Registration Dossier

Administrative data

Description of key information

Skin

Key study:- Shepard (2003) 'Skin sensitization study: local lymph node assay' conducted in line with OECD Guideline 429. The test substance was found to be a non-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:
8 December 2003 to 2 February 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 11 to 12 weeks of age at the start of the induction phase of the study.
- Weight at study initiation: 18.49 to 26.98 grams at the start of the induction phase of the study.
- Housing: Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited vivarium in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). On Days 0-5, the mice were singly housed in suspended, stainless-steel, wire mesh cages. On Day 5 of the study, immediately following injection with PBS containing 20 µCi of [3H-methyl] thymidine 3HTdR), the mice were group housed in polycarbonate shoebox cages. Cages and racks were washed once a week. Absorbent paper, used to collect excreta under the wire mesh cages, was changed at least three times a week. Absorbent bedding used in the polycarbonate shoebox cages was disposed of after the mice were euthanatized.
- Diet: Certified Rodent Diet, pellets, was available ad libitum.
- Water: While the animals were housed in suspended, stainless-steel, wire mesh cages (Days 0-5), water was available ad libitum through an automatic watering system. While the animals were housed in polycarbonate shoebox cages (Day 5), water was supplied to mice in glass bottles equipped with sipper tubes. The source of the water was the local public water system.
- Acclimation period: The animals were isolated upon arrival and allowed to acclimate for a period of 6 days. Animals were judged to be healthy prior to testing.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.8 to 24.0 °C
- Humidity: 38.1 to 47.7 %
- Photoperiod: A photoperiod of 12 hours of light was maintained.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Primary irritation screen: 25 and 50 % w/v test substance
Main study: 10, 25 and 50 % w/v test substance
No. of animals per dose:
Primary irritation screen: 2 (4 in total)
Main study: 6 (25 in total)
Details on study design:
RANGE FINDING TESTS
Primary Irritation Screen:
The primary irritation screen determined the concentrations of the test substance in an acetone/olive oil (4:1 v/v) vehicle, which could-be applied topically without eliciting a strong local or systemically toxic reaction. Mice were treated daily by application of 25 µL of the appropriate preparation to the dorsal surface of each ear for three consecutive days (Days 0, 1, 2). The test substance was administered using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette. Each animal was observed once daily (Days 0-5) for any clinical signs of either local irritation at the application site or of systemic toxicity.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The important calculation in the LLNA is the Stimulation Index (SI). The SI is derived by dividing the mean dpm/mouse within each group by the mean dpm/mouse for the solvent/vehicle control group. The average SI for the vehicle controls is then 1. Usually, a stimulation index of three or greater (SI ≥ 3) is considered a positive sensitization response.

TREATMENT PREPARATION AND ADMINISTRATION
Preparation of test substance:
The test substance concentrations (10, 25 and 50 % w/v) in the vehicle were prepared daily. The vehicle [acetone/olive oil (4:1 v/v)] was prepared once prior to study initiation.

Topical Applications:
Mice were treated daily by application of 25 µL of the appropriate preparation to the dorsal surface of each ear for three consecutive days (Days 0, 1, 2). The test substance was administered using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette.

Preparation of [3H-methyl] thymidine solution:
The 3HTdR was diluted to a radiochemical concentration of 80 µCi/mL using phosphate buffered saline (PBS), pH 7.4 ± 0.1. The specific activity of the 3HTdR was 5 Ci/mmol. The radiochemical concentration was determined by liquid scintillation spectrometry (LSS).

Intravenous Injections:
On Day 5 of the study, mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of 3HTdR. Five mice were allocated to each group in order to yield four mice per group that successfully received injections of 3HTdR. The success of each injection was evaluated at the time of the injection. If the first four mice per group received a full dose of 3HTdR, the fifth mouse was not injected; the fifth mouse in the group was then removed from the study. If any of the first four mice in a group did not receive a full dose of 3HTdR, the fifth mouse was injected. If five mice per group were injected with 3HTdR, the four mice receiving the most successful dose (based on the amount of dose received) continued on study and the fifth mouse was removed from the study.

Necropsy:
Approximately five hours following the administration of 3HTdR, the animals were euthanatized by CO2 asphyxiation and the auricular lymph nodes were collected. The lymph nodes were placed in Petri dishes containing 1 mL of PBS and the remaining carcass was discarded as radioactive waste.

Preparation of Single Cell Suspensions:
A single cell suspension (SCS) of the lymph node cells (LNC) for each group was prepared by gentle mechanical disaggregation of pooled lymph nodes through 200 µm mesh stainless steel gauze. The gauze was washed with approximately 4 mL of PBS and collected into the Petri dish. The LNC were transferred into an appropriate sized round-bottomed centrifuge tube. The Petri dish was rinsed with an additional 5 mL of PBS to remove remaining cells, and this wash was added to the centrifuge tube. The SCS was centrifuged at approximately 1000 rpm for 10 minutes at 4 °C to sediment the cells. The supernatant was removed leaving 2 mL of PBS volume above each cell pellet. The LNC were then resuspended and rinsed using a total of 10 mL PBS.

Following centrifugation and removal of the supernatant, the LNC were resuspended in 3 mL of 5 % trichloroacetic acid (TCA) and incubated at 4 °C for approximately 18 hours.

Deterinination of 3HTdR Incorporation:
After incubation in TCA, the precipitated macromolecules were recovered by centrifugation at approximately 1500 rpm for 10 minutes. The supernatant was removed, the macromolecules were resuspended in 1 mL TCA and transferred to 10 mL of scintillation cocktail. Incorporation of [3H-methyl] thymidine was measured by liquid scintillation spectrometry and reported as dpm for each sample, which represents dpm/group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The stimulation index for the concurrent positive control group was 2.83.
Although the mean stimulation index for the positive control group was less than 3.00, the stimulation index was significantly higher than the negative control group’s stimulation index and falls into the reported stimulation index range of 2.4 - 6.0 for a 25 % concentration of hexyl cinnamic aldehyde.
Key result
Parameter:
SI
Value:
1.12
Test group / Remarks:
10 %
Key result
Parameter:
SI
Value:
0.89
Test group / Remarks:
25 %
Key result
Parameter:
SI
Value:
1.06
Test group / Remarks:
50 %
Cellular proliferation data / Observations:
No mortality was noted during the study.

On Day 5 of the study, animals received intravenous injections of 250 µL of PBS containing 20 µCi of 3HTdR. Approximately five hours later, the auricular lymph nodes were collected, processed and the dpm counted. Table 1 lists the pooled animal dpm as well as the mean dpm per group and the stimulation indices.

In this study, there was no dose-response relationship among the three test substance-exposed groups (10, 25 and 50 % test substance). In addition, the mean stimulation indices for each of the three test substance-exposed groups (1.12, 0.89 and 1.06 with an average of 1.02 for the three test groups) were comparable to the mean stimulation index of the assigned negative control
group (1.00).

Table 1: Pooled dpm, mean dpm per group and the stimulation indices

Dose Group Group Number Group dpm: Repetition Mean Group dpm: Mean ± SD Simulation Index
1 2 3
Vehicle Control 1 2017.4 1955.5 1976.1 1983.0 ± 31.5 1.00
10 % Test Substance 2 2216.7 2218.3 2236.8 2223.9 ± 11.2 1.12
25 % Test Substance 3 1810.7 1714.1 1783.3 1769.4 ± 49.8 0.89
50 % Test Substance 4 2098.7 2070.9 2149.8 2106.5 ± 40.0 1.06
25 % Positive Control 5 5559.3 5651.4 5615.7 5608.8 ± 46.4 2.83
Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of the test, the substance was not considered to be a dermal sensitizer in mice and determined to have low potential for human dermal sensitization.
Executive summary:

In a GLP compliant study conducted in line with OECD Guideline 429, the potential of butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo, tetradecyl ester to cause skin sensitization was investigated using the Local Lymph Node Assay (LLNA).

A primary irritation screen was conducted prior to the sensitization study to determine concentrations of test substance that could be administered without causing systemic toxicity or excessive local irritation. For the primary irritation screen, a single daily dose of 25 µL of a concentration of 25 or 50 % test substance in an acetone/olive oil (4:1 v/v) vehicle was applied to the dorsal surface of each ear of the test animal. No signs of irritation were evident during the screen.

Based on results of the preliminary screen, concentrations of 10, 25 and 50 % test substance in an acetone/olive oil (4:1 v/v) vehicle were selected for the topical applications in the sensitization study. Two additional groups were treated with 25 % of a positive control substance in the same vehicle, or the vehicle alone. Five mice were allocated to each group in order to yield four mice per group that successfully received injections of [3H-methyl] thymidine (3HTdR).

No mortality was noted during the study. For the sensitization study (LLNA), mice were treated daily by application of 25 µL of the appropriate preparation to the dorsal surface of each ear for three consecutive days (Days 0,1,2). Each animal was observed once daily (Days 0-5) for any signs of either local irritation at the application site or systemic toxicity. Individual animal weights were measured on Days 0 and 5. On Day 5 of the study, mice were injected via the tail vein with 250 µL of phosphate buffered saline containing [3H-methyl] thymidine (3HTdR:80 µCi/mL, specific activity 5 Ci/mmol), giving a total of 20 µCi to each mouse.

Approximately five hours following the administration of 3HTdR, the animals were euthanatized and the auricular lymph nodes were collected. The lymph nodes were processed and incorporation of 3HTdR was measured by liquid scintillation spectrometry and reported as disintegrations per minute (dpm). The stimulation indices for the groups of mice treated with 10, 25 or 50 % test substance in vehicle were 1.12, 0.89 and 1.06, respectively. The stimulation index for the concurrent positive control group was 2.83.

In this study, there was no dose-response relationship among the three test substance-exposed groups (10, 25 and 50 % test substance). In addition, the mean stimulation indices for each of the three test substance-exposed groups (1.12, 0.89 and 1.06 with an average of 1.02 for the three test groups) were comparable to the mean stimulation index of the assigned negative control group (1.00). Although the mean stimulation index for the positive control group was less than 3.00, the stimulation index was significantly higher than the negative control group's stimulation index and falls into the reported stimulation index range of 2.4 - 6.0 for a 25 % concentration of hexyl cinnamic aldehyde.

Based on these results, the test substance was not considered to be a dermal sensitizer in mice and has low potential for human dermal sensitization. The test substance requires no label for sensitization by skin contact in line with current EU requirements

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The key study (Shepard, 2003) was performed in compliance with GLP and to OECD Guideline 429 with a sufficient level of detail to assess the quality of the presented data. The study was performed to a good standard in line with an accepted, standardised guideline and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch et al. (1997). The test substance was found to be a non-sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.