1H-Pyrazole-5-carboxamide, 4-amino-1-methyl-3-propyl-, hydrochloride (1:?)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 2015 to 18 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviation.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide hydrochloride

EC Number:

938-723-7

Cas Number:

1688656-88-3

Molecular formula:

C8H14N4O.HCL

IUPAC Name:

4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

Batch 2000533289
Purity/Composition 100.2%

Specific details on test material used for the study:

Batch 2000533289 (taken from label) Purity/Composition 100.2% Test item storage At room temperature Stable under storage conditions until 03 November 2019 (expiry date) (taken from label)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Test System
Species Mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA). Source: Janvier, Le Genest-Saint-Isle, France Number of animals 20 females (nulliparous and non-pregnant), five females per group (main study only). Age and body weight Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean. Identification Tail mark with a marker pen. Health inspection At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.

Animal Husbandry
Conditions Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
Diet Free access to pelleted rodent diet (SM R/M-Z from SSNIFF
® Spezialdiäten GmbH, Soest, Germany).
Water Free access to tap water.
Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 45% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Dimethyl sulphoxide).

No. of animals per dose:

5

Details on study design:

Weight of Evidence Analysis
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to the start of this study. All available information was evaluated (e.g. existing human and animal data, literature, item data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity)). It was concluded by the Study Director that no severe effects were to be expected

Pre-screen Test
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 (see section 6.7) and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; a 25% and 45% concentration. The highest concentration was the maximum concentration that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study except that the application method may have been different (see tables) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation

Main Study
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 NL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 µCi of 3 H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol ® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter: 125 µm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of
Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability Twice daily.
Body weights On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. In addition, a description of all other (local) effects was recorded.

Necropsy No necropsy for gross macroscopic examination was performed according to study plan.

Interpretation
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments.
Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3)

Positive control substance(s):

other: no concurrent positive control group was included in the study.

Statistics:

In case of borderline results, statistical analysis may be performed to determine the dose response relationship and pair wise comparisons between dose groups versus negative control. The methods used will be specified in the raw data and report. The EC3 value (the estimated item concentration that will give a SI=3) may be determined if possible, based on the dose response relationship or calculated using linear interpolation

Results and discussion

Positive control results:

no concurrent positive control group was included in the study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1.8

Test group / Remarks:

10 % test group

Results 2

Key result

Parameter:

SI

Value:

ca. 2.3

Test group / Remarks:

25 % test group

Results 3

Key result

Parameter:

SI

Value:

ca. 1.7

Test group / Remarks:

45 % test group

Cellular proliferation data / Observations:

Pre-screen Test
Very slight erythema was noted for all animals between Days 2 and 4. No signs of systemic toxicity were observed in any of the pre-screen animals. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. White staining of the dorsal surface of the ears by test item remnants was noted all animals throughout the observation period. The staining did not hamper the scoring of the ears. Based on these results, the highest test item concentration selected for the main study was a 45% concentration.

Main Study
Skin Reactions / Irritation
No irritation was observed in any of the animals. White staining of test item remnants on the dorsal surface of the ears of all experimental animals did not hamper scoring for erythema.

Systemic Toxicity
No mortality occurred and body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
Discolouration of the urine was noted for all experimental animals from Day 3 onwards. Since no collaborative findings were found, this was considered to have no toxicologically relevant effect on the lymph nodes.

Macroscopic Examination of the Auricular Lymph Nodes and Surrounding Area
All auricular lymph nodes of the animals treated at 10% and 45% and control group were considered normal in size. The lymph nodes of the animals treated at 25% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 45% were 2428, 3085 and 2195 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1328 DPM. The SI values calculated for the test item concentrations 10, 25 and 45% were 1.8, 2.3 and 1.7, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

A small dose response was noted in the data of the two lowest dose groups. The response of the highest dose group did not follow the dose-response relationship which more often seen in these kind of studies. The response might be less due to differences in skin penetration (less vehicle present), but is in line with the exposure in humans.
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 45%, PF-01049573-01; UK-084443-01 was not considered to be a skin sensitizer.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.
Based on these results, PF-01049573-01; UK-084443-01 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

Assessment of skin sensitization to  PF-01049573-01; UK-084443-01 in the Mouse (Local Lymph Node Assay).

The study was carried out based on the guidelines described in: OECD, Section 4, Health Effects, No.429 (2010), EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay" EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test item concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 45% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Dimethyl sulphoxide).

Three days after the last exposure, all animals were injected with 3 H-methyl  thymidine  and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index  (SI)

was subsequently calculated for each group.

No irritation was observed in any of the animals. White staining of test item remnants on the dorsal surface of the ears of all experimental animals did not hamper scoring for erythema.

All auricular lymph nodes of the animals treated at 10% and 45% and control group were considered normal in size. The lymph nodes of the animals treated at 25% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 45% were 2428, 3085 and 2195 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1328 DPM. The  SI  values calculated for the test item concentrations 10, 25 and 45% were 1.8, 2.3 and 1.7, respectively.

A small dose response was noted in the data of the two lowest dose groups. The response of the highest dose group did not follow the dose-response relationship which more often seen in these kind of studies. The response might be less due to differences in skin penetration (less vehicle present), but is in line with the exposure in humans.

Since there was no indication that the test item elicits a  SI  ≥ 3 when tested up to 45%,

PF-01049573-01; UK-084443-01 was not considered to be a skin sensitizer.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

Based on these results,  PF-01049573-01; UK-084443-01 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).