1-benzhydrylpiperazine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2007-05-15 to 2007-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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:

1-benzhydrylpiperazine

EC Number:

212-667-7

EC Name:

1-benzhydrylpiperazine

Cas Number:

841-77-0

Molecular formula:

C17H20N2

IUPAC Name:

1-(DIPHENYLMETHYL)PIPERAZINE

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-130806-AAA (T000750)
- Physical state: solid (powder)
- Appearance: white to slight beige powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0000008325
- Expiration date of the lot/batch: October 01, 2006 (excluded from the Statement of Compliance)
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was placed in a volumetric flask on a tared balance and the vehicle (acetone:olive oil (4+1)) was quantitatively added. The test item concentrations were prepared individually. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
The preparations were made freshly before each dosing occasion.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: from Harlan Netherlands, B.V. , The Netherlands
- Age at study initiation: 7-8 weeks (beginning of acclimatisation)
- Weight at study initiation: range 18.0 to 21.0 g
- Housing: single caging, in Makrolon type cage, with wire mesh top and granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: acclimatisation was performed under test conditions after health examination, no further data. Only animals without any visible signs of illness were used for the study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-87%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

pre-test (non-GLP): 1, 2.5, 5, 10 % (w/v) in acetone:olive oil (4:1)
main experiment: 1, 5, 10% (w/v) in acetone:olive oil (4:1)

No. of animals per dose:

pre-test (non-GLP): 2 females
main experiment: 4 females (nulliparous and non-pregnant) per group, 3 test concentration and 1 control group, in total 16 females

Details on study design:

PRE-SCREEN TESTS: to determine the highest non-irritant test concentration or the highest technically applicable concentration, a pretest was performed in two mice on three consecutive days.
- Compound solubility: The data showed that the highest test item concentration, which could be technically used was a 10% solution.
- Irritation: After three applications of the test item reddning of the ear skin was observed at concentrations of 5 and 10%, slight reddning was oberved at a concentration of 2.5%, no signs were observed at 1% of the test item. Since the reddning occurred on the third day of application only and since no excessive irritation was observed, concentrations of 1, 5, and 10%
- Systemic toxicity: no data
- Ear thickness measurements: no data
- Erythema scores: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: a test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
first, the exposure resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.).
second, that 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:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1, 5 and 10% (w/v) in acetone: olive oil (4+1). The application volume, 25 µL, was spread over the entire dorsal surface (diameter ~ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for body weight.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation EC3= (a-c) [(3-d)/(b-d)] + c where EC3 is the estimated concentrations of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a,b) and (c,d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:

The stimulation index (SI) was 2.43, 4.07, and 4.88 for the test concentrations 5, 10, and 25%, respectively. An EC3 of 6.7% (w/v) was calculated.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.17

Test group / Remarks:

based on 4 animals of 1% (w/v) group

Results 2

Parameter:

SI

Value:

35.37

Test group / Remarks:

based on 4 animals 10% (w/v) group

Results 3

Parameter:

SI

Value:

19.01

Test group / Remarks:

based on 4 animals 5% (w/v) group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DPM per lymh node (8 nodes in total):
vehicle control group: 396.9
1% (w/v) group: 860.5
5% (w/v) group: 7543.9
10% (w/v) group: 14040.4

DETAILS ON STIMULATION INDEX CALCULATION
See results table above

EC3 CALCULATION
The EC3 value was calculated as follows:
EC3 = (a-c) [(3-d)/(b-d)] + c = 1.2% (w/v)
with a = 1, b = 2.17, c = 5 and d=19.01

CLINICAL OBSERVATIONS:
No deaths occurred during the study period.
Reddning of the ear skin was observed after the third application of the highest test item concentration (10%). At lower concentrations neither local toxicity nor systemic findings were observed.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

The test item was found to be a skin sensitiser under the described conditions. As the calculated EC3 value is < 2% (i.e. 1.2%), the test item is classified as a skin sensitiser category 1A (H317) based on the criteria of the CLP Regulation.