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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
use of 4 control animals instead of 5, no positive control data
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1978
Report date:
1978

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
use of 4 control animals instead of 5, no "reliability check" data.
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time of study completion (1978), the LLNA OECD test method was not adopted.

Test material

Constituent 1
Chemical structure
Reference substance name:
Piperonal
EC Number:
204-409-7
EC Name:
Piperonal
Cas Number:
120-57-0
Molecular formula:
C8H6O3
IUPAC Name:
1,3-benzodioxole-5-carbaldehyde
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Heliotropin
- Analytical purity: Hand-written as 100%

In vivo test system

Test animals

Species:
guinea pig
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: No data
- Age at study initiation: No data
- Weight at study initiation: approximately 320 g
- Housing: No data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: Mixture of 6% acetone, 20% polyethylene glycol 400 and 0.01% saline
Concentration / amount:
1.5 %
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
80 %
Day(s)/duration:
Day 7
Adequacy of induction:
highest technically applicable concentration used
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
80 %
Day(s)/duration:
Day 14 / 24 hours
Adequacy of challenge:
other: maximal achievable concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
80%
Day(s)/duration:
Day 21 / 24 hours
Adequacy of challenge:
other: maximal achievable concentration
No.:
#3
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
80 %
Day(s)/duration:
Day 28 / 24 hours
Adequacy of challenge:
other: maximal achievable concentration
No.:
#4
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
80% or 20 %
Day(s)/duration:
Day 42 / 24 hours
Adequacy of challenge:
other: maximal achievable concentration
No. of animals per dose:
10 animals per dose
Details on study design:
RANGE FINDING TESTS: See Section 7.3.1 Skin Irritation / corrosion.WoE-1978 for details on topical irritation evaluation.

Preliminary irritation tests were conducted to determine the concentrations to be used for the intradermal and epicutenous exposures in the main sensitisation study.

For evaluation of intradermal irritation, 4 previously untreated guinea pigs of the same sex and weighing approximatley 320 g were each injected intradermally on the clipped flanks with 0.1 mL aliquots of a range of concentrations of the test article in a suitable solvent. Concentrations of 0.05, 0.1, 0.25, 0.5, and 1% were evaluated in 4 male animals using 0.01% saline vehicle and concentrations of 1, 2, 3, and 4% were evaluated in a separate group of 4 male animals using a vehicle composed of 6% acetone, 20% polyethylene glycol, and 0.01% saline. Reactions were evaluated 24 hours later by examining for size (2 largets diameters), erythema and oedema. The concentration which produced a definite reaction (10 x 10 mm in size, pale pink, with or without oedema) was selected for the intradermal injection induction concentration. Based on the results of this preliminary test, a concentration of 1.5% was selected for the intradermal induction procedure.

For evaluation of epicutaneous irritation, 8 mm diameter filter paper (Whatman 3MM) patches, in 11 mm "Fintest" aluminum patch test cups, were saturated with a range of concentrations of the test article in acetone and the cups were applied to the shaved flanks of 4 previously untreated guinea pigsweighing approximately 450 g. Concentrations of 5, 10, and 25% were evaluated in 4 male guinea pigs (one of which removed its bandage during the 24-hour period following application) and concentrations of 40, 60, and 80% were evaluated in 4 female guinea pigs. The patches were held in place with adhesive plaster (Poroplast) wound around the trunk. Patches were removed 24 hours later and the reaction sites were examined 24 and 48 hours thereafter for irritation. Scoring for irritation was done on a scale of 0 (no reaction) to +++ (intense erythema (deep pink) and oedema). The concentration giving a definite irritation reaction was to be used for topical induction while the highest non-irritant concentration was to be used for the topical challenge. No irritation was observed in any animal at any evaluation time point. As such, a concentration of 80% was used for both the topical induction and challenge phases of the main study as this concentration was the maximum concentration possible and was also non-irritant.

MAIN STUDY
Sensitisation was induced in 10 previously untreated guinea pigs (4 male and 6 female) weighing approximately 320 g by intradermal injections (0.1 mL) of both the test substance and Complete Freund's Adjuvant within a 2 x 4 cm area of the shoulder region. The induction process was supplemented 7 days later by application of an occluded patch of 80% test article in acetone placed over the injection site. Fourteen (14) days later the animals were challenged on one flank with an occluded patch of 80% test article in acetone for 24 hours. Reactions were recorded 24 and 48 hours following patch removal as follows:

0 = no reaction
+/- = barely perceptible erythema
+ = scattered, mild erythema (faint pink)
++ = moderate and diffuse erythema (pale pink)
+++ = intense erythema (deep pink) and oedema

Additional 24-hour occlusive challenges were performed 1, 2, and 4 weeks after the first challenge and were scored in the same manner. The last challenge included an evaluation of the reaction to test article at concentrations of 20 and 80% in acetone.

The method is intended to follow closely that described by Magnusson and Kligman (Magnusson and Kligman, 1969, 1970). The skin at the site of all intradermal injections is clipped with suitable animal clippers on the morning of the day on which the injections are given. The skin at the site of all patches, including the neck induction patch, is both clipped and shaved, with an electric razor, on the morning of the day on which patches are applied.

References:
Magnusson, B. and Kligman, A.M. 1969. The Identification of Contact Allergens by Animal Assay. The Guinea Pig Maximisation Test.
Magnusson, B. and Kligman, A.M. 1970. Allergic Contact Dermatitis in the Guinea Pig. Identification of Contact Allergens. Thomas, Springfield, Illinois.
Challenge controls:
Positive control not required; Negative control animals were included.
Positive control substance(s):
not required

Results and discussion

Positive control results:
Not required.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
80%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
80%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
positive indication of skin sensitisation
Reading:
other: Challenge 2 (1st reading)
Hours after challenge:
168
Group:
test chemical
Dose level:
80%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
positive indication of skin sensitisation
Reading:
other: Challenge 2 (2nd reading)
Hours after challenge:
192
Group:
test chemical
Dose level:
80%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
positive indication of skin sensitisation
Reading:
other: Challenge 3 (1st reading)
Hours after challenge:
672
Group:
test chemical
Dose level:
80%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
positive indication of skin sensitisation
Reading:
other: Challenge 3 (2nd reading)
Hours after challenge:
693
Group:
test chemical
Dose level:
80%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
positive indication of skin sensitisation
Reading:
other: Challenge 4 (1st reading)
Hours after challenge:
1 008
Group:
test chemical
Dose level:
80%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
no indication of skin sensitisation
Reading:
other: Challenge 4 (1st reading)
Hours after challenge:
1 008
Group:
test chemical
Dose level:
20%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
no indication of skin sensitisation
Reading:
other: Challenge 4 (2nd reading)
Hours after challenge:
1 032
Group:
test chemical
Dose level:
80%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
no indication of skin sensitisation
Reading:
other: Challenge 4 (2nd reading)
Hours after challenge:
1 032
Group:
test chemical
Dose level:
20%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Table 1. Results of Challenge 1

Guinea pigs

Concentration - 80%

No.

Ear no.

Sex

Wt (g)

24 hr

48 hr

1

8544

F

404

±

±

2

8691

F

402

0

0

3

8596

F

468

0

0

4

8567

F

458

++

+++

5

8270

F

472

±

+

6

8488

F

464

±

+

7

8575

M

388

+

+

8

8739

M

424

0

0

9

8915

M

470

±

± / +

10

8750

M

472

0

0

No. of reactions

2

4

0 = no reaction

±= barely perceptible erythema

+ = scattered, mild erythema (faint pink)

++ = moderate and diffuse erythema (pale pink)

+++ = intense erythema (deep pink) and oedema

 

Table 2. Controls for Challenge 1 - Treated controls

Guinea pigs

Concentration - 80%

No.

Ear no.

Sex

Wt (g)

24 hr

48 hr

1

8552

F

444

0

0

2

8257

F

470

0

0

3

8500

F

442

0

0

4

8508

F

474

0

0

0 = no reaction

±= barely perceptible erythema

+ = scattered, mild erythema (faint pink)

++ = moderate and diffuse erythema (pale pink)

+++ = intense erythema (deep pink) and oedema

Table 3. Controls for Challenge 1 - Untreated controls

Guinea pigs

Concentration - 80%

No.

Ear no.

Sex

Wt (g)

24 hr

48 hr

1

8708

M

454

0

0

2

8747

M

504

0

0

3

8740

M

450

0

0

4

8650

M

566

0

0

0 = no reaction

±= barely perceptible erythema

+ = scattered, mild erythema (faint pink)

++ = moderate and diffuse erythema (pale pink)

+++ = intense erythema (deep pink) and oedema

Table 4. Results of Challenge 2

Guinea pigs

Concentration - 80%

No.

Ear no.

Sex

Wt (g)

24 hr

48 hr

1

8544

F

422

0

0

2

8691

F

422

±

±

3

8596

F

478

0

0

4

8567

F

488

+

+++n

5

8270

F

472

+

+

6

8488

F

504

+

+

7

8575

M

436

+

++

8

8739

M

486

±

±

9

8915

M

510

±

±

10

8750

M

530

0

0

0 = no reaction

±= barely perceptible erythema

+ = scattered, mild erythema (faint pink)

++ = moderate and diffuse erythema (pale pink)

+++ = intense erythema (deep pink) and oedema

 

Table 5. Controls for Challenge 2 - Untreated controls

Guinea pigs

Concentration - 80%

No.

Ear no.

Sex

Wt (g)

24 hr

48 hr

1

9372

M

414

0

0

2

9183

M

400

0

0

3

9178

M

428

0

0

4

9030

M

444

0

0

0 = no reaction

±= barely perceptible erythema

+ = scattered, mild erythema (faint pink)

++ = moderate and diffuse erythema (pale pink)

+++ = intense erythema (deep pink) and oedema

Table 6. Results of Challenge 3

Guinea pigs

Concentration - 80%

 

No.

Ear no.

Sex

Wt (g)

24 hr

48 hr

 

 

1

8544

F

444

0

0

 

2

8691

F

460

0

0

 

3

8596

F

526

0

0

 

4

8567

F

510

+

+

 

5

8270

F

524

++

++ 

 

6

8488

F

524

+

+

 

7

8575

M

452

++

++

 

8

8739

M

516

±/+

±/+ 

 

9

8915

M

566

±

± 

 

10

8750

M

588

0

0

0 = no reaction

±= barely perceptible erythema

+ = scattered, mild erythema (faint pink)

++ = moderate and diffuse erythema (pale pink)

+++ = intense erythema (deep pink) and oedema

 

Table 7. Controls for Challenge 3 - Untreated controls

Guinea pigs

Concentration - 80%

 

No.

Ear no.

Sex

Wt (g)

24 hr

48 hr

 

 

1

7856

F

580

0

0

2

7001

F

532

0

0 

3

7337

F

514

0

0 

4

7130

F

516

0

0

0 = no reaction

±= barely perceptible erythema

+ = scattered, mild erythema (faint pink)

++ = moderate and diffuse erythema (pale pink)

+++ = intense erythema (deep pink) and oedema

 

 

Table 8. Results of Challenge 4

Guinea pigs

Concentration - 80%

Concentration - 20%

No.

Ear no.

Sex

Wt (g)

24 hr

48 hr

24 hr

48 hr

1

8544

F

480

0

0

0

0

2

8691

F

466

0

0

0

0

3

8596

F

574

0

0

0

0

4

8567

F

554

+

++

±/+

+

5

8270

F

580

±

±

±/+

+

6

8488

F

560

±

±/+

±/+

+

7

8575

M

484

±/+

+

±

+

8

8739

M

566

0

0

0

0

9

8915

M

580

0

±

0

±

10

8750

M

658

0

0

0

0

0 = no reaction

±= barely perceptible erythema

+ = scattered, mild erythema (faint pink)

++ = moderate and diffuse erythema (pale pink)

+++ = intense erythema (deep pink) and oedema

 

Table 9. Controls for Challenge 4 - Untreated controls

Guinea pigs

Concentration - 80%

Concentration - 20%

No.

Ear no.

Sex

Wt (g)

24 hr

48 hr

24 hr

48 hr

1

10279

M

390

0

0

0

0

2

10280

M

360

0

0

0

0

3

10038

M

400

0

0

0

0

4

10258

M

520

0

0

0

0

0 = no reaction

±= barely perceptible erythema

+ = scattered, mild erythema (faint pink)

++ = moderate and diffuse erythema (pale pink)

+++ = intense erythema (deep pink) and oedema

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test substance induced positive skin sensitisation reactions in 4 of 10 tested guinea pigs when evaluated at an intradermal induction concentration of 1.5% and an epicutaneous induction and challenge concentration of 80% (the highest achievable concentration).
Executive summary:

In this guinea pig maximization test, heliotropin (piperonal) was found to induce positive skin sensitisation reactions in 4 of 10 guinea pigs when evaluated at an intradermal induction concentration of 1.5% and an epicutaneous induction and challenge concentration of 80% (the highest achievable concentration, which also was found to be non-irritant in a preliminary evaluation).