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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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment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:
2008
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008
GLP compliance:
yes (incl. QA statement)
Remarks:
Bioassay, Labor für biologische Analytic GmbH, INF 515, 69120 Heidelberg
Type of study:
mouse local lymph node assay (LLNA)

Test material

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:
- Name of test material (as cited in study report): (+)-Pseudoephedrine
- Test item No.: 02/0251-2
- Purity: 100.0 g/100 g
- Physical state: solid, white
- Storage: ambient temperature, 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: pretest 12 weeks, main test 8 weeks
- Weight at study initiation: mean 21 g
- Housing: 1 animal per cage, Markrolon cage type II; H15005-29 bedding, Ssniff Spezialitäten GmbH (Experimental AnimalDiets Inc., 59494 Soest, Germany)
- Diet: STANRAB (P) SQC; SDS Special Diets Services. 67122 Altrip, Germany
- Water: ad libitum, tap water
- Acclimation period: at least 5 days
- Animals were nulliparous and non-pregnant

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, 10% (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
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. 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. Relevant signs of system toxicity or local skin irritation were not observed at lower concentrations.

MAIN STUDY
- Topical application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item. The application volume 25 μL/ear/day was spread over the entire dorsal surface (Ø ~8 mm) 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.
- Administration of BrdU: Four days after the first topical application (day 5) 0.5 mL of BrdU solution (5 mg/per mouse/injection) were intraperitoneally injected.
- Determination of Incoporated 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 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.
-Observations: In addition to the sensitizing reactions mice were observed daily for sings of toxicity and for mortality. Body weights were measured prior to the application and prior to sacrifice. Ear thickness was determined prior to application, on day 2 and before sacrifice.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
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 per cent level (p < 0.05).However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
A stimulation indice of 2.8 was determined for 25% alfa-hexyl cinnamaldehyde in acetone: olive oil (4:1 v/v) in the current experiment. Stimulation Indices of 3.1, 2.7, 2.7, and 3.8 were determined in 4 previous consecutive positive control experiments, with 25% alfa-hexyl cinnamaldehyde in acetone: olive oil (4:1 v/v).

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
In this study Stimulation Indices (S.I.) of 1.2, 1.7 and 3.1 were determined with the test item at concentrations of 2.5, 5 and 10 % (w/w) in propylene glycol. 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.

Any other information on results incl. tables

No deaths occurred during the study period. 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. 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.

 

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 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.

A statistically significant increase in BrdU labeling was observed for the mid (5%) and high (10%) dose 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).

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test item (+)-Pseudoephedrine is considered to be a skin sensitizer under the test conditions of this study.
Executive summary:

In a local lymph node assay, performed according to OECD Guideline 442B and GLP, three groups of 5 CBA/CaOlaHsd female mice were treated on the dorsal surface of both ears once per day for 3 days with 2.5, 5, 10% of the test substance, with the vehicle alone (propylene glycol) or with the positive control (α-hexyl cinnamaldehyde) at 25%. The mice were observed daily and no irritation at the dosing site or other signs of toxicity were noted. Four days after the first topical application the mice were intraperitoneally injected with BrdU. Approximately 24 hours after intraperitoneally injection, the mice were sacrificed and the draining auricular lymph nodes excised, pooled per animal and immediately weighed. The proliferative capacity of pooled lymph node cells was determined by the incorporation of BrdU measured in a photometer.A statistically significant increase in BrdU labeling was observed for the mid (5%) and high (10%) dose group. This was also observed for all dose groups in lymph node weights and lymph node cell count. Furthermore, 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). In this study Stimulation Indices (S.I.) of 1.2, 1.7 and 3.1 were determined with the test item at concentrations of 2.5, 5 and 10 % (w/w) in propylene glycol. 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. The test item(+)-Pseudoephedrine was found to be a skin sensitizer under the test conditions of this study.