Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP, QUA)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
according to
Guideline:
other: US FDA Title 21 Code of Federal Regulations Part 58; US EPA (FIFRA), Title 40 Code of Federal Regulations Part 160; US EPA (TSCA), Title 40 Code of Federal Regulations Part 792;
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, 59 NohSan, Notifications No.3850; Japanese Ministry of International Trade and Industry, Kanpogyo No.39 Environmental Agency, Kikyoku No.85; Japanese Ministry of Health and Welfare, Ordinance No.21
GLP compliance:
yes
Remarks:
OECD Principles of Good Laboratory Practice
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study was performed two years before the OECD method for the LLNA was adopted.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Name of test material (as cited in study report): FR-6120
- Physical state: white powder
- Analytical purity: ca. 99.5%
- Impurities (identity and concentrations): water (0.27%), Melamine (0.19%) and cyanuric acid (0.012%) (weight%)
- Purity test date: 04 May 2000
- Lot/batch No.: 37432-18-3
- Expiration date of the lot/batch: 22 May 2001
- Stability under test conditions: not indicated
- Storage condition of test material: at room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Himalayan strain, albino guinea pig (SPF-quality) from BRL Ltd., F011insdorf, Switzerland
- Age at study initiation: young adult animals (approx. 4 weeks old) were selected
- Weight at study initiation: 316±21 g in group 1 and 313±26 g for group 2 (see Table 6)
- Housing: group housing of 5 animals per labelled metal cage with wire-mesh floors
- Diet (e.g. ad libitum): free access to standard guinea pig diet, including ascorbic acid (1000 mg/kg); (Charles River Breeding and Maintenance Diet for Guinea Pigs, Altromin, Lage, Germany).
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 50%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
5% at intradermal induction (day 1), 20% at epicutaneous induction (day 8) and 20% at epicutaneous challenge (day 21)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
5% at intradermal induction (day 1), 20% at epicutaneous induction (day 8) and 20% at epicutaneous challenge (day 21)
No. of animals per dose:
5 in the control group (Group 1) and 10 in the test substance group (Group 2)
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main Study. The selection of concentrations was based on the following criteria: (1) the concentrations are well-tolerated by the animals; (2) for the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3); (3) for challenge exposure: the maximum non-irritant concentration.
The test system and procedures were identical to those used during the main study, unless otherwise specified (4 animals selected were between 4 and 9 weeks old).

- Intradermal injections: a series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 ml/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.
- Epidermal application: a series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be applied. 2 different concentrations were applied (0.5 ml each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape which were held in place with Micropore tape and subsequently Coban elastic bandage.
The animals receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance using water. The treated skin areas were assessed for irritation 24 and 48 hours after exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: on days 1 and 8
- Test groups: day 1 (3 pairs of intradermal injections: [1] 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection; [2] test substance at a 5% concentration; [3] 1:1 w/w mixture of the test substance, at twice the concentration used in [2] and Freunds' Complete Adjuvant; day 7 (the scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate in Vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction); day 8 (the 10% SDS treated area between the injection sites was treated with 0.5 ml of a 20% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage)
- Control group: the control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.
- Site: see above
- Frequency of applications: once at intradermal or epicutaneous application
- Duration: 24 hours after epicutaneous application, the dressing was removed and the skin cleaned of residual test substance using water.
- Concentrations: see above

B. CHALLENGE EXPOSURE
- No. of exposures: once
- Day(s) of challenge: on day 21
- Exposure period: the dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water
- Test groups: epidermal application of test substance concentration and the vehicle (0.15 ml each), using Patch Test Plasters. The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
- Control group: same treatment as in the test group
- Site: one flank of all animals was clipped before treatment
- Concentrations: 20%
- Evaluation (hr after challenge): the treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

OTHER: skin reactions were graded according to the OECD numerical scoring systems. Furthermore, a description of all other (local) effects was recorded. Whenever necessary, the treated skin-areas were clipped at least 3 hours before the next skin reading to facilitate scoring.
Challenge controls:
None
Positive control substance(s):
yes
Remarks:
a reliability check is done regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques (last check was done in May/June 2000 with alpha-hexylcinnamicaldehyde, tech. 85% [CAS no. 101-86-0])

Results and discussion

Positive control results:
Positive control substance concentrations selected for this study were: (1) intradermal induction with 5% solution in water (Milli-U, w/w); (2) epidermal induction with undiluted positive control substance; (3) challenge with 10% solution in water (w/w). See Table 5

The skin reactions in the experimental animals observed in response to the 10% test substance concentration in the challenge phase were considered indicative of sensitisation, taking into account the intensity of the response in the control animals. These results lead to a sensitization rate of 100 per cent to the 10% concentration. From these results, it was concluded that the female guinea pig of the albino Himalayan strain is an appropriate animal model for the performance of studies designed to evaluate the sensitising potential of a substance in a Maximisation type of test.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
20%
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: 20%. 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:
20%
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: 20%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 10%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 10%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: None.

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY

Based on the results, the test substance concentrations selected for the main study were a 5% concentration for the intradermal induction and a 20% concentration for the epidermal induction exposure (Table 1).

 

Table 1: Skin reactions after intradermal injection

Animal number

Concentration (%)

24 hours after injection

48 hours after injection

Erythema (grade)

Necrosis (mm)

Erythema (grade)

Necrosis (mm)

188

20*

4

 

 

2

10

3

 

4

 

189

5

3

 

3

 

2

2

 

3

 

*: a 20% test substance concentration was considered the highest concentration that could be prepared homogeneously to a visible acceptable level. Note: white/yellow staining was observed at all epidermally treated skin sites, 24 and 48 hours after exposure

 

No signs of irritation were observed to the highest test substance concentration epidermally tested. Therefore, the test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reaction.

 

Table 2: Skin reactions after epidermal exposure

Animal number

Body weight

(g)

Concentration (%)

24 hours after injection

48 hours after injection

Erythema (grade)

Necrosis (mm)

Erythema (grade)

Necrosis (mm)

186

412

20*

0

0

0

0

10

0

0

0

0

187

434

20*

0

0

0

0

10

0

0

0

0

188

500

5

0

0

0

0

2

0

0

0

0

189

460

5

0

0

0

0

2

0

0

0

0

*: a 20% test substance concentration was considered the highest concentration that could be prepared homogeneously to a visible acceptable level. Note: white/yellow staining was observed at all epidermally treated skin sites, 24 and 48 hours after exposure

 

MAIN STUDY

1) Induction phase (Table 3)

The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

 

Table 3: Induction readings

Experiment group

Animal number

Intradermal injection (Day 3; erythema grade)

Epidermal exposure (Day 10)

D

A

B

C

Erythema

Edema

 

Control

(Group 1)

16

3

0

3

0(a)

0

17

2

1

2

0(a)

0

18

3

0

3

0(a)

0

19

4

1

3

0(a)

0

20

3

1

2

0(a)

0

 

 

 

Experimental

(Group 2)

21

3

4

4

0(a)

0

22

4

4

4

0(a)

0

23

3

4

4

0(a)

0

24

3

3

4

0(a)

0

25

3

3

3

0(a)

0

26

3

3

4

0(a)

0

27

3

4

4

0(a)

0

28

3

3

3

0(a)

0

29

3

4

4

0(a)

0

30

3

4

4

0(a)

0

A: 1:1 Mixture of FCA and water for injection; B: A 5% test substance concentration (Experimental); vehicle (Control); C: 1:1 Mixture of FCA and a 10% concentration (Experimental) or vehicle (Control); D: A 20% test substance concentration (Experimental); vehicle (Control); (a) Small scabs; Note: White/Yellow staining by the test substance was observed at all epidermally test substance treated skin sites.

 

2) Challenge phase

No skin reactions were evident after the challenge exposure in the experimental and control animals (see Table 4). White/yellow staining was observed at the test substance treated skin sites, 24 and 48 hours after challenge. This staining did not hamper the scoring of the skin reactions.

 

Table 4: Challenge

Experiment group

Animal number

Day 23

Day 24

Comments

20%*

Vehicle#

20%*

Vehicle#

 

Control

(Group 1)

16

0

0

0

0

 

17

0

0

0

0

 

18

0

0

0

0

 

19

0

0

0

0

 

20

0

0

0

0

 

 

 

 

Experimental

(Group 2)

21

0

0

0

0

not sensitised

22

0

0

0

0

not sensitised

23

0

0

0

0

not sensitised

24

0

0

0

0

not sensitised

25

0

0

0

0

not sensitised

26

0

0

0

0

not sensitised

27

0

0

0

0

not sensitised

28

0

0

0

0

not sensitised

29

0

0

0

0

not sensitised

30

0

0

0

0

not sensitised

*: test substance concentration; #: corn oil

 

Table 5: Skin reactions in the challenge phase (Number of animals with skin reactions)

Experiment group

Animal number

alpha-hexylcinnamicaldehyde (concentration)

10% (24/48*)

0% (Vehicle; 24/48*)

 

Experimental group (10 females)

Score 3

1/1

0/0

Score 2

9/8

0/0

Score 1

0/1

0/0

No reactions

0/0

10/10

Conrol group (5 females

Score 1

5/0

0/0

No reactions

0/5

5/5

*: time (hours) after the challenge exposure.

 

3) Toxicity / Mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

 

3) Body Weights

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period (see Table 6).

Table 6: Mean body weight (g)

Group / Sex (Treatment)

Mean body weight ± standard deviation (g) at indicated time period

Day 1

Day 24

Group 1 / Females (Control)

316 ± 21

432 ± 25

Group 2 / Females (Experimental)

313 ± 26

438 ± 33

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information