Registration Dossier

Administrative data

Description of key information

The LLNA was performed with 1-hexyl-1H-pyrazole-4,5-diamine dihydrochloride, i.e. the dihydrochlorde instead of the hemisulfate salt. 1-Hexyl-1H-pyrazole-4,5-diamine

dihydrochloride was positive in the LLNA with an EC3 value of 8.6%. The sensitising potential and potency are attributable to the free base component. Therefore, the results of

the LLNA carried out with the dihydrochloride salt can be used for the hazard assessment of 1-hexyl 4,5-diamine pyrazole sulfate. The EC3 value of the hemisulfate salt was calculated

from the dihydrochloride salt by using the conversion factor of 0.91 to account for the different molecular weights. Therefore, the calculated EC3 value for 1-hexyl 4,5-diamine

pyrazole sulfate is 7.8%. Based on the calculated EC3 value of 7.8%, 1-hexyl 4,5-diamine pyrazole sulfate is a moderate skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 3, 2009 to June 12, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP, the study was performed on a structural analogue
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Remarks:
OECD Principles of Good Laboratory Practice
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: CBA/J mouse were obtained from Jackson Laboratories, Bar Harbor, ME, USA- Age at study initiation: 8 to 12 wk- Weight at study initiation: 18-22 g at Day 1 of the study- Housing: Animals were individually housed upon receipt and during the study in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals". The room in which the animals were kept was documented in the study records. No other species were kept in the same room.- Diet: Certified Rodent Chow 7012C, ad libitum. No contaminants were known to be present in the certified diet at levels that would be expected to interfere with the results of this study. - Water: Tap water, ad libitum, via water bottles. The water is routinely analyzed for contaminants as per Calvert SOP's. No contaminants were known to be present in the water at levels that would be expected to interfere with the results of this study. - Acclimation period: 6 d. All animals used in this study were assessed as to their general health by a member of the technical staff or other authorized personnel. During the acclimation period, each animal was observed at least once daily for any abnormalities or for the development of infectious disease. Only animals that were determined to be suitable for use were assigned to this study.Mice were selected and randomly assigned to study groups based on body weight and apparent good health. Mice were given the identification numbers and identified by tail mark with an indelible marker. ENVIRONMENTAL CONDITIONS- Temperature: 24.4-28.3˚C- Humidity: 17-61%- Air changes: Not reported- Photoperiod: 12 h dark/12 h lightIN-LIFE DATES: From: June 03, 2009 To: June 08, 2009
Vehicle:
other: 70% ethanol (vehicle group1) and Acetone:Olive oil, 4:1 (AOO) (vehicle group 2)
Concentration:
Dose concentration: 0, 1, 3, 10, and 15% (w/v) Positive control: 35% hexylcinnamaldehyde (HCA) in Acetone:Olive oil (AOO) (4:1)
No. of animals per dose:
Five female animals/dose group
Details on study design:
RANGE FINDING TESTS: No range-finding study was performedMAIN STUDYANIMAL ASSIGNMENT AND TREATMENT- Name of test method: Local Lymph Node Assay (LLNA)- Criteria used to consider a positive response: The criterion for a positive response was that one or more concentrations of a test substance elicited a 3-fold or greater increase in isotope incorporation relative to the vehicle control.TREATMENT PREPARATION AND ADMINISTRATION:- Dose preparation: On each day of dosing, the test substance was prepared at the appropriate concentrations (w/v) in volumetric flasks by diluting the appropriate amount of test substance in the appropriate vehicle. All preparations were vortexed to mix. Test substance dosing formulations were dosed as clear colorless to light pink liquids.The positive control (HCA) was prepared daily as a 35% (v/v) solution in AOO. It was dosed as a clear yellow liquid.- Rationale for the choice of test material concentrations: The dose levels were specified by the Sponsor.- Administration of the test preparations: The test/control substance dosing solutions were applied using a mechanical pipette to the dorsal surfaces of both ears (25 µL/ear) once a day for 3 consecutive days (Days 1, 2, and 3) to each animal. OBSERVATIONS: - Morbidity/mortality: All animals were observed at least once daily on Days 1 to 6.- Clinical signs: Daily observations were made of the test and control substance application sites, immediately prior to dose administration and immediately post-dose on Days 1 through 3 and once daily on Days 4 through 6. Any significant alterations (e.g., erythema and edema) to the application sites, and the general appearance of the pinnae, including any build up of test article, was recorded.- Body weight: All animals were weighed on Days 1 and 6- Evaluation of cell proliferation: On Day 6, the mice were injected, i.v., with 20 µCi of 3H-thymidine in 250 µL of sterile saline. 5 h later the mice were euthanized with CO2 and the draining auricular lymph nodes removed. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8°C. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to 12 ml of scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and it was also transferred to the 12 mL of scintillation fluid. Incorporation of 3H-thymidine was measured in a β-scintillation counter (Beckman LS 6000 SC).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis of the disintegrations per minute (DPM) data was performed using SAS version 6.12. Individual DPM values were analyzed by log transformation (base 10) of the data. Comparisons with the control group were based on the least significant difference criterion. All statistical tests were conducted at a 1%, two-tailed probability level.Body weight and change in body weight means and SEM were calculated for each group. Evaluation of equality of means were made by a one way analysisof variance using the F distribution to assess statistical significance using SYSTAT version 9.01 (SPSS, Inc.).
Positive control results:
Mean DPM: 2722 ± 327Mean Stimulation Indices (SI): 10.6 (The mean SI is greater than 3 thus validated the experimental conditions of OECD 429)
Key result
Parameter:
SI
Value:
ca. 0.7
Test group / Remarks:
1%
Remarks on result:
other: The mean stimulation indices at the concentrations of 1%, 3%, 10% and 15% in 70% ethanol were 0.7, 1, 3.5 and 3.1, respectively. The response was thus considered to be positive at 10 and 15% test concentration.
Key result
Parameter:
SI
Value:
ca. 1
Test group / Remarks:
3%
Remarks on result:
other: The mean stimulation indices at the concentrations of 1%, 3%, 10% and 15% in 70% ethanol were 0.7, 1, 3.5 and 3.1, respectively. The response was thus considered to be positive at 10 and 15% test concentration.
Key result
Parameter:
SI
Value:
ca. 3.5
Test group / Remarks:
10%
Remarks on result:
other: The mean stimulation indices at the concentrations of 1%, 3%, 10% and 15% in 70% ethanol were 0.7, 1, 3.5 and 3.1, respectively. The response was thus considered to be positive at 10 and 15% test concentration.
Key result
Parameter:
SI
Value:
ca. 3.1
Test group / Remarks:
15%
Remarks on result:
other: The mean stimulation indices at the concentrations of 1%, 3%, 10% and 15% in 70% ethanol were 0.7, 1, 3.5 and 3.1, respectively. The response was thus considered to be positive at 10 and 15% test concentration.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM: 212 ± 27, 293 ± 59, 1008 ± 261 and 890 ± 279 at 1, 3, 10 and 15% test substance concentration respectively. For details, refer to’ Table 1’ under ‘Any other information on results’ section.

Mortality: No mortality was observed during the study

Clinical signs: All animals appeared clinically normal throughout the study with 2 exceptions. One mouse treated with the test substance at a concentration of 3% and one at a concentration of 15% had orange staining on the fur on Days 4 -6. The application sites of one mouse treated with the test substance at a concentration of 3% had orange staining on the ears on Days 4 and 5. Another mouse treated with the test substance at 15% had orange staining on the ears on Day 4. The ears of the mice treated with HCA appeared wet on Days 2 -6. There were no other findings.

Body weight: There were no statistically significant differences observed between any of the treatment groups. Therefore, the test article did not appear to cause any overt toxicity.

Lymph node size: At termination, the lymph nodes from the mice in the vehicle treated groups and all test substance treated groups were normal in size and appearance. Enlarged lymph nodes relative to the vehicle-treated mice were observed in the HCA treated group.

Table 1: Local lymph node assay with 4,5 -Diamino-1 -Hexyl-1H-Pyrazole, Dihydrochloride (Study # 65871)

 Treatment Group

Dose

DPM (mean± SEM)

SI (Test/control ratio)

Results1

Vehicle group 1(70% EtOH)

-

285 ± 51

-

-

Vehicle group 2 (AOO)

-

258 ± 42

-

-

Treatment groups

 

 

 

1%

212 ± 27

0.7

-

3%

293 ± 59

1

-

10%

1008 ± 261 **

3.5

+

15%

890 ± 279 **

3.1

+

Positive control group (HCA)

35% (v/v)

2722 ± 327 ***

10.6

+

1Test/control ratio of 3.0 or greater represents a positive result

**Statistically significant difference when Log DPM compared to the corresponding vehicle control group (p < 0.01)

***Statistically significant difference when Log DPM was compared to the corresponding vehicle control group (p = 0.0001).

The solubility and stability of the test item in the solvent was considered sufficient. None of the animals died during the study, bodyweights were not altered and all animals appeared

clinically normal throughout the study. Some orange staining on the ears and on the fur occurred, which was considered to be related to the staining properties of 4,5-diamino-1-

hexyl-1H-pyrazole dihydrochloride. There were no treatment-related effects on bodyweight or bodyweight gain.

The positive control, HCA, at a concentration of 35.0% in acetone: olive oil, induced a 10.6 -fold increase in isotope incorporation in the draining auricular lymph nodes relative to the vehicle control. The mean stimulation index above 3 validated the experimental conditions.

4,5-Diamino-1-hexyl-1H-pyrazole dihydrochloride was positive in the LLNA, as there was a statistically significant increase in isotope incorporation in the draining auricular lymph

nodes relative to the vehicle at 10% and 15%, which were greater than a 3-fold increase.

The mean stimulation indices were 0.7, 1.0, 3.5 and 3.1 at the concentrations of 1%, 3%, 10% and 15%, respectively. The EC3 value was calculated to be 8.6%.

4,5-Diamino-1-hexyl-1H-pyrazole dihydrochloride is a moderate skin sensitiser under the defined experimental conditions, with a calculated EC3 value of 8.6%.

Calculation for the hemisulfate salt of 1-hexyl-1H-pyrazole-4,5-diamine:

The EC3 value of the hemisulphate salt was calculated from the dihydrochloride salt by using the conversion factor of 0.91 to account for the different molecular weight.

EC3 value dihydrochloride: 8.6%

8.6% = 8.6 g/100 mL x 0.91 (conversion factor) = 7.8 g /100 mL = 7.8%

EC3 value hemisulfate: 7.8%

Therefore, the calculated EC3 value for 1-hexyl-1H-pyrazole-4,5-diamine hemisulfate is 7.8%. Based on this calculation, 1-hexyl-1H-pyrazole-4,5-diamine hemisulfate is considered a moderate skin sensitiser according to the relative skin sensitisation potency classification scheme published in the ECETOC technical report on contact sensitisation

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Migrated informationCriteria used for interpretation of results: expert judgment
Conclusions:
4,5-Diamino-1-Hexyl-1H-Pyrazole, dihydrochloride, was considered as sensitizer in Local Lymph Node Assay (LLNA) at test concentration of 10 and 15%. The calculated EC3 value for 1-hexyl-1H-pyrazole-4,5-diamine hemisulfate is 7.8%. Based on this calculation, 1-hexyl-1H-pyrazole-4,5-diamine hemisulfate is considered a moderate skin sensitiser according to the relative skin sensitisation potency classification scheme published in the ECETOC technical report on contact sensitisation
Executive summary:

The in-vivo skin corrosion test of 4,5-Diamino-1-Hexyl-1H-Pyrazole, dihydrochloride, was determined following OECD 429 guideline (Skin Sensitisation: Local Lymph Node Assay).

A total of 35CBA/Jfemale mice (8-12 wk old)weighing 18-22 g were acclimated for 6 d for this study. The feed used was Certified Rodent Chow (ad libitum). Animals were individually housed upon receipt and during the study in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals". Animals were maintained under standard laboratory conditions (temperature: 24.4 ± 28.3°C, humidity: 17 ± 61%; lighting: 12 h light/12 h dark cycle). Mice were selected and randomly assigned to study groups based on body weight and apparent good health.

The test substance was prepared at different test concentrations in vehicle (70% ethanol and Acetone:Olive oil, 4:1 (AOO)). Positive control animals received 35% (v/v) Hexylcinnamic aldehyde (HCA) in Acetone:Olive oil (AOO) (4:1). The animals received 25 µL of the appropriate formulations or vehicle on dorsal surface of each ear on Days 1 to 3. On Day 6, the mice were injected, i.v., with 20 µCi of3H-thymidine in sterile saline. 5 h later, the animals were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were precipitated with 5 % trichloroacetic acid (TCA) and the pellets counted in a β-scintillation counter (Beckman LS 6000 SC) to determine incorporation of3H-thymidine. Following treatment groups were employed in the study:

Group 1 (vehicle group): 70% Ethanol

Group 2 (vehicle group): AOO

Group 3 (treatment group): 1%

Group 4 (treatment group): 3%

Group 5 (treatment group): 10%

Group 6 (treatment group): 15%

Group 7 (Positive control): 35% v/v HCA

There was no mortality and all animals appeared clinically normal throughout the study. One mouse treated with the test substance at a concentration of 3% and one at a concentration of 15% had orange staining on the fur on Days 4-6. The application sites of one mouse treated with the test substance at a concentration of 3% had orange staining on the ears on Days 4 and 5. Another mouse treated with the test substance at 15% had orange staining on the ears on Day 4. The ears of the mice treated with HCA appeared wet on Days 2-6. Other treatment-related findings were not observed.

Mean body weights at Day 1 and Day 6 and mean changes in body weights were evaluated. There were no statistically significant differences observed between any of the treatment groups. Therefore, the test article did not appear to cause any overt toxicity.

The positive control, 35% (v/v) HCA in AOO, resulted in a stimulation index (SI) of 10.6. A 3-fold or greater increase in proliferative activity relative to the concurrent AOO vehicle control is considered a positive response.

The test substance at concentrations of 1, 3, 10, and 15% (w/v) resulted in SI of 0.7, 1.0, 3.5, and 3.1, respectively. Statistically significant differences were found between the groups treated with the test substance at 10 and 15% and the 70% ethanol vehicle control.

Treatment with 4,5-Diamino-1-Hexyl-1H-Pyrazole, dihydrochloride, at concentrations of 10 and 15% resulted in stimulation indices greater than 3. Therefore, 4,5-Diamino-1-Hexyl-1H-Pyrazole, dihydrochloride, is considered to have skin sensitizing activity.

4,5-Diamino-1-Hexyl-1H-Pyrazole, dihydrochloride, was considered as sensitizer in Local Lymph Node Assay (LLNA) at test concentration of 10 and 15%, with a calculated EC3 value of 8.6%.

Calculation for the hemisulfate salt of 1-hexyl-1H-pyrazole-4,5-diamine: The EC3 value of the hemisulphate salt was calculated from the dihydrochloride salt by using the conversion factor of 0.91 to account for the different molecular weight.

EC3 value dihydrochloride: 8.6%

8.6% = 8.6 g/100 mL x 0.91 (conversion factor) = 7.8 g /100 mL = 7.8%

EC3 value hemisulfate: 7.8%

Therefore, the calculated EC3 value for 1-hexyl-1H-pyrazole-4,5-diamine hemisulfate is 7.8%. Based on this calculation, 1-hexyl-1H-pyrazole-4,5-diamine hemisulfate is considered a moderate skin sensitiser according to the relative skin sensitisation potency classification scheme published in the ECETOC technical report on contact sensitisation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

Based on the calculated EC3 value of 7.8%, 1-hexyl 4,5-diamine pyrazole sulfate is a moderate skin sensitiser and should be classified as skin sensitizer Category 1B.