Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-09-07 - 2001-10-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Pyridine-2-ethanol
- Physical state: light brown liquid
- Lot/batch No.: 1280031
- Storage condition of test material: room temperature, in the dark until 31 July 2001, thereafter at approximately 4°C in the dark
- Other: date receied: 10. July 2001

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: approximately eight to twelve weeks
- Weight at study initiation: 300 to 450g,
- Housing: housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): Free access to food (Certified Guinea Pig Diet (Code 5026) supplied by PMI Nutrition International, Nottingham, UK) was allowed throughout the study. The diet was routinely analysed and was considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study. The drinking water was routinely analysed and was considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06:00 to 18:00) and twelve hours darkness

OTHER:
- each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black indelible marker-pen
- the animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: distilled water
Concentration / amount:
Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as:
Intradermal Induction: 1% w/w in distilled water
Topical Induction: undiluted
Topical Challenge: undiluted and 75% w/w in distilled water
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: distilled water
Concentration / amount:
Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as:
Intradermal Induction: 1% w/w in distilled water
Topical Induction: undiluted
Topical Challenge: undiluted and 75% w/w in distilled water
No. of animals per dose:
Ten test and five control animals
Details on study design:
RANGE FINDING TESTS:
Selection of Concentrations for Main Study (Sighting Tests) :
The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:
Selection of Concentration for Intradermal Induction :
Intradermal injections (0.1 ml/injection site) were made on the clipped shoulder of two guinea pigs, using concentrations of 1% and 5% v/v in distilled water. The degree of erythema at the injection sites was assessed approximately 24, 48, 72 hours and 7 days after injection . One animal was killed for humane reasons following the 48-hour observation. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The highest concentration that caused only mild to moderate skin irritation, and which was well tolerated systemically, was selected for the intradermal induction stage of the main study.
Selection of Concentration for Topical Induction :
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant fifteen days earlier) were treated with the undiluted test material and three preparations of the test material (75%, 50% and 25% v/v in distilled water). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. One animal was found dead 24 hours after dressing removal, the cause of death was not established and the absence of this animal was considered not to affect the purpose or integrity of the study. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.
Selection of Concentration for Topical Challenge :
The undiluted test material and three preparations of the test material (75%, 50% and 25% v/v in distilled water) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal injection
Shortly before treatment on Day 0 the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 mL each) was made on each side of the mid-line into a 20 mm x 40 mm area. The injections were:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 1% w/w formulation of the test material in distilled water
c) a 1% w/w formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites (ie. injection site b) was evaluated
- No. of exposures: 1
- Exposure period: 24 / 48
- Test groups: 10 males
- Control group: 5 males
- Site: an area approximately 40 mm x 60 mm on the shoulder region of each animal
- Frequency of applications: once
- Duration: 24 / 48
- Concentrations: 1 % w/w formulation of the test material in distilled water and 1 % w/w formulation of the test material in a 1:1 prepartion of Freud's Complete Adjuant plus distilled water

Topical induction
On Day 7 the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the undiluted test material. A filter paper patch (WHATMAN No. 4: approximate size 40 mm x 20 mm), saturated with the undiluted test material was applied to the prepared skin and held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.
- No. of exposures: one
- Day(s) of induction: day 7 after induction
- Exposure period: 48 hours
- Site: the same area on the shoulder region used previously for intradermal injections
- Concentrations: undiluted test material
- Evaluation (hr after topical induction): The degree of erythema and oedema was quantified one and twenty-four hours following removal of the patches

B. CHALLENGE EXPOSURE
Shortly before treatment on Day 21, an area of approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers. A square filter paper patch (WHATMAN No. 4: approximate size 20 mm x 20 mm), saturated with the undiluted test material was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 75% w/w in distilled water was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal.
After 24 hours, the dressing was carefully removed and discarded. The challenge sites were swabbed with cotton wool soaked in distilled water to remove residual material. The position of the treatment sites was identified by using a black indelible marker-pen.
Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.
- No. of exposures: one
- Day(s) of challenge: day 21 after induction
- Exposure period: 24 hours
- Site: an area of approximately 50 mm x 70 mm on both flanks of each animal
- Concentrations: undiluted test material on the right flank; 75 % w/w formulation in distilled water on the left flank
- Evaluation (hr after challenge): Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified

OTHER: Determination by analysis of the concentration, homogeneity and stability of the test material preparations was not conducted.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: Reading 24 hours after intradermal induction
Group:
test group
Dose level:
1% w/w formulation in distilled water
Clinical observations:
Discrete or patchy to moderate and confluent erythema was noted at the intradermal induction sites of test group animals
Remarks on result:
other: see Remark
Remarks:
Reading: other: Reading 24 hours after intradermal induction. Group: test group. Dose level: 1% w/w formulation in distilled water. Clinical observations: Discrete or patchy to moderate and confluent erythema was noted at the intradermal induction sites of test group animals.
Reading:
other: Reading 24 hours after intradermal induction
Group:
negative control
Dose level:
distilled water
Clinical observations:
Discrete or patchy erythema was noted at the intradermal induction sites of control group animals
Remarks on result:
other: Reading: other: Reading 24 hours after intradermal induction. Group: negative control. Dose level: distilled water. Clinical observations: Discrete or patchy erythema was noted at the intradermal induction sites of control group animals.
Reading:
other: Reading 1 hour after topical induction
Group:
test group
Dose level:
undiluted and 75% w/w formulation in distilled water
Clinical observations:
Discrete or patchy to moderate and confluent erythema was noted at the topical induction sites of test group animals
Remarks on result:
other: see Remark
Remarks:
Reading: other: Reading 1 hour after topical induction. Group: test group. Dose level: undiluted and 75% w/w formulation in distilled water. Clinical observations: Discrete or patchy to moderate and confluent erythema was noted at the topical induction sites of test group animals.
Reading:
other: Reading 1 hour after topical induction
Group:
negative control
Clinical observations:
No skin reactions were noted at the topical induction sites of control group animals
Remarks on result:
other: Reading: other: Reading 1 hour after topical induction. Group: negative control. Clinical observations: No skin reactions were noted at the topical induction sites of control group animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
undiluted and 75% w/w formulation in distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: undiluted and 75% w/w formulation in distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
undiluted and 75% w/w formulation in distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: undiluted and 75% w/w formulation in distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Any other information on results incl. tables

Skin Reactions Observed After Intradermal Induction :

 

- Discrete or patchy to moderate and confluent erythema was noted at the intradermal induction sites of test group animals.

- Discrete or patchy erythema was noted at the intradermal induction sites of control group animals

Table 1 : Intradermal Sighting Test - Summary of Results

VEHICLE : Distilled Water

Animal
Identification
Time of Observation Concentration of
Test Material
(% v/v)
Grade of Erythema at
Injection Sites
Evidence of Systemic Toxicity
A 24 Hours
48 Hours
72 Hours
7 Days
1 2
2
1
0
None
None
None
None
B 24 Hours
48 Hours
5 NE
*
None
None

The concentration of the test material selected for the intradermal induction stage of the main study was 1% v/v in distilled water

N = Necrosis

E = Eschar

* = No data; animal killed for humane reasons due to the severity of the reactions

Table 2 : Intradermal Induction - Individual Skin Reactions

Group Animal
Number
Grade of Erythema at Observation Site
24 Hours 48 Hours
Left Side Right Side Left Side Right Side
TEST  1 2 2 1 1
  2 2 2 1 1
  3 2 2 1 1
  4 2 2 1 1
  5 2 2 1 1
6 2 2 1 1
  7 2 2 1 1
  8 2 2 1 1
  9 2 2 1 1
  10 2 2 1 1
CONTROL  11 1 1 0 0
  12 1 1 0 0
13 1 1 0 0
  14 1 1 0 0
  15 1 1 0 0

Skin Reactions Observed After Topical Induction :

- Discrete or patchy to moderate and confluent erythema was noted at the topical induction sites of test group animals.

- No skin reactions were noted at the topical induction sites of control group animals.

Table 3 : Topical Sighting Test for Induction Application (48-Hour Exposure) - Individual Skin Reactions

VEHICLE: Distilled Water

Animal
Identification
Concentration of
Test Material
(% v/v)
Skin Reactions (Hours After Removal of Patches)
1 24 48
Er Oe Other Er Oe Other Er Oe Other
C 100 1 0 - *          
75 0 0 - *          
50 0 0 - *          
25 0 0 - *          
D 100 0 0 - 0 0 - 0 0 -
75 0 0 - 0 0 - 0 0 -
50 0 0 - 0 0 - 0 0 -
25 0 0 - 0 0 - 0 0 -

The undiluted test material was selected for the main study topical induction

Er = Erythema

Oe = Oedema

- = No other reactions noted

* = No data; animal found dead

Table 4 : Topical Induction - Individual Skin Reactions

Group Animal
Number
Skin Reactions (Hours After Removal of Dressing)
1 Hour 24 Hours
Er Oe Other Er Oe Other
TEST 1 1 0 - 0 0 -
2 1 0 - 0 0 -
3 1 0 - 1 0 -
4 1 0 - 0 0 -
5 2 0 - 1 0 -
6 1 0 - 1 0 -
7 1 0 - 0 0 -
8 1 0 - 0 0 -
9 1 0 - 1 0 -
10 1 0 - 0 0 -
CONTROL 11 0 0 - 0 0 -
12 0 0 - 0 0 -
13 0 0 - 0 0 -
14 0 0 - 0 0 -
15 0 0 - 0 0 -

Er = Erythema

Oe = Oedema

- = No other reactions noted

Skin Reactions Observed After Topical Challenge :

- Undiluted as Supplied : No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48-hour observations.

- 75%v/v in Distilled Water : No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48-hour observations.

Table 5 : Topical Sighting Test for Challenge Application (24-Hour Exposure) - Individual Skin Reactions

VEHICLE: Distilled Water

Animal
Identification
Concentration of
Test Material
(% v/v)
Skin Reactions (Hours After Removal of Patches)
1 24 48
Er Oe Other Er Oe Other Er Oe Other
E 100 1 0 - 0 0 - 0 0 -
75 1 0 - 0 0 - 0 0 -
50 1 0 - 0 0 - 0 0 -
25 1 0 - 0 0   0 0 -
F 100 1 0 - 0 0 - 0 0 -
75 1 0 - 0 0 - 0 0 -
50 1 0 - 0 0 - 0 0 -
25 1 0 - 0 0 - 0 0 -

The undiluted test material and a 75% v/v concentration of the test material in distilled water were selected for the main study topical challenge

Er = Erythema

Oe = Oedema

- = No other reactions noted

Table 6 : Individual Skin Reactions at Challenge

CHALLENGE CONCENTRATIONS : 100% and 75%

VEHICLE: Distilled Water

Group Animal
Number
SkinReactions (Hours after Removal of Dressings) 
24 Hours 48 Hours
75% 100% 75% 100%
Er Oe Other Er Oe Other Er Oe Other Er Oe Other
TEST 1 0 0 - 0 0 - 0 0 - 0 0 -
2 0 0 - 0 0 - 0 0 - 0 0 -
3 0 0 - 0 0 - 0 0 - 0 0 -
4 0 0 - 0 0 - 0 0 - 0 0 -
5 0 0 - 0 0 - 0 0 - 0 0 -
6 0 0 - 0 0 - 0 0 - 0 0 -
7 0 0 - 0 0 - 0 0 - 0 0 -
8 0 0 - 0 0 - 0 0 - 0 0 -
9 0 0 - 0 0 - 0 0 - 0 0 -
10 0 0 - 0 0 - 0 0 - 0 0 -
CONTROL 11 0 0 - 0 0 - 0 0 - 0 0 -
12 0 0 - 0 0 - 0 0 - 0 0 -
13 0 0 - 0 0 - 0 0 - 0 0 -
14 0 0 - 0 0 - 0 0 - 0 0 -
15 0 0 - 0 0 - 0 0 - 0 0 -

Er = Erythema

Oe = Oedema

- = No other reactions noted

Table 6 : Summary of Positive Control Data for the Magnusson and Kligman Maximisation Study

Project
Number
Date Start Date End Number of Animals and
Sex*
Positive Control Material Concentration Incidence of Sensitisation
Induction Challenge
Test Control Intradermal Topical
039/333 22.12.1998 5.2.1999 10 Female 5 Female 2-Mercaptobenzothiazole 10% in
arachis oil BP
50% in
acetone:PEG 400
(70:30)
50% and 25% in
acetone:PEG 400
(70:30)
90% (9/10)
039/370 14.6.1999 17.7.1999 10 Male 5 Male 2-Mercaptobenzothiazole 5% in
arachis oil BP
50% in
acetone:PEG 400
(70:30)
50% and 25% in
acetone:PEG 400
(70:30)
100% (10/10)
039/422 12.1.2000 5.2.2000 10 Female 5 Female 2-Mercaptobenzothiazole 5% in
arachis oil BP
50% in
acetone:PEG 400
(70:30)
50% and 25% in
acetone:PEG 400
(70:30)
100% (10/10)
039/444 29.6.2000 22.7.2000 10 Male 5 Male 2-Mercaptobenzothiazole 5% in
arachis oil BP
50% in
acetone:PEG 400
(70:30)
50% and 25% in
acetone:PEG 400
(70:30)
100% (9/9)
039/446 28.6.2000 6.8.2000 10 Male 5 Male a-Hexylcinnamaldehyde 5% in
arachis oil BP
100% 100% and 75%in
arachis oil BP
50% (5/10)
039/458 25.1.2001 25.2.2001 10 Male 5 Male a-Hexylcinnamaldehyde 5% in
arachis oil BP
100% 100% and 75%in
arachis oil BP
40% (4/10)

* All animals supplied by David Hall Ltd, Burton-on-Trent, Staffordshire, UK

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information (Under the conditions of the test, the test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitiser to guinea pig skin) Criteria used for interpretation of results: EU
Conclusions:
The GLP-study was performed according to the OECD Guideline 406,guinea pig maximisation method according to Magnusson and Kligman without deviations and is considered to be reliable without restrictions (reliability Klimisch 1). The test material did not induce any response. The test material was considered to be not sensitising under the conditions of the test since no evidence of sensitizing or allergenic properties was found.
Executive summary:

The test item was tested for its sensitising potential according to OECD Guideline 406 in guinea pigs (Safepharm, 2001). The procedure used was performed according to the guinea pig maximisation method - Magnusson and Kligman. The test included three phases: an intradermal induction phase, when test material (0.1mL of a 1 % w/w formulation of the test material in distilled water and 1 % w/w formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water) is applied intradermally to the test animals; after an one-week rest period, a topical induction with the undiluted test material for 48 hours was conducted. Finally after a two-week rest period, a challenge was performed (on day 21 after intradermal induction) with the undiluted test material or 75 % w/w formulation in distilled water for 24 hours. 10 test animals and 5 control animals were used in the study. Treated skin sites were scored according scale as described in the Guinea Pig Maximisation Test of Magnusson B & Kligman. 24- and 48-hour readings were performed after the challenge applications. Under the conditions of the test, the test material produced a 0% (0/10) sensitisation rate. Therefore the substance is not considered to be as skin sensitizer and was classified as a non-sensitizer to guinea pig skin.