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Description of key information

The Kligman guinea pig maximization test was conducted using 10 albino Dunkin/Hartley guinea pigs for test chemicalPhenethyl salicylate(CAS No:87-22-9) atGivuadan Roure SA Ecotoxicological Laboratory, Switzerland (SSS (Sustainability Support Services) has a letter of access,1981)..Preliminary irritation tests were carried out in guinea pigs to determine the concentration of test substances suitable for induction of sensitization and for sensitization challenge.As a result of the preliminary studies, the concentration selected for skin sensitization test were 0.5% forintradermal induction, 50% for topical induction and 10% for challenge.Induction consisted of intradermal injections followed 1week later by a 48 h occluded patch. Three pairs of intradermal injections were made as follows: two 0.1 ml injections of 0.5% phenethyl salicylate in 6% acetone/20% PEG400/0.01% Tween 80/saline; two 0.1 ml injections of 0.5% phenethyl salicylate in 50% FCA; and two 0.1 ml injections of 50% FCA. Seven days later, an occluded patch with 50% phenethyl salicylate in acetone was applied over the shoulder injection sites for 48 h. After a 14-day rest period, the animals were challenged with 10% phenethyl salicylate in acetone applied for 24 h under occlusion using an 11 mm aluminum patch test cup. Reactions were read 24 and 48 h after patch removal.Second challenge was conducted 1 week after on the opposite flank exactly as per the primary challenge. Further challenges and cross challenges maybe made on alternate flanks at weekly or greater intervals as required with controls included.Sensitization reactions were observed after both challenges. Cross-challenge applications with 10% benzyl salicylate and 10% phenyl salicylate were then made at weekly intervals. Cross-reactions were observed after each challenge.Approximately 3 out of 10 guinea pigs showed positive skin sensitization reaction after 2 challenges. Hence the chemicalPhenethyl salicylate(CAS No:87-22-9) was considered to as weak sensitizer on albino Dunkin/Hartley guinea pigs.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental reports
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
The Kligman guinea pig maximization test was conducted using 10 albino Dunkin/Hartley guinea pigs for test chemical Phenethyl salicylate (CAS No: 87-22-9).
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
not specified
Test material information:
Composition 1
Specific details on test material used for the study:
- IUPAC name: 2-phenylethyl 2-hydroxybenzoate
- Common name: Phenethyl Salicylate
- Molecular formula: C15H14O3
- Molecular weight : 242.2726 g/mol
- Smiles notation : c1ccc(cc1)CCOC(=O)c2ccccc2O
- InChl: 1S/C15H14O3/c16-14-9-5-4-8-13(14)15(17)18-11-10-12-6-2-1-3-7-12/h1-9,16H,10-11H2
- Substance type:Organic
- Physical state: Solid
- Physical apperance: white crystals
Species:
guinea pig
Strain:
other: Albino Dunkin/Hartley
Sex:
male/female
Details on test animals and environmental conditions:
- Weight at study initiation: 320 g
- Diet (e.g. ad libitum): RGP pellets, hay cabbage ad libitum
- Water (e.g. ad libitum): water ad libitum
Route:
intradermal
Vehicle:
other: 6% acetone/20% PEG400/0.01% Tween 80/saline
Concentration / amount:
•two 0.1 ml injections of 0.5% phenethyl salicylate in 6% acetone/20% PEG400/0.01% Tween 80/saline
•two 0.1 ml injections of 0.5% phenethyl salicylate in 50% FCA; and
•two 0.1 ml injections of 50% FCA
Day(s)/duration:
48 hours
Adequacy of induction:
not specified
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
50% phenethyl salicylate in acetone
Day(s)/duration:
24 hours
Adequacy of induction:
not specified
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
10% phenethyl salicylate in acetone
Day(s)/duration:
24 hours
Adequacy of challenge:
not specified
No. of animals per dose:
10 (6 males and 4 females or vise versa)
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL APPLICATION
- No. of exposures: 1
- Exposure period: 7 days
- Test groups: 10
- Control group: no data
- Site: 2X4 cm area of skin in the dorsal shoulder region
- Frequency of applications: no data
- Duration: 1 week
- Concentrations:
• two 0.1 ml injections of 0.5% phenethyl salicylate in 6% acetone/20% PEG400/0.01% Tween 80/saline
• two 0.1 ml injections of 0.5% phenethyl salicylate in 50% FCA; and
• two 0.1 ml injections of 50% FCA

A. II.INDUCTION EXPOSURE: TOPICAL APPLICATION
- No. of exposures: 1
- Exposure period: 48 hours
- Test groups: 10 guinea pigs
- Control group: no data
- Site: same shoulder injection site
- Frequency of applications: no data
- Duration: 48 hours
- Concentrations: 50% phenethyl salicylate in acetone



B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: after 13-14 days
- Exposure period: 24 hours
- Test groups: 10 guine pigs
- Control group: 4 guinea pigs
- Site: clipped and shaved flank
- Concentrations: 10% phenethyl salicylate in acetone
- Evaluation (hr after challenge): 24 and 48 hours

Second challenge was conducted 1 week after on the opposite flank exactly as per the primary challenge. Further challenges and cross challenges maybe made on alternate flanks at weekly or greater intervals as required
Challenge controls:
Two types of controls are used
Treated control: they are given a mock induction treatment at the same time and in the same way as for the test animals except that test substance is omitted from the injection and application preparations. (4 same sex)

Untreated control: at every challenge in the test 4 previously –untreated animals of the same sex and weighing approximately the same as the test animals at the challenge are treated inexactly the same as the test animals.
Positive control substance(s):
not specified
Key result
Reading:
other: Challenge 1
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
Weak sensitizer
Remarks on result:
positive indication of skin sensitisation
Reading:
other: Challenge 1
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Weak sensitizer
Remarks on result:
no indication of skin sensitisation
Reading:
other: Challenge 2
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
Weak sensitizer
Remarks on result:
positive indication of skin sensitisation
Reading:
other: Challenge 2
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
Weak sensitizer
Remarks on result:
positive indication of skin sensitisation
Cellular proliferation data / Observations:
Sensitization reactions were observed after both challenges.

Table 1.Skin Reaction Scoring System

SKIN REACTIONS

SCORES

No reaction

0

Barely perceptible erythema

±

Scattered, mild erythema (faint pink)

+

Moderate and diffuse erythema (pale pink )

++

Intense erythema (deep pink) and oedema

+++

 

 

 

Table 2.1.RESULTS OF CHALLENGE 1 (CONCENTRATION: 10%)

Scores

(Hours)

No. of animals

1

2

3

4

5

6

7

8

9

10

Sex

F

F

F

F

F

F

M

M

M

M

24 HOURS

±

±

++

±

+++

±

+

±/+

±

+

48 HOURS

±

±

++

0

+++

0

+

++

±

±

No of positive reactions

24 hours:3+?2

48 hours:2+?1

 

Table 2.2 Treated Controls of Challenge 1(Concentration: 10%)

 Scores

(Hours)

No. of animals

1

2

3

4

Sex

F

F

F

F

24 HOURS

0

0

±

0

48 HOURS

0

0

0

0

 

 

Table 2.3. Untreated Controls of Challenge 1 (Concentration: 10%)

 

 Scores

(Hours)

No. of animals

1

2

3

4

Sex

F

F

F

F

24 HOURS

0

0

0

±

48 HOURS

0

0

±

±

 

 

Table 3.1.RESULTS OF CHALLENGE 2 (CONCENTRATION: 10%)

Scores

(Hours)

No. of animals

1

2

3

4

5

6

7

8

9

10

Sex

F

F

F

F

F

F

M

M

M

M

24 HOURS

+/++

±

++

±

++/+++

0

++

+

0

+

48 HOURS

±/+

0

++

0

+++

0

+

±

0

±

 

Table 3.2. Untreated Controls of Challenge 2 (Concentration: 10%)

 

 Scores

(Hours)

No. of animals

1

2

3

4

Sex

F

F

F

F

24 HOURS

0

0

0

±

48 HOURS

0

0

±

±

 

 

Interpretation of results:
other: sensitizing
Conclusions:
The test material Phenethyl salicylate (CAS No: 87-22-9) was considered to be sensitizing on albino Dunkin/Hartley guinea pigs in the Kligman guinea pig maximization test.
Executive summary:

The Kligman guinea pig maximization test was conducted using 10 albino Dunkin/Hartley guinea pigs for test chemicalPhenethyl salicylate(CAS No:87-22-9).

 

Preliminary irritation tests are carried out in guinea pigs to determine the concentration of test substances suitable for induction of sensitization and for sensitization challenge.As a result of the preliminary studies, the concentration selected for skin sensitization test were 0.5% forintradermal induction, 50% for topical induction and 10% for challenge.

 

Induction consisted of intradermal injections followed 1week later by a 48 h occluded patch. Three pairs of intradermal injections were made as follows: two 0.1 ml injections of 0.5% phenethyl salicylate in 6% acetone/20% PEG400/0.01% Tween 80/saline; two 0.1 ml injections of 0.5% phenethyl salicylate in 50% FCA; and two 0.1 ml injections of 50% FCA. Seven days later, an occluded patch with 50% phenethyl salicylate in acetone was applied over the shoulder injection sites for 48 h. After a 14-day rest period, the animals were challenged with 10% phenethyl salicylate in acetone applied for 24 h under occlusion using an 11 mm aluminum patch test cup. Reactions were read 24 and 48 h after patch removal.

 

Second challenge was conducted 1 week after on the opposite flank exactly as per the primary challenge. Further challenges and cross challenges maybe made on alternate flanks at weekly or greater intervals as required with controls included.

 

Sensitization reactions were observed after both challenges. Cross-challenge applications with 10% benzyl salicylate and 10% phenyl salicylate were then made at weekly intervals. Cross-reactions were observed after each challenge.

 

Approximately 3 out of 10 guinea pigs showed positive skin sensitization reaction after 2 challenges. Hence the chemicalPhenethyl salicylate(CAS No:87-22-9) was considered to as weak sensitizer on albino Dunkin/Hartley guinea pigs.

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

SKIN SENSITIZATION:

Various studieshas been investigated for the test chemicalPhenethyl salicylate(CAS No:87-22-9)to observe the potential for skin sensitization to a greater or lesser extent. The studies are based on in vivo experiments in guinea pigsand humans for target chemicalPhenethyl salicylate(CAS No:87-22-9)which have beensummarized as below;

 

The Kligman guinea pig maximization test was conducted using 10 albino Dunkin/Hartley guinea pigs for test chemicalPhenethyl salicylate(CAS No:87-22-9) atGivuadan Roure SA Ecotoxicological Laboratory, Switzerland (SSS (Sustainability Support Services) has a letter of access,1981)..Preliminary irritation tests were carried out in guinea pigs to determine the concentration of test substances suitable for induction of sensitization and for sensitization challenge.As a result of the preliminary studies, the concentration selected for skin sensitization test were 0.5% forintradermal induction, 50% for topical induction and 10% for challenge.Induction consisted of intradermal injections followed 1week later by a 48 h occluded patch. Three pairs of intradermal injections were made as follows: two 0.1 ml injections of 0.5% phenethyl salicylate in 6% acetone/20% PEG400/0.01% Tween 80/saline; two 0.1 ml injections of 0.5% phenethyl salicylate in 50% FCA; and two 0.1 ml injections of 50% FCA. Seven days later, an occluded patch with 50% phenethyl salicylate in acetone was applied over the shoulder injection sites for 48 h. After a 14-day rest period, the animals were challenged with 10% phenethyl salicylate in acetone applied for 24 h under occlusion using an 11 mm aluminum patch test cup. Reactions were read 24 and 48 h after patch removal.Second challenge was conducted 1 week after on the opposite flank exactly as per the primary challenge. Further challenges and cross challenges maybe made on alternate flanks at weekly or greater intervals as required with controls included.Sensitization reactions were observed after both challenges. Cross-challenge applications with 10% benzyl salicylate and 10% phenyl salicylate were then made at weekly intervals. Cross-reactions were observed after each challenge.Approximately 3 out of 10 guinea pigs showed positive skin sensitization reaction after 2 challenges. Hence the chemicalPhenethyl salicylate(CAS No:87-22-9) was considered to as weak sensitizer on albino Dunkin/Hartley guinea pigs.

 

G. Klecak (1985) and A. Lapczynski , D. McGinty, L. Jones, S. Bhatia, C.S. Letizia, A.M. Api (2007)conducted an Open Epicutaneous Test (OET) on guinea pigs to assess the skin sensitization potential of test chemical Phenethyl salicylate (CAS No: 87-22-9). On day 1 during induction, 0.1 ml of the Phenethyl salicylate (CAS No: 87-22-9) was applied at concentrations of 100%, 30%, 10%, 3%, 1%, or 0.3% in vehicle to an area measuring 8 cm2 on the clipped flank skin of the guinea pigs. The applications are repeated daily for 3 weeks or done 5 times weekly during 4 weeks, usually on the same skin sites. The application sites were left uncovered and the reactions, if continuous daily applications were performed, can be read 24 h after each application, or at the end of each week. To determine whether or not contact sensitization was induced, all groups of guinea pigs previously treated for 21 days, as well as 10 untreated, or only pretreated with the vehicle, controls are tested on days 21 and 35 on the contralateral flank with the test material. This test was performed by applying with a pipette 0.025 ml of each concentration to skin areas measuring 2 cm2. The reactions were read after 24, 48 and/or 72h. It was observed that the guinea pigs induced contact sensitization at challenge concentration of 8%.Thus the chemicalPhenethyl salicylate (CAS No: 87-22-9) was considered to be sensitizing on skin of guinea pigs at concentration of 8% in an Open Epicutaneous Test (OET).

 

The G. Klecak , H. Geleick and J. R. Frey (1977) and A. Lapczynski , D. McGinty, L. Jones, S. Bhatia, C.S. Letizia, A.M. Api (2007) carried out maximization test (MT) of chemicalPhenethyl salicylate(CAS No:87-22-9) in Himalayan white-spotted guinea pigs. Duringinduction, on day 0the animals were injected intradermally with 0.1 ml of a   5% solution of the compound tested, with 0.1 ml of a 5 % emulsion of the same compound in Freund's complete adjuvant (FCA) and with 0.1 ml of FCA alone. On day 8 the animals were induced epicutaneously with 250 ml of 25% test compound in dissolved in petrolatum on clipped skin area of the neck under occlusion for 2 days. On day 21, animals were challenged with the compound at a subirritant concentration in petrolatum was applied to the flank for 24 h. The reactions were read 24 and 48 h after removing the patch.Since positive results were observed during the 24 hours, the chemicalPhenethyl salicylate(CAS No:87-22-9)was considered to be sensitizingto the skin of Himalayan white-spotted guinea pigs.

 

G. Klecak (1985) and A. Lapczynski , D. McGinty, L. Jones, S. Bhatia, C.S. Letizia, A.M. Api (2007) performed another FREUND'S COMPLETE ADJUVANT (FCAT) in Himalayan white-spotted guinea pigs (male and female) of Phenethyl salicylate(CAS No:87-22-9)).In this test, two groups of 10-20 guinea pigs each were involved: (a) the experimental group, and (b) the control group. Additionally, 4 guinea pigs were used for determination of the primary irritating concentration of the test substance so that nonirritating concentrations may be chosen for challenge. For induction, the scapula region of test and control animals is shaved. During days 1-9 the animals of the experimental group are treated with 0.1 ml of the test material in FCA intradermally into the scapula region three times (days I, 5 and 9) on an area of 6 x 2 cm. The animals of the control group are treated with 0.1 ml of FCA only. To determine whether or not contact allergy was induced, both animal groups, the experimental and the control one, are challenged on days 21 and 35 on the contralateral flanks with the same compound at the minimal irritating and some lower nonirritating concentrations. The tests are performed by applying with a pipette 0.025 ml of each concentration to skin areas measuring 2 cm2. The reactions are read 24, 48 and 72 h after application. Skin reactions were observed during 72 hours observation period. Thus the chemicalPhenethyl salicylate(CAS No:87-22-9)was considered to be sensitizingto the skin of Himalayan white-spotted guinea pigs.

 

A. Lapczynski , D. McGinty, L. Jones, S. Bhatia, C.S. Letizia, A.M. Api (2007) and D. Belsito, D. Bickers, M. Bruze, P. Calow, H. Greim,J.M. Hanifin, A.E. Rogers, J.H. Saurat, I.G. Sipes, H. Tagami (2007) carried out an LLNA test in 25 female CBA/Ca female mice for test chemical Phenethyl salicylate (CAS No: 87-22-9) to determine its sensitization efficacy. During the test, each animal received a daily topical application of 25 25µl of 1.0%, 2.5%, 5.0%, 10% or 25% test material in EtOH:DEP (3:1) on the dorsal surface of each ear for three consecutive days. Control animals were treated with the vehicle alone. Three days after the third topical application all mice were injected intravenously through the tail vein with 250 µl sterile saline (PBS) containing 20 µCi 3H-methylthymidine (3H-thymidine). All mice were sacrificed 5 h after the intravenous injection. Draining auricular lymph nodes were excised and were pooled for each experimental group. Single cell suspensions were then prepared, washed with PBS, suspended in trichloroacetic acid (TCA) and left overnight at 4 °C. The samples were then resuspended in TCA and then transferred to a scintillation cocktail. Incorporation of 3H-TdR was then measured by β-scintillation counting and stimulation indices were determined for each experimental group. Also the EC3 value was calculated.The EC3 value calculated for Phenethyl salicylate was 2.1% (525 µg/cm2). Thus on the basis of calculated EC3 value the chemical Phenethyl salicylate (CAS No: 87-22-9) was considered to as skin sensitizer.

 

A. Lapczynski , D. McGinty, L. Jones, S. Bhatia, C.S. Letizia, A.M. Api (2007) and D. Belsito, D. Bickers, M. Bruze, P. Calow, H. Greim,J.M. Hanifin, A.E. Rogers, J.H. Saurat, I.G. Sipes, H. Tagami (2007) carried out anopen epicutaneous test (OET)ofPhenethyl salicylate(CAS No:87-22-9) was conducted in Himalayan white-spotted guinea pigs (male and female) to determine the minimal sensitizing concentration necessary to induce contact hypersensitivity and the minimal eliciting concentration necessary to cause a positive reaction.Each Guinea pigs received 21 daily open applications to an 8 cm 2 area on the clipped flank with neat phenethyl salicylate and phenethyl salicylate at concentrations of100%, 30%, 10%, 3%, 1%(in a suitable vehicle). Reactions were read 24 h after each application.On days 21 and 35, guinea pigs were challenged by an open application with 0.025 ml of phenethyl salicylate applied to a skin area measuring 2 cm2on the contralateral flank. The skin reactions were examined after 24, 48 and/or 72 hours.The treated animals showed positive skin reactions. HencePhenethyl salicylate(CAS No:87-22-9)was considered to be not sensitizingon Himalayan white-spotted guinea pigs. The minimum sensitizing concentration necessary to induce contact hypersensitivity was 30% and the minimum eliciting concentration necessary to cause a positive reaction was 0.03%.

 

Another Open Epicutaneous Test (OET) was performed on guinea pigs by G. Klecak , H. Geleick , J. R. Frey (1977) and A. Lapczynski , D. McGinty, L. Jones, S. Bhatia, C.S. Letizia, A.M. Api (2007) of test chemical Phenethyl salicylate (CAS No: 87-22-9). On day 1 during induction, 0.1 ml of the Phenethyl salicylate (CAS No: 87-22-9) was applied at concentrations of 100%, 30%, 10%, 3%, 1%, or 0.3% in vehicle to an area measuring 8 cm2 on the clipped flank skin of the guinea pigs. The applications are repeated daily for 3 weeks or done 5 times weekly during 4 weeks, usually on the same skin sites. The application sites were left uncovered and the reactions, if continuous daily applications were performed, can be read 24 h after each application, or at the end of each week. To determine whether or not contact sensitization was induced, all groups of guinea pigs previously treated for 21 days, as well as 10 untreated, or only pretreated with the vehicle, controls are tested on days 21 and 35 on the contralateral flank with the test material. This test was performed by applying with a pipette 0.025 ml of each concentration to skin areas measuring 2 cm2. The reactions were read after 24, 48 and/or 72h. It was observed that none of the guinea pigs induced contact sensitization at challenge concentration of 8%.Thus the chemicalPhenethyl salicylate (CAS No: 87-22-9) was considered to be not sensitizing on skin of guinea pigs at concentration of 8% in an Open Epicutaneous Test (OET).

 

The G. Klecak , H. Geleick and J. R. Frey (1977) and A. Lapczynski , D. McGinty, L. Jones, S. Bhatia, C.S. Letizia, A.M. Api (2007) carried out Draize test (DT).In theDraize test, the 6-8 guinea pigs received a dose of 0.05 ml of a 0.1 % solution of the chemical tested in isotonic saline intradermally on day 0 and further doses of 0.1 ml each were injected on 9 alternate days.The treated animals and untreated controls were challenged intradermally with 0.05 ml of a 0.1 per cent solution on days 35 and 49. The evaluation criterion was the mean diameter of the popular reactions.None of the treated animals showed positive skin reactions. HencePhenethyl salicylate(CAS No:87-22-9)was considered to be not sensitizing to the skin of Himalayan white-spotted guinea pigs.

A. Lapczynski , D. McGinty, L. Jones, S. Bhatia, C.S. Letizia, A.M. Api (2007) and D. Belsito, D. Bickers, M. Bruze, P. Calow, H. Greim,J.M. Hanifin, A.E. Rogers, J.H. Saurat, I.G. Sipes, H. Tagami (2007) carried out A Closed Epicutaneous Tests (CET) was conducted on eight guinea pigs for test chemical Phenethylsalicylate(CAS No:87-22-9) under occlusive condition. Induction consisted of six, 48 h occluded patch applications which were made to the shaved nape using Torii’s patch plaster and adhesive tape. The applications were made three times a week for 2 weeks with 30% phenethyl salicylate. On day 28, a 48 occluded challenge application with 1% phenethyl salicylate was made to the shaved flank. Reactions were read at patch removal and 24 and 48 h after patch removal. Since no sensitization reactions were observed, the chemical Phenethyl salicylate(CAS No:87-22-9) was considered to be not sensitizing to the skin of guinea pigs.

 

Although negative skin reactions were observed in few studies but on the basis of available dataand majority of results obtained in various studies, the chemicalPhenethyl salicylate(CAS No:87-22-9) can considered as sensitizing.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The skin sensitization potential of test substancePhenethyl salicylate(CAS No:87-22-9) were observed in various studies. On the basis of available dataand majority of results obtained in various studies, the chemicalPhenethyl salicylate(CAS No:87-22-9) can considered as sensitizing and hence can be classified as a skin sensitizer.