Phenylephrine hydrochloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Remarks:

Bioassay Labor für biologische Analytik GmbH INF 515, 69120 Heidelberg

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Pseudoephedrine

EC Number:

202-018-6

EC Name:

Pseudoephedrine

Cas Number:

90-82-4

IUPAC Name:

2-(methylamino)-1-phenylpropan-1-ol

Details on test material:

- Test Item: (+)-Pseudoephedrine
- Physical state, colour: Solid / white
- Homogeneity: The test item was homogeneous by visual inspection.
- Storage conditions: ambient (RT); dry storage; under light exclusion

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 12 weeks (pretest), 8 weeks (main test)
- Weight at study initiation: Mean: 21.0g
- Housing: Individually in Makrolon cages, type II with H 15005-29 bedding; Ssniff.Spezialitäten GmbH (Experimental AnimalDiets Inc., 59494 Soest, Germany)
- Diet: STANRAB (P) SQC; SDS Special Diets Services. 67122 Altrip, Germany
- Water: Tap water ad libitum
- Acclimation period: At least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

2.5, 5, and 10% (w/w)

No. of animals per dose:

6 (pre-test), 5 (main test)

Details on study design:

- Vehicle and Dose Selection: A solubility experiment was performed according to the recommendations given by OECD 442B. The highest test item concentration, which could be technically used, was a 25 % (w/w) dilution in propylene glycol. The test item preparation was heated approx. 15 minutes at 100°C. The preparation was administrated hand warm. To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, a pre-test was performed in six animals (2 animals per test item concentration and vehicle, each). Six mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 25 and 10% (w/w) and with the vehicle propylene glycol once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined using a micrometer (C110TKroeplin. 36381 Schlüchtern. Germany). Additionally the ears were punched after sacrifice (day 5) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 2 or day 5.The measured ear weight was also considered in this evaluation. At the test item concentration of 25 % (w/w) signs of local skin irritation such as incrustations, test item residues and an increase in ear weight (> 25 % in one animal) were observed. At the test item concentration of 10% (w/w) the animals did not show relevant signs of system toxicity or local skin irritation. From day 0 to day 5, both treated animals did not show an erythema of the ear skin, but on day 5 in one animal each scaling or incrustation was observed, respectively. (for detailed results see Annex 1).Measurement of ear thickness exceeded in one animal 25%, but as this was only observed in one of two animals on day 2, not accompanied by erythema and not confirmed by ear weight this was considered to be incidental and of no biological relevance. The test item in the main study was, with consent of the sponsor, assayed at 2.5, 5, and 10% (w/w).
- Test Item Preparation: The test item preparations were produced on a weight per weight (w/w) basis. For better handling the test item preparations were heated approx. 15 minutes at 100°C and were soluble in the vehicle propylene glycol. The preparations were administrated hand warm. The positive control preparation was produced on a weight per weight (w/w) basis shortly before application by stirring with a magnetic stirrer. After stirring with a magnetic stirrer the positive control was soluble in the vehicle AOO.
- Topical Application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% (w/w) in propylene glycol. The application volume 25 μL/ear/day was spread over the entire dorsal surface of each ear once daily for three consecutive days. The control test group of mice was treated with an equivalent volume of the relevant vehicle alone. The positive control test group of mice was treated with 25 % α-hexyl cinnamaldehyde dissolved in acetone: olive oil (4:1 v/v).
- Administration of BrdU: BrdU was purchased from Roche Diagnostics GmbH. For injection it was dissolved in DPBS (10 mg/mL) before administration and stored in a refrigerator until use. Four days after the first topical application (day 5) 0.5 mL of BrdU solution (5mg/per mouse/injection) were intraperitoneally injected.
- Determination of Incorporated BrdU: Approximately 24 hours after treatment with BrdU the mice were sacrificed and the draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a 70 μm nylon mesh. The lymph node cells were resuspended in 6 mL DPBS. After cell count with a cell counter (Casy Cell Counter, Innovatis), cell suspensions of 100 000 cells/mL were adjusted. The incorporation of BrdU into lymph node cells was determined using a commercial cell proliferation assay kit (Roche Applied Science. Mannheim): 100 μL of the lymph node cell suspension (100 000 cells/mL) were added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the lymph node cells with FixDenat (included inCell Proliferation Elisa Kit), anti BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution TMB (included inCell Proliferation Elisa Kit) was then added and allowed to produce chromogen. After stopping the substrate reaction the absorbance was measured at 450 nm with a reference wavelength of 690 nm.
- Determination of Lymph Node Weight and Cell Count: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned in section 9.3.3 was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (Casy Cell Counter, Innovatis). The values obtained were taken down manually.
- Determination of Ear Weights: After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
- Interpretation of Raw Data: The proliferative response of the lymph node cells is expressed as the mean SI. The SI is derived by dividing the mean BrdU labeling index/mouse within each test substance group as well as for the positive control group by the mean BrdU labeling index for the vehicle group. A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled: First, that exposure to at least one concentration of the test item resulted in an incorporation of BrdU at least 1.6-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. Furthermore, an index was calculated for the lymph node weight and -cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. Furthermore, according to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation.
- Observations: In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period: Mortality / Viability: At least once daily from experimental start to necropsy. Body weights: prior to the first application and prior to sacrifice. Ear thickness: in the pre-test prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Ear weights: in the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear. Lymph node weights: after excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Lymph node cell count: the lymph node cell count was determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a cell counter. Clinical signs (local/systemic): Clinical signs were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

Statistical Analysis: The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the BrdU values. The EC1.6 value was calculated according to the equation EC1.6 = (a-c) [(1.6-d)/(b-d)] + c where EC1.6 is the estimated concentration of the test item required to produce a 1.6-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 1.6 on the local lymph node assay dose response plot. A statistical analysis was conducted on the BrdU values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations Statistica (Version 10) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test or the Student Newman Keuls test. Statistical significance was set at the five percent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

In the positive control an S.I. of 2.8 was observed.

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Control: 1.0, 2.5%: 1.2, 5%: 1.7, 10%: 3.1 Based on the S.I.s obtained with 2.5 and 5% test item concentration, an EC1.6 value of 4.5% (w/w) was calculated. A clear dose response was observed. A technical outlier was found in the high dose group (animal No.19), but as neither inclusion or exclusion of the outlier changed the overall study result, the results of this animal were included in the calculations.

Any other information on results incl. tables

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

- Clinical Signs: At the test concentration of 5% and 10% (w/w) one animal of the 5% and 3 animals of the 10% test group showed scaling on day 5 only, in one animal with incrustation. No systemic findings were observed during the study period.

- Body Weights: The body weight of the animals recorded prior to the first application and prior to sacrifice were within the range commonly recorded for animals of this strain and age. Nevertheless, in all groups a slight decrease in body weight was observed, but as that occurred in the vehicle control and positive control group also, this was considered not to be biologically relevant.

- Lymph Node Weights and Cell Counts: The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant increase in lymph node weights and - cell counts was observed in all dose groups in comparison to the vehicle control group. The cut-off-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in the high dose group (index of 2.4).

- Ear Weights: The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights were observed for the low (2.5%) and the high (10%) doses groups. According to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was not exceeded in any test item treated group.

 Lymph Node Weights

	Mean lymph node weight (mg)	SD
Vehicle	4.7	0.7
2.5%	6.1*	0.5
5%	6.6*	0.4
10%	9.5*	1.5
Positive control	12.3*	2.1

*statistically significant increase vs. control group (p<0.05)

 

Lymph Node Cell Counts

	Mean lymph node cell counts (x 1e6)	SD
Vehicle	10.5	1.8
2.5%	14.3*	2.7
5%	15.0*	1.1
10%	25.1*	6.0
Positive control	34.3*	5.4

*statistically significant increase vs. control group (p<0.05)

 Ear Weights

	Mean ear weight (mg)	SD
Vehicle	28.9	1.1
2.5%	30.8*	1.5
5%	31.8	2.3
10%	32.0*	2.6
Positive control	35.0*	1.9

*statistically significant increase vs. control group (p<0.05)