Loperamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-02-22 to 2006-03-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted according to the following guidelines: OECD Guideline for the Testing of Chemicals, Guideline 429: Skin Sensitization: Local LymphNode Assay (adopted 24 April 2002), Commission Directive 2004/73/EC, B.42: Skin Sensitization: Local Lymph Node Assay, 29 April, 2004 and Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.2600 "Skin Sensitization" EPA 712-C-03-197, March 2003, without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

Loperamide

EC Number:

258-416-5

EC Name:

Loperamide

Cas Number:

53179-11-6

Molecular formula:

C29H33ClN2O2

IUPAC Name:

4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-N,N-dimethyl-2,2-diphenylbutanamide

Details on test material:

- Name of test material (as cited in study report): T1001, 4-(4-chlorophenyl)-4hydroxy-N,N-dimethyl-alpha, alpha-diphenyl-1-piperidinebutanamide- Physical state: solid- Analytical purity: 100%- Lot/batch No.: 00456462 RT001001G1B591- Expiration date of the lot/batch: 2006-06-30- Stability under test conditions: no data- Storage condition of test material: at room temperature (range of 20±5 °C), light protected

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaHsdRcc(SPF)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: RCC Ltd., Laboratory Animal Service, CH-4414 Fullinsdorf/Switzerland- Age at study initiation: 8-12 weeks (beginning of acclimatization)- Weight at study initiation: 16 g-24 g (ordered)- Housing: Individually in Makrolon Type 2 cages with standard soft wood bedding ("Lignocel", Schill AG, CH-4132 Muttenz)- Diet: pelleted standard Kliba 3433, batch no. 76/05 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum- Water: community tap water from Itingen, available ad libitum- Acclimation period: 2006-02-22 to 2006-03-01ENVIRONMENTAL CONDITIONS- Temperature (deg C): 22±3 °C- Humidity (%): 30-70%- Air changes (per hr): 10-15 air changes per hour.- Photoperiod (hrs dark / hrs light): There was a 12-hour fluorescent light/ 12-hour dark cycle with at least 8 hours of music played during the light period.IN-LIFE DATES: From: 2006-03-02 To: 2006-03-07

Study design: in vivo (LLNA)

Vehicle:

other: N,N-Dimethylformamide (DMF)

Concentration:

Main experiment: 1, 2.5, 5% (w/v) 5% was the highest technically applicable concentration in the chosen vehicle.

No. of animals per dose:

Main experiment: 4 females (nulliparous and non-pregnant)

Details on study design:

RANGE FINDING TESTS: - Compound solubility: In non-GLP solubility pre-test, the test substance was tested in different vehicles: acetone/olive oil (4/1, v/v), ethanol/water (7/3, v/v) and DMF. DMF was found to be a suitable vehicle and was selected and used in the main test. 5% was the highest technically applicable concentration in the chosen vehicle. - Irritation: A non-GLP local toxicity pre-test was performed for determination of concentrations for the main test. Three single animals were each treated with one of three different concentrations: 1, 2.5 and 5%, in both ears on three consecutive days. Neither clinical signs nor findings were observed at these concentrations one day after each single application. 5% was the highest dosing concentration in the main tests.- Lymph node proliferation response: no dataMAIN STUDYANIMAL ASSIGNMENT AND TREATMENT- Name of test method: LLNA- Criteria used to consider a positive response: First, that exposure to at least one concentration of the test substance results in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index. 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: -Test substance Preparation: The test substance was placed into a volumetric flask on a tared Mettler balance and the vehicle DMF was quantitatively added. Test substance formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test substance inthe vehicle was maintained until start of treatment using an appropriate homogenizer. Concentrations were in terms of material as supplied. -Topical Application: 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 substance concentrations of 1, 2.5, and 5% (w/v) in N,N-dimethylformamide (DMF). 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).- Administration of 3H-Methyl Thymidine: Five days after the first topical application, all mice were administered with 250 µL of PBS containing 77.22 µCi/mL 3HTdR (corresponds to 19.3 µCi 3HTdR per mouse) by intravenous injection via a tail vein.- Determination of Incorporated 3HTdR: Approximately five hours after treatment with 3HTdR all mice were euthanised by inhalation of CO2 (dry ice).The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing twice with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to glass scintillation vial with 10 mL of 'Irga-Safe Plus' scintillation liquid and thoroughly mixed.The level of 3HTdR incorporation was then measured on a B-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1mL-aliquots of 5% trichloroacetic acid. The B-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (dpm).

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for body weight.

Results and discussion

Positive control results:

Disintegrations per minute: Vehicle control: 47135% w/v: 851310% w/v: 1363625% w/v: 29235Stimulation Index: Vehicle control: not applicable5% w/v: 1.810% w/v: 2.925% w/v: 6.2EC3 (estimated concentration for a stimulation index (SI) of 3 was calculated to be 10.5%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Vehicle control: not applicable1% w/v: 1.42.5% w/v: 1.35% w/v: 1.2

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle control: 41611% w/v: 57772.5% w/v: 55635% w/v: 4823

Any other information on results incl. tables

Table 1: Results of the Positive Control Study
Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM- BG^a)	Number of lymph node	DPM per lymph node^b)	S.I.
---	BG I	31	---	---	---	---
---	BG II	20	---	---	---	---
---	CG 1	4713	4687	8	586
5	TG 2	8513	8487	8	1061	1.8
10	TG 3	13636	13610	8	1701	2.9
25	TG 4	29235	29209	8	3651	6.2

BG = Background (1mL 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II.

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

 

	Test item concentration % (w/v)	S.I.
Group 3	10.0 (a)	2.9 (b)
Group 4	25.0 (c)	6.2 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c =10.5 %(w/v)

EC3 = Estimated concentration for a S.I. of 3

 

Table 2: Main Study: Calculation and Results of Individual Data
Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM- BG^a)	Number of lymph node	DPM per lymph node^b)	S.I.
---	BG I	28	---	---	---	---
---	BG II	33	---	---	---	---
---	CG 1	4161	4130	8	516
1	TG 2	5777	5746	8	718	1.4
2.5	TG 3	5563	5532	8	692	1.3
5	TG 4	4823	4792	8	599	1.2

BG = Background (1mL 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II.

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

No positive dose-response relationship was observed.

Calculation of the EC 3 value was not performed because no test concentrations produced a S.I. of 3 or higher.

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: Neither clinical/local signs nor other findings were observed in any animals of the control group of Group 2 (1%). One day after the first topical application, the residual test substance was found at all the dosing sites in all mice of Group 3 (2.5%) and Group 4 (5%), persisting for a total of four days or for the remainder of the in-life phase of the study.

Body Weights: The body weight of the animals, recorded prior to the first application and prior necropsy, was within the range commonly recorded for animals of this strain and age.

Size of The Draining Lymph Nodes: No findings were observed on the size of the draining lymph nodes.

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated informationCriteria used for interpretation of results: EU

Conclusions:

The purpose of this Local Lymph Node assay was to identify the contact allergenic potential of the test substance when administered to the dorsum of both ear lobes of mice. A test substance is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the S.I.In this study, S.I. values of 1.4, 1.3, and 1.2 were determined with the test substance at concentrations of 1%, 2.5%, and 5%, respectively, in DMF. The test substance was found to be a non-sensitizer when tested up to the highest applicable concentration of 5% in DMF.