1-Piperazineacetamide, N-(2,6-dimethylphenyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 November 2014 - 02 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 429. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

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:

N-(2,6-dimethylphenyl)-2-(piperazin-1-yl)acetamide

EC Number:

610-916-8

Cas Number:

5294-61-1

Molecular formula:

C14H21N3O

IUPAC Name:

N-(2,6-dimethylphenyl)-2-(piperazin-1-yl)acetamide

Details on test material:

- Name of test material (as cited in study report): N-(2,6-dimethylphenyl)-1-(piperazineacetamide)
- Physical state: White solid
- Analytical purity: 99.8 %

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE (France)
- Age at study initiation: 10 weeks old
- Weight at study initiation: 20.1-21.8 g
- Housing: Group caging / mice were provided with glass tunnel-tubes. Cage type II, polypropylene / polycarbonate.
- Diet (e.g. ad libitum): ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”, ad libitum.
- Water (e.g. ad libitum): tap water, ad libitum.
- Acclimation period: 20 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours of light daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

10, 25 and 50%.

No. of animals per dose:

4 animals / group.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test: AOO (acetone:olive oil 4:1 (v:v)
mixture), N,N-dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic® PE9200 (1% Pluronic) were tested. The best vehicle was considered to be PG. The highest achievable concentration of the test item was 50 % (w/v).
- Preliminary Irritation/Toxicity Test:
2 animals per dose were exposed to test item concentrations of 50 and 25 % (w/v) in PG. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. Mortality and signs of systemic toxicity were observed. No marked body weight loss was detected on the mean body weight values of the groups. There were no indications of any irritancy at the site of application. The revealing ear punch weights were within the historical control range. The draining auricular lymph nodes of the animals were visually examined: they were normal for one-one animal in both dose groups and they were slightly enlarged for one-one animal in the groups (subjective judgement by analogy with observations of former experiments). Based on these results, the 50 and 25 % (w/v) doses were considered to be acceptable for the main test. Therefore, 50, 25 and 10 % (w/v) doses were examined in the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
1) That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
2) The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 mL of sterile PBS (phosphate buffered saline) containing
approximately 20 mCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes). Five hours later, the mice were euthanized and the auricular lymph nodes were extracted from the animals. A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and the samples were examined in a scintillation counter.

OBSERVATIONS
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection).

EVALUATION OF RESULTS
Radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Since the test item gave a positive response and data permitted, the EC3 value of the test item was calculated (EC3 means the effective chemical concentration required for SI=3).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The stimulation index values were 4.2, 2.3 and 1.7 at concentrations of 50, 25 and 10 % (w/v), respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Test item 50 % (w/v) in PG: 8636.5 DPM Test item 25 % (w/v) in PG: 4661.5 DPM Test item 10 % (w/v) in PG: 3463.5 DPM

Any other information on results incl. tables

Clinical observations:

No mortality or signs of systemic toxicity were observed during the study. Minimal amount of test item precipitate was observed on the ears of the animals in the 50 % (w/v) dose group on Days 3-5.

Body weight measurement:

No treatment related effects were observed on the body weight changes of the experimental animals.

Proliferation assay:

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5 % (w/v) TCA)	30 31	-	-	-	-
Negative (vehicle) control (PG)	2091	2060.5	8	257.6	1.0
Test item 50 % (w/v) in PG	8667	8636.5	8	1079.6	4.2
Test item 25 % (w/v) in PG	4692	4661.5	8	582.7	2.3
Test item 10 % (w/v) in PG	3494	3463.5	8	432.9	1.7
Positive control (25 % (w/v) HCA in PG)	12700	12669.5	8	1583.7	6.1

The stimulation index values were 4.2, 2.3 and 1.7 at concentrations of 50, 25 and 10 % (w/v), respectively.

The obtained data are compatible with a conventional dose response and allow the calculation of the EC3 value according to an adequate scientific method. EC3 means the effective chemical concentration required for SI=3. The calculated EC3 value of the test substance is 34.2 % (w/v).

The appearance of the lymph nodes was normal in the negative (vehicle) control group and in the 10 % (w/v) group. Slightly enlarged lymph nodes were detected in the 50 and 25 % (w/v) groups. Larger than normal lymph nodes were observed in the positive control group.

The DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 6.1) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. The stimulation index values were 4.2, 2.3 and 1.7 at concentrations of 50, 25 and 10 % (w/v), respectively. The calculated EC3 value was 34.2 % (w/v).

Executive summary:

The skin sensitisation test following dermal exposure was performed according to OECD Guideline 429 and EU method B.42, following the Principles of GLP. Based on the results of the Preliminary Compatibility Test, the test item was formulated in Propylene glycol (PG) at a highest achievable concentration of 50% (w/v). The Preliminary Irritation / Toxicity Test was performed in CBA/J Rj mice using two doses: 50 and 25 % (w/v) in PG. Based on the observations recorded in the preliminary test, the 50 % (w/v) was selected as top dose for the main test. In the main assay, twenty female CBA/J Rj mice were allocated to five groups of four animals each: three groups received the test substance formulated in PG at 50, 25 and 10 % (w/v) concentrations; the negative control group received the vehicle (PG); the positive control group received 25 % (w/v) HCA (dissolved in PG). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or signs of systemic toxicity were observed during the study. Minimal amount of test item precipitate was observed on the ears of the animals in the 50 % (w/v) dose group on Days 3-5. No treatment related effects were observed on the body weight changes of the experimental animals. The stimulation index values were 4.2, 2.3 and 1.7 at concentrations of 50, 25 and 10 % (w/v), respectively. The calculated EC3 value was 34.2 % (w/v). All validity criteria were fulfilled. In conclusion, under the conditions of the present assay the test item, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.