Pseudoephedrine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted according to OECD Guideline and under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD TG 442 B

GLP compliance:

yes

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

Molecular formula:

C10H15NO

IUPAC Name:

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

Details on test material:

- name of test item: (+)-Pseudoephedrine
- Test item number: 02/0251-2
- Batch number: 023513AX20
- CAS number: 90-82-4
- Purity: 99.9%
- Physical state, colour: solid/white
- homogeneity: homogenous by visual inspection
- Storage: ambient (RT); dry storage; light exclusion
- Expiration date: december 02, 2012

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 (pretest) and 8 (main test) weeks
- Weight at study initiation: 19.1-21.3 g
- Housing: single housing
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (6 am - 6 pm/6 pm - 6 am)

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0; 2.5; 5 and 10 %

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 25 % (w/w) in propylene glycol was the highest feasible concentration
- Irritation: 25 % was the lowest irritating concentration
- Lymph node proliferation response: incorporation of BrdU into lymph node cells

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: 1.6 fold greater increase in incorporation of BrdU compared to controls, as indicated by the Stimulation Index. The estimated concentration of test item required to produce a S.I. of 1.6 is referred to as the EC1.6 value.

TREATMENT
Topical application
- test item concentrations: 2.5; 5 and 10 % (w/w) in propylene glycol.
- application volume: 25 µL/ear/day [3 consecutive days]
- dorsal surface: approx. 8mm
- positive control: 25 % alpha-hexyl cinnamaldehyde in acetone:olive oil (4:1 v/v).

Administration
For injection bromodeoxyuridine (BrdU) 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.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations were calculated for body weights, ear weights, lymph node weights, lymph node cell counts and BrdU values.
The EC1.6 value was calculated according to the equation: EC1.6 = (a-c) x [(1.6-d)/(b-d)] + c. The 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.

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).

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Individual S.I. values are given below.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Not part of OECD TG 442 B, since no radiolabelled material was used. Investigation of BrdU incorporation by an ELISA approch.

Any other information on results incl. tables

Test Group	Animal No.	ABS450-ABS blank450	ABS690-ABS blank690	BrdU labeling Index	Mean BrdU labeling Index	S.I.^b)	Group S.I.
1 Vehicle Control	1	0.065	-0.011	0.076	0.077	1.0	1.0
	2	0.048	-0.012	0.060		0.8
	3	0.060	-0.011	0.071		0.9
	4	0.052	-0.033	0.085		1.1
	5	0.087	-0.005	0.092		1.2
2  2.5%	6	0.110	-0.020	0.130	0.095	1.7	1.2
	7	-0.013	-0.035	0.022		0.3
	8	0.146	-0.036	0.182		2.4
	9	0.035	-0.036	0.071		0.9
	10	0.038	-0.033	0.071		0.9
3 5%	11	0.106	-0.042	0.148	0.127*	1.9	1.7
	12	0.078	-0.041	0.119		1.6
	13	0.118	-0.036	0.154		2.0
	14	0.095	-0.038	0.132		1.7
	15	0.056	-0.029	0.084		1.1
4 10%	16	0.223	-0.037	0.260	0.241*	3.4	3.1
	17	0.158	-0.039	0.197		2.6
	18	0.228	-0.037	0.265		3.5
	19	0.127	-0.041	0.168		2.2
	20	0.276	-0.038	0.314		4.1
5 Positive Control   25%HCA	21	0.270	-0.037	0.307	0.279*	3.1	2.8**
	22	0.372	-0.026	0.397		4.0
	23	0.053	-0.041	0.094		0.9
	24	0.290	-0.039	0.329		3.3
	25	0.230	-0.038	0.267		2.7

The vehicle control group was propylene glycol.

S.I.     = Stimulation Index (values of the test item groups related to the mean value of the control group)

ABS   =  absorbance

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

**       = Data in relation to historical vehicle control AOO (Mean BrdU labelling Index = 0.0996)

Signs of irritation were observed with the 25% concentration. Therefore, the main study was conducted using 2.5, 5 and 10% (w/w). The animals did not show any relevant signs of systemic toxicity or local skin irritaion during the course of the main study and no cases of mortality were observed.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

Based on the result of this studie, the test item (+)-Pseudoephedrin needs to be classified R43 and Skin Sens. Cat. 1B, according to EEC/67/548 and EEC/1272/2008, respectively.