1-Hexyl 4,5-diamino pyrazole hemisulfate

Skin irritation / corrosion

Administrative data

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

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 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

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: 4,5-Diamino-1-hexyl-1H-pyrazole, hemisulfate (Code: A0021277)- TSIN: WR804146- Substance type: Pure active substance- Physical state: White feathery crystals- Storage condition of test material: At room temperature, in dark

Test animals

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

Test system

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.

Results and discussion

In vivo

Results

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)

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.