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Description of key information

Piperonal was concluded to be a moderate sensitiser (positive in 4 of 10 animals) in a guinea pig maximization test in which it was evaluated at an intradermal induction concentration of 1.5%, a topical induction concentration of 80%, and a topical challenge concentration of 80% (Unilever Research Laboratory, 1978). Other guinea pig studies, none of which reported quantitative data, have reported positive findings (maximization test), conflicting findings (open epicutaneous tests), or negative findings (Draize test). No evidence of skin sensitization by piperonal was observed in 3 human studies, including 2 repeat insult patch tests (RIPTs) (Hill Top Research, 1964; Harrison Research Laboratories, 2011) and a maximization test (Greif, 1967). Based on the positive findings from a reliable guinea pig maximization test, piperonal is considered to possess moderate potency as a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
Reason / purpose for cross-reference:
reference to same study
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.
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
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
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
Positive control results:
Not required.
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

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

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).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

No evidence of sensitisation was observed in the available human studies.

In the RIPT conducted by Hill Top Research (1964), 41 subjects received a 0.5 mL aliquot of heliotropin (piperonal) (5% in ethanol) applied to a Webril pad which was then affixed to an adhesive elastic bandage. The patches were applied to the upper arm of each subject for 24 hours under semi-occlusion. The applications were conducted on a Monday-Wednesday-Friday schedule. Nine induction applications were made over a period of 3 weeks. After a rest period of approximately 2 weeks, a 24-hour semi-occluded challenge application was made to a virgin site. Reactions were evaluated at 24 and 72 hours after patch removal. No evidence of skin sensitization was observed in any of the subjects.

The RIPT conducted by Harrison Research Laboratories (2011), which was conducted under the supervision of a Board-certified dermatologist, enrolled a total of 120 subjects (47 males and 73 females) to evaluate the sensitizing potential of 3 test materials: heliotropin (piperonal) (2.5% in a 1:3 ethanol:diethyl phthalate vehicle; designated HRIPT-146-1), vehicle (1:3 ethanol:diethyl phthalate; designated HRIPT-146-3), and saline (; designated HRIPT-146-2). A total of 112 subjects completed the study (the other 8 subjects withdrew due to personal reasons; none withdrew due to test material reactions). In the induction phase, a volume of 0.3 mL of each test material was applied to designated 25 mm patches that were adhered occlusively to 1 side of the back of each subject (i.e., each subject received a patch of each test material). For heliotropin (piperonal), this resulted in a dose of 2,953µg/cm2. Patches were applied as such on Mondays, Wednesdays, and Fridays for 3 consecutive weeks (9 total applications), and were removed approximately 24 hours after each application (on Tuesdays, Thursdays, and Saturdays). Patches were repeatedly applied to the same sites, which were evaluated on Mondays, Wednesdays, and Fridays for possible reactions. A 2-week rest period followed the induction phase, after which subjects were challenged with a similar application of 3 patches (1 for each test material) on the opposite side of the back. Patches were removed 24 hours later at which time scoring for reactions was conducted; reaction scoring also was conducted 48, 72, and 96 hours post-patching. No evidence of skin sensitization was observed in any of the subjects.

 

Greif (1967) reported on a maximization test conducted in 25 human volunteers (sex not reported). Occluded applications with 6% heliotropin (piperonal) in an unspecified vehicle were made to the same site on the subjects' forearms for five 48 -hour periods, each of which was preceded by a 24 -hour occluded exposure to 5% aqueous sodium lauryl sulfate (SLS). These exposures were accomplished over 15 days. After a 10-day rest period, occluded challenge patches were applied to fresh sites for 48 hours. The sites were pretreated with 10% aqueous SLS for 1 hour under occlusion. The reactions were evaluated at the time of patch removal and also at 24 and 48 hours after the patch removal. No evidence of skin sensitization was observed in any of the subjects.

 

Positive results were reported in a key guinea pig maximization test conducted by Unilever Research Laboratory (1978). On Day 1 of this study, 10 guinea pigs (4 males and 6 females) were administered 3 pairs of intradermal injections according to the method of Magnusson and Kligman. Heliotropin (piperonal) was evaluated at an induction concentration of 1.5% in a vehicle composed of 6% acetone, 20% PEG 400, and 0.01% Dobs saline. The topical induction phase was conducted 1 week later using an occluded patch with 80% heliotropin (piperonal) in acetone which was applied to the clipped and shaved neck. The duration of the topical induction period was not reported. Two weeks after the topical induction, a 24-hour occluded challenge patch was applied to the clipped and shaved flank region, using a concentration of heliotropin (piperonal) of 80% in acetone (this was the maximum feasible concentration and was determined to be non-irritating in a preliminary evaluation). Reactions were evaluated at 24 and 48 hours after patch removal. Two animals had positive responses at the 24-hour time point while 4 animals were positive at the 48-hour time point. Additional 24-hour occluded patch challenges were conducted 1, 2, and 4 weeks later. The same 4 animals had positive responses following the challenges conducted 1 and 2 weeks later, while 2 of these animals had positive responses following the challenge conducted 4 weeks later. Heliotropin (piperonal) was concluded to be a moderate sensitiser under the conditions of this study.

 

Klecak et al.(1977) reported on an open epicutaneous test that was conducted in guinea pigs. During each day of the induction phase (study Days 0 to 20), guinea pigs (5 to 8/group; males and females, but proportions of each not reported) were administered 0.1 mL of heliotropin (piperonal) epicutaneously to an area measuring 8 cm2on the clipped flanks which was left uncovered; the heliotropin (piperonal) concentration was 0, 3, 10, 30, or 60% in ethanol vehicle. A challenge application was performed on Day 21 and Day 35 of the study to animals of all 5 study groups. A volume of 0.025 mL of test material in ethanol was applied on the contralateral flank (2 cm2area) at a concentration of up to 10% (the minimal irritating concentration, which caused minimal irritation after a single administration). Reactions were determined after 24, 48, and/or 72 hours. The test material was considered to be allergenic at a given concentration when at least 1 out of the 5 or 8 animals of the given concentration group showed positive reactions with non-irritant concentrations used for challenge. At a challenge concentration of 3% in ethanol (i.e., a non-irritant concentration), heliotropin (piperonal) produced positive sensitization reactions on challenge Days 21 and 35 in animals that had been pre-treated with 30% (5/5 animals) or 60% (4/5 animals) concentrations once daily for 21 days, but not in animals that had been pre-treated with concentrations of 3 or 10% once daily for 21 days. A challenge concentration of heliotropin (piperonal) of 1% in ethanol produced positive sensitization reactions at challenge in 3 of 5 animals that had been pre-treated with the 60% concentration once daily for 21 days. Under the conditions of the study, which were extreme in terms of the number of induction applications (21 applications over 21 days), heliotropin (piperonal) was considered to be a weak local sensitizer in the guinea pig open epicutaneous test.

 

Klecak (1985) subsequently reported that heliotropin (piperonal) was negative in a similarly-conducted open epicutaneous test in guinea pigs. No sensitization was reported at a heliotropin (piperonal) challenge concentration of 6% (it was not specified if this was the minimal irritating concentration or one of the lower primary non-irritating concentrations).

 

Klecak et al.(1977) also reported on a maximization test and a Draize test in guinea pigs. In the maximization test, Himalayan white-spotted guinea pigs (sex and number of animals/group not reported) were administered 3 pairs of intradermal injections during the induction phase: 0.1 mL of Freund's Complete Adjuvant (FCA), 0.1 mL of heliotropin (piperonal) at 5% (vehicle not reported), and 0.1 mL of heliotropin (piperonal) at 5% emulsified in FCA. The topical induction phase was conducted 1 week later using a 48-hour occluded patch with 25% heliotropin (piperonal) in petrolatum which was applied to the clipped neck. Two weeks after the topical induction, a 24-hour occluded challenge patch was applied with a sub-irritant concentration of heliotropin (piperonal) in petrolatum (concentration not reported). Reactions were evaluated at 24 and 48 hours after patch removal. Sensitization was observed; further details were not provided (Klecak et al., 1977).

 

In the Draize test, Himalayan white-spotted guinea pigs (sex and number of animals/group not reported) were administered intradermal injections of heliotropin (piperonal) at 0.1% in isotonic saline. The first injection was 0.05 mL and the remaining 9 injections were 0.1 mL, which were administered on 9 alternate days. On days 35 and 49, an intradermal challenge was conducted with 0.05 mL of a 0.1% solution of heliotropin (piperonal) in isotonic saline. No reactions were observed; further details were not provided (Klecak et al., 1977).

 

Based on the positive findings from a reliable guinea pig maximization test, heliotropin (piperonal) is considered to possess moderate potency as a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the key GPMT study, greater than 30% of guinea pigs had an incidence of sensitisation (i.e. 4/10) with an intradermal induction of greater than 1% (i.e. 1.5%). As a result, the substance does meet the criteria for Category 1B skin sensitisation classification according to Regulation (EC) No 1272/2008, Annex I section 3.4.