBS and gently shaken to remove residual test material (repeated three times). Finally, each tissue was rinsed once again using the wash bottle. The rinsed tissues were transferred to the appropriate wells of a 12-well plate previously prepared with 2 mL of maintenance medium. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 ± 1 hour. The negative and positive control groups were rinsed using the same process.MTT Loading/Formazan Extraction (Day 3):- Following 42 hour post-exposure incubation, each 12-well plate was placed onto a plate shaker for 15 ± 2 minutes to homogenise the released mediators in the maintenance medium.- MTT viability assay: 3 tissues for each treatment group, including negative and positive controls, were transferred to 12-well plates containing 2 mL of 0.3 mg/mL MTT solution, freshly prepared in assay medium. Any excess maintenance medium from the bottom of the tissue inserts was removed by blotting on absorbent paper. 1.6 mL of the maintenance medium from beneath each tissue of the negative and positive controls and test item treated viable tissues was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30°C for possible inflammatory mediator determination. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. After the 3 hour incubation period, each tissue was placed onto absorbent paper to dry. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability) using the MTT Visual Scoring scheme.Following qualitative evaluation of tissue viability, a total biopsy of the epidermis was made using the EpiSkin biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.- MTT Colour Correction: 3 test material treated tissues for each group were used for MTT color correction purposes. The tissues were treated identically to the tissues placed in the MTT solution except that they were transferred to 12-well plates containing 2 mL of assay medium in a sufficient number of wells. Similarly, 3 tissues treated with the negative control were transferred to 12-well plates containing 2 mL of assay medium in a sufficient number of wells to determine and correct background colour in the tissues. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment to allow the extraction of residual colour out of the tissues.- Absorbance/Optical Density Measuremts (Day 6): At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For the MTT treated groups and the colour correction groups, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone were added to the two wells designated as ‘blanks’. All wells were examined and any air bubbles removed. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.HISTOLOGY: The tissues retained for histopathology were fixed in 10% Formalin. Histopathological processing was performed by the test site Histologix Ltd, BioCity, Pennyfoot Street, Nottingham, NG1 1GF, UK (Principal Investigator: Dorinda Wright). Details on histological score and interpretation are provided in the study report.CLASSIFICATION OF IRRITATION POTENTIAL: The test material was classified based on MTT viability analysis in a prediction model:i) Relative MTT true viability (with % negative control) ≤ 50: Irritantii) Relative MTT true viability (with % negative control) > 50: Non - irritant

Irritation / corrosion parameter:

other: other: Relative tissue viability based on MTT assay (%)

Value:

119.4

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes . Remarks: Neat test material relative to sterile water control. (migrated information)

Irritant / corrosive response data:

The skin irritancy of the test material was evaluated according to the prediction model based on MTT viability analysis (%MTT viability > 50% for non-irritancy):- Neat concentration of the test material did not induce any significant cytotoxicity in the MTT assay indicative of skin irritation (Non-irritant).- Compared to the negative control tissues, the MTT relative viability of the test item treated tissues after a 15-Minute exposure period and a 42 ± 1 hour post-treatment incubation period was 119.4%

Other effects:

- Direct reduction of MTT by test material relative to negative control: showed a negligible degree of direct reduction of MTT. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.- MTT color correction assay: The color of the test material was considered not to have caused interference with the MTT test (no absorbance measured in the colour correction tissues).- Histology: The negative control did not show pathological changes.The positive control showed Marked necrosis/disruption with some basal layers intact orNecrosis/disruption with some basal layers intact.The test item showed minimal degranulation/swelling superficial, minimal vacuolation superficial or Occasional vacuolation/necrotic cells.Histological evaluation revealed that the test material induced minimal epidermal effects (score 1) compared to the negative control (score 0) confirming an absence of marked epidermal effects in histopathology.

Quality Criteria

a) Negative control: The mean OD562 for the negative control treated tissues was 0.712 and the standard deviation value of the percentage viability was 4.6%. The negative control acceptance criterion was therefore satisfied.

b) Positive control: The relative mean tissue viability for the positive control treated tissues was 12.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 3.5%. The positive control acceptance criterion was therefore satisfied.

 

c) Test substance: The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 10.6%. The test item acceptance criterion was therefore satisfied.

Table 1: Mean OD₅₄₀Values and Percentage Viabilities for the Negative Control, Positive Control and Test material

Treatment	Mean OD540of triplicate tissues	± SD of OD540	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control (Sterile water)	0.712	0.033	100*	4.6
Positive Control (Sodium dodecyl sulfate)	0.086	0.025	12.1	3.5
Test substance	0.850	0.076	119.4	10.6

SD= Standard deviation

*=The mean viability of the negative control tissues is set at 100%

Interpretation of results:

not classified

Remarks:

Migrated informationCriteria used for interpretation of results: EU

Conclusions:

1-hexyl-1H-pyrazole-4,5 diamine sulfate (2:1) was classified as a non-irritant to the skin (RHE model) when applied as neat (% MTT viability > 50%) according to the prediction model based on MTT viability analysis. The test substance was classified as ‘Not classified for Irritation’ according to EU DSD & CLP and as ‘Not classified for Irritation (category 3 cannot be determined)’ as per UN GHS.

Executive summary:

The in-vitro skin irritation potential of 1-hexyl-1H-pyrazole-4,5 diamine sulfate (2:1) was determined following methods comparable to the OECD Guideline 439 (In Vitro Skin Irritation).

The skin irritation potential of the test material was determinedcubation period by using the EpiSkin Reconstructed Human Epidermal (RHE) prediction model based on the colorimetric MTT viability assay. Three RHEs were exposed to 10 ± 2 mg neat test material for an exposure period of 15 minutes followed by a rinsing step and a 42 ± 1 hour post-treatment incubation period.

The negative control (sterile water) and positive control (SDS 5.0%w/v aqueous solution) were measured in triplicate.

In this study two endpoints, cytotoxicity in the MTT assay, expressed as percentage viability of treated cultures in comparison to negative controls, as well as morphological changes identified by histological examination at the end of the treatment, were evaluated, for skin irritation potential of 1-hexyl-1H-pyrazole-4,5 diamine sulfate (2:1).

Tissue viability was analyzed by visual examination and evaluation of the degree of MTT stain and the color of tissue (qualitative) and the viability was measured by the colorimetric MTT reduction assay (quantitative) based on a prediction model (% MTT viability >50%: Non irritant).

A decrease in MTT reduction capacity and changes in tissue morphology were used as indicators of potential irritancy.

The color-corrected MTT relative viabilities of test material treated tissues after a 15 minute exposure period and a 42 ± 1 hour post-treatment incubation were compared to the mean of the negative control tissues. The relative tissue viability calculated was 119.4%.

The test material was considered not to have the ability to directly reduce MTT. Histological evaluation revealed that the test material induced minimal epidermal effects (score 1) compared to the negative control (score 0) confirming the absence of cytotoxicity in the MTT assay. 

The quality criteria required for acceptance of the results in the test were satisfied.

The neat concentrations of 1-hexyl-1H-pyrazole-4,5 diamine sulfate (2:1)did not induce cytotoxicity in the MTT assay indicative of skin irritation according to the prediction model.

Based on above, 1-hexyl-1H-pyrazole-4,5 diamine sulfate (2:1) was classified as a non-irritant to the skin (RHE model) when applied as neat (% MTT viability > 50%) according to the prediction model based on MTT viability analysis.

The test substance was classified as ‘Not classified for Irritation’ according to EU DSD & CLP and as ‘Not classified for Irritation (category 3 cannot be determined)’ as per UN GHS.

This in vitro skin irritation study is classified as acceptable, and satisfies the guideline requirements of OECD 439 method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

other information

Study period:

From Mar. 26, 2010 to May 10, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline GLP

Qualifier:

according to guideline

Guideline:

other: 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

Species:

other: chicken

Strain:

not specified

Vehicle:

water

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied: 30 mg (for solid) and 30 µL (for liquids)- Concentration: Neat (undiluted) and 5% w/v aqueous solution- POSITIVE CONTROL: NaOH (for solid test substance) and 5% w/v benzalkonium chloride (for liquid test substance)- NEGATIVE CONTROL: Saline

Duration of treatment / exposure:

10 sec

Observation period (in vivo):

Observed at approx. 0, 30, 75, 120, 180 and 240 min after treatment. Fluorescein retention was only scored at approx. 30 min 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 v.d. Bor, Amersfoortseweg 118, Nijkerkerveen, The Netherlands. The eyes were dissected in the test facility for experiment.- Age of animal: 7 wk old- Weight of animal: Approx. 1.5 - 2.5 kg- Collection and transportation of chicken heads: The heads of animal were removed 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 were maintained at ambient temperature.EXPERIMENTAL PROCEDURE:- Determination of eye damage: Eye (corneal surface) was treated with small drop of Fluorescein sodium BP 2% w/v and subsequently rinsed off with isotonic saline. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp BP 900, Haag-Streit AG, Liebefeld-Bern, Switzerland), to ensure that the cornea was not damaged.- Positioning of eye in superfusion apparatus: Undamaged eye was further dissected from the head (without damaging the eye or cornea), without cutting off the optical nerve too short. 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). 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 approx. 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 approx. 32°C (water pump set at 34°C, Thermomix 1441, B. Braun Melsungen AG, Melsungen, Germany). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged.- Measurement of corneal thickness prior to treatment: An accurate measurement was taken at the corneal apex of each eye. After 45-60 min equilibration period, corneal thickness was measured to determine the zero reference value for corneal swelling calculations. Corneal thickness was measured by using Depth Measuring Attachment no. I for the Haag-Streit slit-lamp microscope. Thickness of the cornea was expressed in instrument units. - Criteria for eye selection: Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, or 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.- Number of test eyes: 3 per concentration - Number of control eyes: 1 for negative control and 3 eyes for each positive control- Application of test material: The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards and test substance was applied to corneal surface. Prior to treatment, each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention.- Histological evaluation: After final examination, all treated eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at 5 µM and stained with PAS (Periodic Acid-Schiff). Histopathological examination was performed by light microscopy.REMOVAL OF TEST SUBSTANCE- Washing: After exposure, eyes were rinsed thoroughly with 20 mL of isotonic saline (ambient temperature).- Time after start of exposure: 10 secSCORING SYSTEM: The scoring is as follows:1. Corneal swelling: Corneal swelling, 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) × 1002. Corneal opacity: Opacity degree 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 iscernible.....…...…….……….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, the thickness assessment will not be possible. Intermediate scores can also be assigned.3. Fluorescein retention 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. Intermediate scores can also be assigned.4. Morphological effects: 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.5. Microscopic effects: Corneal lesions were determined by microscopic 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 was subject to the interpretation of the investigator.TOOL USED TO ASSESS SCORE: All observations were carried out with Slit lamp microscope (Slit-lamp BP 900, Haag-Streit AG, Liebefeld-Bern, Switzerland)

Irritation parameter:

other: Irritation index

Basis:

mean

Time point:

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

Score:

33

Max. score:

200

Remarks on result:

other: 5% concentration of test substance in water

Irritation parameter:

other: Irritation index

Basis:

mean

Time point:

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

Score:

136

Max. score:

200

Remarks on result:

other: Neat test substance

Irritant / corrosive response data:

- The 5% aqueous test solution caused no swelling, very slight or slight opacity and very slight or slight fluorescein retention. The calculated Irritation Index was 33. - The neat test substance caused slight swelling, severe opacity and severe fluorescein retention. The calculated Irritation Index was 136.

Other effects:

HISTOPATHOLOGICAL EFFECTS: In eyes treated with 5% test solution, microscopic examination of the corneas revealed minor epithelial effects. In eyes treated with neat test substance, microscopic examination of the corneas only revealed very slight to moderate epithelial effects.Details on individual results are provided in the study report.

Table 1. Maximum mean score for corneal swelling, opacity and fluorescein retention, irritation categories by slit-lamp examination after 10 sec treatment with 4,5-Diamino-1-hexyl-1H-pyrazole dihydrochloride (Study # 66833)

Treatment	Maximum mean score for			Irritation categories^1	Irritation Index^2	Irritation classification
	% swelling	Opacity	Fluorescein retention
5% test solution	3	0.8	0.7	I;II;II	33	Not classified (NC)^5
Neat test substance(Undiluted)	16	3.0	3.0	II;IV;IV	136	Category 1^3,4
Saline (negative control)	0	0.0	0.0	NA^7	NA	NA
BAC (5% w/v)(Positive control)	34	3.0^8	3.0	IV; IV; IV	154	Category 1^3,4
NaOH (Positive control)	n.d.^9	4.0	3.0	?; IV; IV	>140	Category 1^3,4

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

2: Irritation index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)

3: EU-CLP: NC = not classified; Category 2 = Irritating to eyes; Category 1 = irreversible effects on the eye/serious damage to the eye. Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006.

4: GHS: Category 2B = mild irritant; Category 2A = irritant; Category 1 = irreversible effects on the eye/serious damage to the eye. United Nations-Economic Commission for Europe (UN/ECE) (2003). Globally Harmonised System of Classification and Labelling of Chemicals (GHS). UN, New York and Geneva, 2007.

5: Not irritating, but borderline case with Category 2B

6: Individual values based on one eye

7: NA =not applicable

8: Blistering or detachment (severe loosening) of epithelium

9: n.d. = not discernable because of the very severe opacity; all eyes showed iris constriction immediately after exposure

RESULT OF CONTROL GROUPS:

NEGATIVE CONTROL: The control eye did not show any corneal effect. Microscopic examination of the corneas did not reveal abnormalities.

POSITIVE CONTROL:

- Benzalkonium chloride (BAC 5%) caused severe corneal swelling, severe opacity and severe fluorescein retention. The calculated Irritation Index was 154.

- NaOH caused very severe opacity and severe fluorescein retention. Corneal thickness could not be measured, because of the presence of very severe opacity. The calculated Irritation Index was higher than 140.

Microscopic examination of the cornea treated with Benzalkonium chloride and NaOH confirmed the corneal severity.

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated informationat neat test concentrationCriteria used for interpretation of results: other: EU-CLP and OECD GHS classification of ICE

Conclusions:

4,5-Diamino-1-hexyl-1H-pyrazole dihydrochloride (Hexylpyrazole) was classified as Category 1 (irreversible effects on the eye/serious damage to the eye) according to EU-CLP and OECD GHS classification when applied to the isolated chicken eye (ICE) at neat concentration for 10 sec.4,5-Diamino-1-hexyl-1H-pyrazole dihydrochloride (Hexylpyrazole) was categorized as not classified according to EU-CLP and OECD GHS classification when applied to the isolated chicken eye (ICE) at 5% w/v test concentration for 10 sec.

Executive summary:

The in-vitro eye irritation of 4,5-Diamino-1-hexyl-1H-pyrazole dihydrochloride (Hexylpyrazole) was determined by following the methods of the OECD guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants).

The eyes were isolated from either male or female spring chicken heads (ROSS) of 7 wk old (approx. 1.5-2.5 kg) obtained from poultry slaughterhouse v.d. Bor, Amersfoortseweg 118, Nijkerkerveen, The Netherlands. The isolated chicken eyes were exposed to a single application of the following test concentrations for 10 sec using an application of:

5% in water (30 μL) and neat (undiluted solid; 30 mg)

In test substance and positive control group, 3 corneas/ treatment were used. One cornea was used for negative control. Saline served as negative control. NaOH (for solid test substance) and 5% w/v benzalkonium chloride served as positive controls. 

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

The 5% aqueous test solution caused no swelling, very slight or slight opacity and very slight or slight fluorescein retention. The calculated Irritation Index was 33.

The neat test substance caused slight swelling, severe opacity and severe fluorescein retention. The calculated Irritation Index was 136.

In eyes treated with 5% test solution, microscopic examination of the corneas revealed minor epithelial effects. In eye treated with neat test substance, microscopic examination of the corneas revealed very slight to moderate epithelial effects.

In negative control group (saline), microscopic examination of the corneas did not reveal abnormalities.

Benzalkonium chloride (BAC 5%) caused severe swelling, severe opacity and severe fluorescein retention. The calculated Irritation Index was 154.

NaOH caused very severe opacity and severe fluorescein retention. Corneal thickness could not be measured, because of the presence of very severe opacity. The calculated Irritation Index was higher than 140.

Microscopic examination of the cornea treated with Benzalkonium chloride and NaOH confirmed the corneal severity. 

Based on above, 4,5-Diamino-1-hexyl-1H-pyrazole dihydrochloride (Hexylpyrazole) was classified as Category 1 (irreversible effects on the eye/serious damage to the eye) according to EU-CLP and OECD GHS classification when applied to the isolated chicken eye (ICE) at neat concentration for 10 sec.

4,5-Diamino-1-hexyl-1H-pyrazole dihydrochloride (Hexylpyrazole) was categorized as not classified according to EU-CLP and OECD GHS classification when applied to the isolated chicken eye (ICE) at 5% w/v test concentration for 10 sec.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)