Registration Dossier

Administrative data

Description of key information

Based on the available data, piperonal is not considered to be a skin or eye irritant in humans.
Skin irritation:
A total of 4 studies conducted in human volunteers have demonstrated minimal irritation potential for piperonal (Katz, 1946; Hill Top Research, 1964; Fujii et al., 1972; Harrison Research Laboratories, 2011). No irritation was reported in a rat acute dermal toxicity study in which animals were exposed to piperonal at a dose of 5,000 mg/kg body weight (Cosmopolitan Safety Evaluation, 1980), although irritation scores were not provided. Data obtained in the preliminary portion of a guinea pig maximization test supports a lack of skin irritation for piperonal when administered under occlusion for 24 hours at a concentration of up to 80% in acetone (Unilever Research Laboratory, 1978). A concentration of piperonal of 3% in ethanol was not irritating to the skin of guinea pigs after a single exposure and was considered very slightly irritating after 21 daily exposures (Givaudan, 1977b; Klecak et al., 1977). Higher concentrations (≥10%) were reported to be irritating after a single exposure (degree of irritation not reported) or repeated exposures (slightly irritating).
Eye irritation:
No reactions were observed and the primary irritation score was 0 at all time points in a GLP-compliant eye irritation study in New Zealand albino rabbits (Cosmopolitan Safety Evaluation, 1980). Another rabbit study reported intense conjunctival irritation with chemosis and discharge that decreased in intensity over time; no corneal opacity or iris congestion was noted at any time point, and all eyes were normal by Day 7 (Leberco Laboratories, 1963). In a study for which reliability could not be assessed, slight irritating effects were observed following instillation of an unknown quantity of piperonal crystals into the eyes of 3 rabbits that resolved by 14 days post-instillation (Eastman Kodak, 1982).

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from February to June 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Purity of the tested substance was not provided
Qualifier:
according to guideline
Guideline:
other: protocol #RE(7/23/79) supplied by IFF, based on the procedure originally suggested by Draize.,H.J. "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics", Assoc. of Food and Drug Officials of the U.S., Austin, Texas (1959).
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
-Purity of the substance was not provided; No observation made at 1 hour post-dosing.
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Six healthy young adult albino New Zealand type rabbits with body weights in the range of 2.0 to 3.5 kg at the preliminary eye examination were used. The animals were individually numbered and the identification was marked in the ear of each rabbit by a metal tag as well as being written on the cage card. Animals were assigned to the study at random and the entire group was indicated by colour code on the cage card.

The animals were housed singly under standard laboratory conditions. They were fed 100 grams of Purina Rabbit Chow supplemented by whole oats daily, and were allowed water ad libitum. The criteria used for housing and maintenance were those specified in the ‘Guide for the Care and Use of Laboratory Animals’ (DHEW Publication No. (NIH) 78—23, Revised 1978). No extraneous material, such as sawdust or woodchips was used in the cages. The suspended pans beneath the cages were littered with prepared bedding (Easi Litter).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
The test article was crushed into a powder with a mortar in a pestle for this study.
0.1 g of the test article 80-004-02 was applied once to the right eye of each of six rabbits.
Duration of treatment / exposure:
Single exposure.
Observation period (in vivo):
24, 48 and 72 hours and at 4, 7 days after dosing. In addition any persistent changes observed on day 7 were re-evaluated on day 10 using a hand held ophthalmoscope and the fluorescein method.
Number of animals or in vitro replicates:
six albino New Zealand rabbits
Details on study design:
Twenty-four hours prior to dosing, both eyes were examined using a hand held ophthalmoscope and then by fluorescein staining. One drop of Fluorescein Sodium Ophthalmic Solution USP was dropped onto the cornea which was then irrigated with Sodium Chloride Solution USP. The eyes were examined for corneal ulceration using an ultraviolet light. Animals used in the study were selected to be free of apparent ocular defects.
On the day of dosing, the lower lid was everted from the eyeball of the right eye to form a cup into which the test article was placed at the specified dose. The eyelid was then gently held together for one second. The eye was not washed after application of the test article. Any untoward reaction to application of test substance was noted and the animal was returned to its cage.
Both eyes were scored according to the Draize Method at 24, 48 and 72 hours and at 4 and 7 days after dosing. In addition at 24 hours and 7 days, both eyes were examined with fluorescein stain under an ultraviolet light.

CORNEA
A. Opacity—degree of density (area most dense taken for reading)
No opacity = 0
Scattered or diffuse area, details of iris clearly visible = 1
Easily discernible translucent areas, details of iris slightly obscured = 2
Opalescent areas, no details of iris visible, size of pupil barely discernible = 3
Opaque, iris invisible = 4

B. Area of cornea involved
One quarter (or less) but not zero = 1
Greater than one quarter, but less than half = 2
Greater than one half, but less than three quarters = 3
Greater than three quarters, up to whole area = 4
A X B X 5 Total maximum = 80

IRIS
A. Values
Normal...0
Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive) = 1
No reaction to light, hemorrhage, gross destruction (any or all of these) = 2
A X 5 Total maximum = 10

CONJUNCTIVAE
A. Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Vessels normal = 0
Vessels definitely injected above normal = 1
Vessels diffuse, deeper crimson red, individual vessels not easily discernible = 2
Diffuse beefy red = 3

B. Chemosis
No swelling = 0
Any swelling above normal (includes nictitating membrane) = 1
Obvious swelling with partial eversion of lids = 2
Swelling with lids about half closed = 3
Swelling with lids about half closed to completely closed = 4

C. Discharge
No discharge = 0
Any amount different from normal (does not include small amounts observed in inner canthus of normal animals) = 1
Discharge with moistening of the lids and hairs just adjacent to lids = 2
Discharge with moistening of the lids and hairs, and considerable area around the eye = 3
(A + B + C) X 2 Total maximum = 20

Classification Scale:
0.0-0.5 non-irritating
0.6-10.0 practically non-irritating
10.1-25.0 mildly irritating
25.1-50.0 moderately irritating
50.1-110.0 extremely irritating
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #2, #3, #4, #5 and #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: #1, #2, #3, #4, #5 and #6
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal: #1, #2, #3, #4, #5 and #6
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal: #1, #2, #3, #4, #5 and #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
animal: #1, #2, #3, #4, #5 and #6
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
None of the dosed eyes had any reaction during the 7 day observation period. All eyes were negative for fluorescein retention.

The mean ocular irritation scores were:
0 at 24 hours
0 at 48 hours
0 at 72 hours
0 at 4 days
0 at 7 days
Other effects:
None.

The Primary Ocular Irritation Scores were 0 at all readings; therefore, the test article was classified as non-irritating to the eye of the albino rabbit.

Interpretation of results:
not irritating
Remarks:
Migrated information under CLP REGULATION (EC) No 1272/2008 Criteria used for interpretation of results: other: REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL
Conclusions:
The Primary Ocular Irritation Scores were 0 at all readings; therefore, the test article was classified as non-irritating to the eye of the albino rabbit.
Executive summary:

The results of this study demonstrated that administration of heliotropin (piperonal) (0.1 g) to the eyes of rabbits is not irritating; the primary ocular irritation score was 0 for all animals at all evaluation timepoints up to and including 7 days post-dosing.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation/Corrosion

From the cumulative data from several human and animal studies, in can be concluded that heliotropin (piperonal) has minimal potential to cause skin irritation. Therefore, further in vivo skin irritation testing was deemed unnecessary.

 

The HRIPT 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. During the induction phase, 3 subjects exhibited faint, minimal erythema after the first (1 subject) or second (2 subjects) application. A fourth subject exhibited faint, minimal erythema after applications 5, 6, and 7 and had erythema and edema after application 8. The application site was changed and this subject again had faint, minimal erythema after application 9. Comparatively, a total of 10 subjects exhibited faint, minimal erythema after 2 or more applications of saline. Thus, heliotropin (piperonal) demonstrated minimal potential to induce irritation in this study.

In the HRIPT 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. During the induction phase, 3 subjects had slight erythema on 1 occasion (1 subject after each of the second, third, and eighth administrations). A fourth subject had a moderately strong response that was reported as erythema and papules after the third administration and very strong edema and/or papules after the seventh administration; no such reactions were observed at the challenge application. Thus, heliotropin (piperonal) demonstrated minimal potential to induce irritation in this study.

 

Two other human patch test studies reported minimal irritation due to heliotropin (piperonal) (Katz, 1946; Fujiiet al., 1972). Both of these studies were judged to be unreliable due to methodological and reporting deficiencies. The study by Katz (1946) reported a “positive reaction” in 1 of 20 adult subjects exposed to a saturated alcoholic solution of heliotropin (piperonal) for 24 hours. A “positive reaction” was considered to be visible inflammation, irritation, formation of a popular rash, or any abnormal condition of the application area. No information was provided on the purity or dose level/concentration of heliotropin (piperonal), the sex of the test subjects, the type and severity of reaction that occurred, the time point at which the reaction was observed, or the reversibility of the noted reaction. The study by Fujiiet al.(1972) reported that heliotropin (piperonal) caused no irritation in adult subjects when evaluated at concentrations of 0.1% (81 subjects) or 20% (28 subjects). At a concentration of 2%, 3 of 26 subjects had slight erythema (suspected positive reaction) and 1 of 26 subjects had erythema (positive reaction). Limitations of this study include the use of only a single evaluation time point and the lack of information on the purity of heliotropin (piperonal), the vehicle that was used for each subject, the sex of the test subjects, the duration of exposure for each subject, and the reversibility of the reactions.

 

The acute dermal toxicity of heliotropin (piperonal) was evaluated in a GLP-compliant study by Cosmopolitan Safety Evaluation (1980) that was conducted in conformity with IFF Protocol No. DLD (9/7/79) supplied by International Flavors and Fragrances. Sixteen Sprague-Dawley CD rats (8/sex) weighing between 180 and 280 grams were clipped on the day prior to dosing and the test substance, prepared in alcohol, was applied onto the skin of the back at a dose of 5,000 mg/kg body weight. The volume of solution administered was 10 mL/kg body weight and the area of skin to which the dose was applied was reported to comprise less than 30% of the body surface. The test article was allowed to remain in contact with the skin and open to the air for 24 hours after which excess material was removed with a clean cloth. Animals were observed 1, 3, 5, and 24 hours post-dosing and twice daily (once daily on weekends) for the remainder of the 14-day observation period. None of the animals showed any clinical signs indicative of systemic toxicity and there was no indication of effects on the skin, although it is acknowledged that skin irriation scores were not reported. All animals remained healthy throughout the study and gained weight in a normal manner, except for one female which lost 10 grams over the course of the study. At necropsy, none of the animals had any signs indicative of systemic toxicity or effects on the skin (as evaluated by examination of skin and fur).

 

Unilever Research Laboratory (1978) reported on a guinea pig maximization test in which the skin irritation potential of heliotropin (piperonal) was evaluated prior to the conduct of the sensitization portion of the study. Heliotropin (piperonal) was dissolved in acetone and applied under occlusion for 24 hours to the shaved flank region of naive animals. Concentrations of 5%, 10%, and 25% were evaluated in 3 male animals (another male animal removed its bandage during the application period and therefore was not evaluated) while concentrations of 40%, 60%, and 80% were evaluated in 4 female animals. Irritation was scored on a scale of 0 (no reaction) to +++ (intense erythema and oedema) at 24 and 48 hours after removal of the patches. No reaction was observed at any test concentration in any animal at either evaluation time point.

 

Klecaket al.(1977) reported on an open epicutaneous test that was conducted in guinea pigs; the same study was reported by Geleick (1977) but in insufficient detail, thus rendering the study report unreliable. During the induction phase (study Days 0 to 20), guinea pigs (6 to 8/group; males and females, but proportions of each were 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 3, 10, 30, or 60% in ethanol vehicle. During the induction phase, the highest non-irritant concentration for a single application was 3%. After 21 days of daily administration, very slight irritation (3% concentration) or slight irritation (10, 30, and 60% concentrations) was noted. Details were not provided on how many animals at each dose level exhibited the findings or on what constituted a determination of “very slight” or “slight” irritation. Therefore, the results of this study are considered reliable but with restrictions.

 

Two other guinea pig studies cannot be judged for reliability due to insufficient reporting of experimental details and results (Quest, 1977; Eastman Kodak, 1982). Quest (1977)reported no observations in 3 of 4 female guinea pigs exposed to 10, 25, or 50% heliotropin (piperonal) in ethanol; the fourth animal had barely perceptible erythema at both 24 and 48 hours after removal of the test patch with 50% heliotropin (piperonal) in ethanol (no reactions were observed at the 10 or 25% concentrations). Eastman Kodak (1982) reported gross edema, slight to severe erythema, necrosis, and other findings at various time points after dermal exposure to heliotropin (piperonal) at a dose level of 0.25 to 1.0 g/kg body weight. A total of 8 guinea pigs were studied but no details were provided on the dose administration protocol, exposure period, criteria used for evaluation of reactions, individual animal findings and other methodological aspects.

 

In summary, reliable studies of high quality indicate no or minimal skin irritation potential for heliotropin (piperonal) in humans, rats, and guinea pigs.

 

Eye Irritation

Based on the available information, which includes a key GLP-compliant rabbit study and two supporting rabbit studies, it is concluded that heliotropin (piperonal) is not an eye irritant.

 

In a GLP-compliant study, the eye irritation potential of heliotropin (piperonal) was evaluated in 6 New Zealand albino rabbits (sex not reported) (Cosmopolitan Safety Evaluation, 1980). A 0.1 g aliquot of neat heliotropin (piperonal) was instilled into the right eye of each rabbit and the untreated left eye served as a control for each animal. Eyes were not washed following application of the test article. Reactions were scored according to Draize at 24, 48, and 72 hours and at 4 and 7 days post-administration. No reactions were observed and the primary irritation score was 0 at all time points.

 

An earlier eye irritation study was conducted by Leberco Laboratories (1963). In this supporting study, a 0.1 mL aliquot of heliotropin (piperonal) (concentration and vehicle not reported) was instilled into the right eye of 3 albino rabbits (sex and strain not reported). Both the untreated left eyes and the treated right eyes were examined and scored according to Draize every 24 hours for 4 days, and again at 7 days post-administration. An intense conjunctival irritation with chemosis and discharge that decreased in intensity over time was observed in all 3 rabbits. These effects could not be attributed directly to heliotropin (piperonal) given the lack of information on the concentration of heliotropin (piperonal) that was assessed and the vehicle that was used in this study. Notably, no corneal opacity or iris congestion was noted at any time point, and all eyes were normal by Day 7.

 

A study by Eastman Kodak (1982) reported slight irritating effects following instillation of an unknown quantity of heliotropin (piperonal) crystals into the eyes of 3 rabbits that resolved by 14 days post-instillation; however, the reliability of the study could not be determined due to insufficient reporting of experimental details and results.

 


Justification for selection of skin irritation / corrosion endpoint:
Weight of evidence for skin irritation / corrosion is based on information from human and animal studies.

Justification for selection of eye irritation endpoint:
A reliable eye irritation study is available.

Justification for classification or non-classification

From the cumulative data from several human and animal studies, in can be concluded that piperonal has minimal potential to cause skin irritation. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.2.

Based on the available information, which includes a key GLP-compliant rabbit study and two supporting rabbit studies, it is concluded that piperonal is not an eye irritant. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.3.

The classification for respiratory irritation is not available for data missing.