Phenethyl salicylate

Endpoint summary

Description of key information

Skin sensitisation:

The Kligman Guinea pig maximization test was conducted using 10 albino Dunkin/Hartley guinea pigs for test chemical. 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% for intradermal 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% test chemical in 6% acetone/20% PEG400/0.01% Tween 80/saline; two 0.1 ml injections of 0.5% test chemical in 50% FCA; and two 0.1 ml injections of 50% FCA. Seven days later, an occluded patch with 50% test chemical in acetone was applied over the shoulder injection sites for 48 h. After a 14-day rest period, the animals were challenged with 10% test chemical 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 may be 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 chemical was considered to as weak sensitizer on albino Dunkin/Hartley guinea pigs and classified under "Category 1" as per CLP Regulation.

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Principles of method if other than guideline:

According to OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

not specified

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% test chemical in 6% acetone/20% PEG400/0.01% Tween 80/saline
Two 0.1 ml injections of 0.5% test chemical 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% test chemical in acetone

Day(s)/duration:

24 hours

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: acetone

Concentration / amount:

10% test chemical in acetone

Day(s)/duration:

24 hours

Adequacy of challenge:

not specified

No. of animals per dose:

10 (6 males and 4 females or vice 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% test chemical in 6% acetone/20% PEG400/0.01% Tween 80/saline
Two 0.1 ml injections of 0.5% test chemical 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% test chemical 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% test chemical 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

Positive control results:

No Data Available

Key result

Reading:

other: Challenge 1

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

Weak sensitizer

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

other: Challenge 1

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

Weak sensitizer

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

other: Challenge 2

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

Weak sensitizer

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

other: Challenge 2

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

Weak sensitizer

Remarks on result:

positive indication of skin sensitisation

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:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on all the observations and results, it was concluded that the test chemical 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 chemical. 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% for intradermal 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% test chemical in 6% acetone/20% PEG400/0.01% Tween 80/saline; two 0.1 ml injections of 0.5% test chemical in 50% FCA; and two 0.1 ml injections of 50% FCA. Seven days later, an occluded patch with 50% test chemical in acetone was applied over the shoulder injection sites for 48 h. After a 14-day rest period, the animals were challenged with 10% test chemical 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 may be 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 chemical was considered to as weak sensitizer on albino Dunkin/Hartley guinea pigs.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Various studies have been reviewed to evaluate the degree of dermal sensitization caused by the test chemical. These include in vivo experimental studies performed on guinea pigs, mice as well as humans for the test chemical. The results are mentioned below:

The Kligman Guinea pig maximization test was conducted using 10 albino Dunkin/Hartley guinea pigs for test chemical. 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% for intradermal 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% test chemical in 6% acetone/20% PEG400/0.01% Tween 80/saline; two 0.1 ml injections of 0.5% test chemical in 50% FCA; and two 0.1 ml injections of 50% FCA. Seven days later, an occluded patch with 50% test chemical in acetone was applied over the shoulder injection sites for 48 h. After a 14-day rest period, the animals were challenged with 10% test chemical 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 may be 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 chemical was considered to as weak sensitizer on albino Dunkin/Hartley guinea pigs.

This result is supported by a LLNA study was conducted in 25 female CBA/Ca female mice to determine the sensitization efficacy of the test chemical. During the test, each animal received a daily topical application of 25µlof 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 250µlsterile saline (PBS) containing 20µCi 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 the test chemical was 2.1% (525 µg/cm2). Thus on the basis of calculated EC3 value the chemical was considered as a skin sensitizer.

 

The results obtained from the LLNA study are supported by a guinea pig maximization test was conducted in Himalayan white-spotted guinea pigs (male and female) to determine the skin sensitization potential of the test chemical. During induction, 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 sub irritant 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 test chemical was considered to be sensitizing to the skin of Himalayan white-spotted guinea pigs.

These results are lent support by a Freund's Complete Adjuvant test was conducted in Himalayan white-spotted guinea pigs (male and female) to determine the skin sensitization potential of the test chemical. 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 non-irritating 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 non-irritating concentrations. The tests are performed by applying with a pipette 0.025 ml of each concentration to skin areas measuring 2 sq.cm. The reactions are read 24, 48 and 72 h after application. Skin reactions were observed during 72 hours observation period. Based on all the observations and results, it was concluded that the test was sensitizing to Himalayan white-spotted guinea pigs in Freund's Complete Adjuvant (FCAT) test.

The above results are further supported by an Open Epicutaneous Test (OET) was performed on guinea pigs to assess the skin sensitization potential of test chemical. On day 1 during induction, 0.1 ml of the 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 pre-treated 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 chemical was considered to be sensitizing on skin of guinea pigs at concentration of 8% in an Open Epicutaneous Test (OET).

These results are also supported by a Draize test was conducted in Himalayan white-spotted guinea pigs (male and female) to determine the skin sensitization potential of the test chemical. In this 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. Based on all the observations and result, it was concluded that the test chemical was considered to be not sensitizing in a Draize test was conducted on Himalayan white-spotted guinea pigs.

All of the above results are also supported by a Closed Epicutaneous Tests (CET) was conducted on eight guinea pigs for test chemical 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% of the test chemical. On day 28, a 48 occluded challenge application with 1% test chemical 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 test chemical was considered to be not sensitizing to the skin of guinea pigs.

Even though, the results of some of the studies conducted on guinea pigs failed to demonstrate any dermal reactions, but majority of the studies mild to moderate dermal reactions were observed. Hence, based on the majority results, the test chemical can be considered to be sensitizing to skin and classified under the category "Category 1" as per CLP Regulation.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Even though, the results of some of the studies conducted on guinea pigs failed to demonstrate any dermal reactions, but majority of the studies mild to moderate dermal reactions were observed. Hence, based on the majority results, the test chemical can be considered to be sensitizing to skin and classified under the category "Category 1" as per CLP Regulation.