Registration Dossier

Administrative data

Description of key information

DPRA (OECD 442c): Not reactive

Kerationsens (OECD 442d): Negative

h-CLAT (OECD 442e): Positive

OVERALL In vitro conclusion: NEGATIVE

GPMT : Not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
8 Oct 2018 to 12 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 442c and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
Principles of method if other than guideline:
​Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP. In addition this Givaudan laboratory at Dubendorf devised the KeratinoSens testing protocol and instigated the assay acceptance with ECHA and the OECD. The Givaudan laboratory at Dubendorf also was a member of the DPRA acceptance testing program of laboratories and has over 5 years experience with both the KeratinoSens and DPRA assays.
GLP compliance:
no
Remarks:
Conducted within the "spirit" of GLP (See Principles of method if other than guideline)
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
Test material name (as stated in the report): AMYL SALICYLATE (pentyl 2-hydroxybenzoate )
Batch No.: VE00541093
Purity>=98%
Expiry date: 17 Apr 2019
Details on study design:
BASIS OF THE METHOD
Skin sensitizing chemicals have the ability to covalently modify skin proteins or to be biotically or abiotically activated to become protein-reactive. Chemical-modified proteins are recognized by the immune system as foreign and trigger a specific T-cell mediated immune response. A key step in the skin sensitization process is therefore the formation of a covalent adduct between the skin sensitizer and endogenous proteins and/or peptides in the skin. Based on this wellestablished toxicity mechanism, the most straightforward approach to predict skin sensitization involves the measurement of the reactivity of a test compound towards peptides and proteins. Gerberick et al. [2, 4] therefore developed a peptide depletion assay using different heptapeptides (later coined the DPRA or ‘direct peptide reactivity assay’) to assess a chemicals ability to react with and deplete a test peptide. Depletion is measured as the loss of the peptide signal as determined by HPLC-UV.

EXPERIMENTAL DESCRIPTION
Test System(s):
The Lys-peptide Ac-RFAAKAA is incubated at a final concentration of 0.5 mM in an ammonium acetate buffer at pH 10.5 in presence of a final level of 25% acetonitrile and in presence of a 50-fold excess of the test substance (25 mM) dissolved in acetonitrile.
The Cys-peptide Ac-RFAACAA is incubated at a final concentration of 0.5 mM in phosphate buffer at pH 7.5 in presence of a final level of 25% acetonitrile and in presence of a 10-fold excess of the test substance (5 mM) dissolved in acetonitrile.

Endpoint & Endpoint Detection:
24 h after start of the incubation the remaining peptide is quantified with HPLC-UV.

HPLC-Conditions:
▪ LC-System: Agilent 1100 Series (quat. low pressure gradient pump)
▪ Column: Zorbax SB-C18, 3μm, 2.1mm x 100mm
▪ DAD-Detector: 220nm
▪ Inj.Vol.: 7μl, Temp.: 30°C, Flow: 0.35ml/min
▪ Mobile Phase: ACN + 0.085% TFA / H2O + 1% TFA
▪ Gradient: 0min: 10% ACN / 90% H2O
10min: 25% ACN / 75% H2O
11min: 90% ACN / 10% H2O
13min: 90% ACN / 10% H2O
13.5min - 20min: 10% ACN / 90% H2O (conditioning)

Endpoint Value:
The endpoint is expressed as % peptide depletion.

Positive control:
In each test Cinnamic aldehyde (EC: 203-213-9) (purity >99%) (supplied by Aldrich) is included as positive control.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the SOP.

Prediction Model:
Based on the average peptide depletion values for both the Cysteine and the Lysine peptide, a reactivity class is attributed to the substances. Average depletion below 6.38% indicates minimal reactivity, substances in this class are predicted as non-sensitizers by the DPRA.

The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals is described in Natsch, A., Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the Givaudan testing facility. Givaudan Red Corner Report, RCR 153’453, 2015.
Positive control results:
Cinnamic aldehyde
Key result
Parameter:
other: Cys-peptide depletion
Run / experiment:
Peptide Depletion Test sample, Average (%)
Value:
1.1
Vehicle controls validity:
valid
Remarks:
Acetonitrile
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Cinnamic aldehyde
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Lys-peptide depletion
Run / experiment:
Peptide Depletion Test sample, Average (%)
Value:
0
Vehicle controls validity:
valid
Remarks:
Acetonitrile
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Cinnamic aldehyde
Remarks on result:
no indication of skin sensitisation
Remarks:
co-elution occured, Cysteine 1:10 prediction model was used to conclude
Other effects / acceptance of results:
Acceptance criteria: The standard deviation for Cys-peptide depletion should be < 14.9% and for Lys-peptide depletion < 11.6%. These criteria were fulfilled (0.3 % and 2.2% SD, respectively).
The co-elution controls indicated no co-elution with an UV-absorbing component.

The test substance gave 1.1 %depletion of the Cys-peptide. With the Lys-peptide, co-elution was observed with a shoulder between RT 6 - 6.5 min and accurate quantification is thus not possible. Still the peak of the Lys peptide at the true retention time in the original height can be observed, indicating that no Lysine peptide depletion occurred. Manuel integration confirmed this (values given in brackets in Table 4), indicating negative depletion values due to partially overlapping peaks. Since quantification is not accurate, the Cysteine 1:10 prediction model was applied as indicated in the OECD test guideline. According this prediction model, the test substance is attributed to the “minimal” reactivity class (Cysteine-peptide depletion). In this Cysteine only model, chemicals are rated as sensitizers if they lead to >13.89% depletion of the Cysteine peptide.

Interpretation of results:
GHS criteria not met
Conclusions:
The result of the DPRA assay should be used as part of an integrated approach for testing and assessment (IATA). A parallel test in the KeratinoSens™ assay may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of chemicals, two congruent results in these two tests give a good prediction of the sensitizer hazard [3-5] particularly when predicting human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.
AMYL SALICYLATE was non-reactive and classified into the minimal reactivity class according to the Cysteine 1:10 prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.
Executive summary:

The Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of test a substance towards peptides. This assay has been validated for a broad range of low-molecular weight chemicals and it was found to detect reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).

The test substance AMYL SALICYLATE was dissolved in acetonitrile and mixed with a Cysteine and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by AMYL SALICYLATE was determined by HPLC-UV.

AMYL SALICYLATE was non-reactive with the Cys-peptide. Co-elution occurred with the Lyspeptide. It was thus rated with the Cysteine 1:10 prediction model according to the guideline and classified into the minimal reactivity class. It is therefore considered a non-sensitizer according to the DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 Sep 2018 to 28 Sep 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 442d and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
Principles of method if other than guideline:
​Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP. In addition this Givaudan laboratory at Dubendorf devised the KeratinoSens testing protocol and instigated the assay acceptance with ECHA and the OECD. The Givaudan laboratory at Dubendorf also was a member of the DPRA acceptance testing program of laboratories and has over 5 years experience with both the KeratinoSens and DPRA assays.
GLP compliance:
no
Remarks:
Conducted within the "spirit" of GLP (See Principles of method if other than guideline)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Test material name (as stated in the report): Amyl Salicylate
Batch No.: VE00541093
Purity >=98%
Expiry date: 17 Apr 2019
Details on study design:
The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response.
This assay has been validated for a broad range of low-molecular weight chemicals and it was found to respond to skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA). It forms part of OECD guideline 442d.
The only feature all skin sensitizers have in common is their intrinsic electrophilicity or their potential to be metabolically transformed to electrophilic chemicals. The signaling pathway with the repressor protein Keap1(Kelch-like ECH-associated protein 1) and the transcription factor Nrf2 (nuclear factor (erythroid-derived 2)-like 2), which binds to the antioxidant / electrophile response element (ARE / EpRE), is known to respond to electrophilic chemicals and it was found to be a valuable cellular endpoint to detect skin sensitizers in vitro [10]. This result was confirmed by independent laboratories [11-16].
Cells are grown for 24 h in 96-well plates. The medium is then replaced with medium containing a final level of 1% of the solvent DMSO containing the test substance. Each compound is tested at 12 concentrations in the range from 0.98 to 2000 μM. Each test plate contains six wells with the solvent control, 1 well with no cells for background value and 5 wells with a dose response of the positive control cinnamic aldehyde. In each repetition, three parallel replicate plates are run with this same set-up, and a forth parallel plate is prepared for cytotoxicity determination.




Positive control results:
Cinnamic aldehyde
Key result
Parameter:
other: Luciferase induction: Imax (fold induction)
Remarks:
Given is the Imax values indicating maximal fold-induction up to a concentration of 1000 µM
Run / experiment:
Replicate 1
Value:
1.07
Vehicle controls validity:
valid
Remarks:
DMSO
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Cinnamic aldehyde
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Luciferase induction: Imax (fold induction)
Remarks:
Given is the Imax values indicating maximal fold-induction up to a concentration of 1000 µM
Run / experiment:
Replicate 2
Value:
0.88
Vehicle controls validity:
valid
Remarks:
DMSO
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Cinnamic aldehyde
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Luciferase induction: Imax (fold induction)
Remarks:
Given is the Imax values indicating maximal fold-induction up to a concentration of 1000 µM
Run / experiment:
Replicate 3
Value:
1.16
Vehicle controls validity:
valid
Remarks:
DMSO
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Cinnamic aldehyde
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Luciferase induction: Average Imax (fold induction)
Remarks:
Given is the Imax values indicating maximal fold-induction up to a concentration of 1000 µM
Run / experiment:
Mean of all 3 replicates (SD 0.14)
Value:
1.04
Vehicle controls validity:
valid
Remarks:
DMSO
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Cinnamic aldehyde
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This requirement was fulfilled in all three repetitions. The induction at 64 µM and the EC 1.5 for cinnamic aldehyde were also calculated. The targets are: (i) Average induction in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8, and (ii) the EC 1.5 value should be between 7 µM and 30 µM. At least one of these two numerical criteria must be met in order to accept a repetition. Both criteria were fulfilled in all three repetitions.
Thus all three repetitions were valid for the positive control.

As second performance criterion, the variability of the solvent control must be below 20%. Table 9 lists the results of the three repetitions. Two out of three repetitions were valid for the solvent control. The high variability of the blanks in rep3 was not due to a high variability within the plates but due to overall lower luciferase values in one of the three replica plates of rep3. Repetition still did pass the positive control criteria. In addition, the result is also conclusive if only considering repetition 1 and 2, therefore this deviation is considered acceptable.
Interpretation of results:
GHS criteria not met
Conclusions:
The result of the KeratinoSens™ assay should be used as part of an integrated approach for testing and assessment (IATA)[9]. A parallel test in the DPRA may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of substances, two congruent results in these two tests give a good prediction of the sensitizer hazard [5, 7, 18], in particular when comparing against human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.
In all three repetitions, no induction of the luciferase above the threshold of 1.5 was observed. According to the prediction model of the KeratinoSens™ assay, the test substance is rated as a non-sensitizer.
Executive summary:

The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response. This assay has been validated for a broad range of low-molecular weight chemicals and it was found to respond to skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA). It forms part of OECD guideline 442d.

The test substance Amyl Salicylate was dissolved in DMSO and tested according to the standard operating procedure of the KeratinoSensTM assay at 12 concentrations in three repetitions, each time in three replicates. After a 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.

Amyl Salicylate was moderately toxic to the KeratinoSens™ cells. It did not significantly induce the luciferase gene above a threshold of 1.5 in any of the three repetitions. It is therefore considered a non-sensitizer according to the prediction model of the KeratinoSens™ assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 Mar 2018 to 22 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 442e and in compliance with GLP.
Qualifier:
according to
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation Assays addressing the Key Event on Activation of Dendritic Cells on the Adverse Outcome Pathway for Skin Sensitisation : U937 cell line activation Test (U-SENS™) test method)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
Test material name (as stated in the report): AMYL SALICYLATE (pentyl 2-hydroxybenzoate )
Batch No.: VE00527069
Purity: 99.99%
Expiry date: 16 Feb 2019
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422E – In Vitro Skin Sensitisation
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442E.
- The test article was dissolved in dimethyl sulfoxide (DMSO) and a dose finding assay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75). 8 stock solutions were prepared by 1.2-fold serial dilutions using DMSO to give 8 doses ranging from 0.335 x CV75 to 1.2 x CV75. These stock solutions were then diluted 250-fold into the culture medium (working solutions).Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 cells per well. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 24±0.5 hours.
After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4°C for 15 minutes.
After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate, centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4°C for 30 minutes.
The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.
The relative fluorescence intensity (RFI) values for the test article were calculated.
- Cell line used:
The study was conducted to investigate the potential of Amyl Salicylate to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). Human monocytic leukemia cell line, THP-1 (an immortalised human monocytic leukemia cell line, used as a surrogate for dendritic cells), supplied by American Type Culture Collection (ATCC), Manassas, VA 20110 USA.

- Acceptability criteria:
1. The cell viabilities of medium and solvent control should be higher than 90%
2. In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%)
3. For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%
4. In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability
should be more than 50%
5. For the test article, the cell viability should be more than 50% in at least 4 tested doses in each run.
All assay acceptance criteria were met.

Prediction Model
An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the
h-CLAT prediction is considered NEGATIVE:
• The RFI of CD86 is ≥150% at any tested concentration (with cell viability ≥50%)
• The RFI of CD54 is ≥200% at any tested concentration (with cell viability ≥50%).


Positive control results:
DNCB
Key result
Parameter:
other: EC150
Remarks:
CD 86 (μM)
Run / experiment:
Test item
Value:
34.79
Vehicle controls validity:
valid
Remarks:
DMSO
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
DNCB
Key result
Parameter:
other: EC200
Remarks:
CD54 (μM)
Run / experiment:
Test item
Value:
35.81
Vehicle controls validity:
valid
Remarks:
DMSO
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
DNCB
Other effects / acceptance of results:
In both experiments, RFI values >150% were obtained for CD86 and values >200% were obtained for CD54. The test article therefore gave a positive prediction in this assay.
The EC150 for CD86 was 34.79 μg/mL and the EC200 for CD54 was 35.81 μg/mL.
The test article, Amyl Salicylate, was considered to give a positive prediction in the human Cell Line Activation Test.

Acceptability criteria:
1. The cell viabilities of medium and solvent control should be higher than 90%
2. In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%)
3. For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%
4. In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability
should be more than 50%
5. For the test article, the cell viability should be more than 50% in at least 4 tested doses in each run.
All assay acceptance criteria were met.
Interpretation of results:
study cannot be used for classification
Remarks:
Need a weight of evidence with other key event in vitro investigation to allow an overall conclusion
Conclusions:
The test article, Amyl Salicylate, was considered to give a positive prediction in the human Cell Line Activation Test.
Executive summary:

The study was conducted to investigate the potential of Amyl Salicylate to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test article was dissolved in dimethyl sulfoxide (DMSO) and a dose finding assay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75).

Eight stock solutions were prepared by 1.2-fold serial dilutions using DMSO to give eight doses ranging from 0.335 x CV75 to 1.2 x CV75. These stock solutions were then diluted 250-fold into the culture medium (working solutions).

Aliquots of 500 μL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 cells per well. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 24±0.5 hours.

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 μL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4°C for 15 minutes.

After blocking, the cells were split into three aliquots of 180 μL into a 96-well plate, centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4°C for 30 minutes.

The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

In both experiments, RFI values >150% were obtained for CD86 and values >200% were obtained for CD54. The test article therefore gave a positive prediction in this assay.

The EC150 for CD86 was 34.79 μg/mL and the EC200 for CD54 was 35.81 μg/mL.

The test article, Amyl Salicylate, was considered to give a positive prediction in the human Cell Line Activation Test.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11.06.80- 12.11.81
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards and acceptable for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
according to Magnusson, B. and Kligman, A.M. 1970. Allergic Contact Dermatitis in the Guinea Pig: Identification of Contact Allergens. C.C. Thomas, Springfield, Illinois, U.S.A
Principles of method if other than guideline:
Sensitisation was induced in guinea pigs by intradermal injections of both test substance and Complete Freunds Adjuvant and the induction process supplemented 7 days later by test substance applied to the shoulder injection sites under occluded patch: further challenges were made at weekly intervals as required.
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
LLNA not available at time of testing
Specific details on test material used for the study:
- Name of test material (as cited in study report): amyl salicylate
- Structural formula attached as image file (if other than submission substance): see Fig.
Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Environmental Safety Division
- Weight at study initiation: approximately 344 g- 364 g
- Water (e.g. ad libitum): ad libitum
Route:
intradermal and epicutaneous
Vehicle:
other: Injection induction: 0.01 % dobs/ saline. Application induction and application challenge: acetone.
Concentration / amount:
Induction (intradermal injection): 1%
Route:
intradermal and epicutaneous
Vehicle:
other: Injection induction: 0.01 % dobs/ saline. Application induction and application challenge: acetone.
Concentration / amount:
Induction (covered patch application): 40%
Route:
epicutaneous, occlusive
Vehicle:
other: Injection induction: 0.01 % dobs/ saline. Application induction and application challenge: acetone.
Concentration / amount:
challenge (covered patch application): 10%
No. of animals per dose:
6 males and 4 females
Details on study design:
RANGE FINDING TESTS: Animals were treated by intradermal injections in the shoulder region to induce sensitisation and 7 days later the sensitisation was boosted by an occluded patch placed over the injection site. Fourteen days later the animals were challenged on 1 flank by occluded patch. Seven days after this a further confirmatory challenge was given on the opposite flank using the same method.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2, intradermal injection followed 1 week later by topical application
- Exposure period: topical application 48 hrs
- Test groups: 1
- Control group: 2 types of control groups are used. Treated controls: 4 animals of the same sex are selected and treated controls for the first challenge. 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. Untreated control: 4 previously untreated animals of the same sex and weighing approximately the same as the test animals.
- Site: 2x4 cm are in the dorsal shoulder region.
- Frequency of applications: Intradermal injection: 3 pairs of intradermal injections are made within the site as follows (i) two 0.1 mL injections of 50 % Freund's complete adjuvant (FCA) in the solvent chosen for the test substance. (ii) two 0.1 mL injections of test substance at the concentration selected for the induction from the preliminary irritation test. (iii) two 0.1 mL injections of the test substance in solvent mixed 50/50 with FCA such that the final concentration of test substance injected is the same as that in (ii). Topical application: 1 week after the injections the same 2x4 cm area was clipped and shaved. A 2x4 cm filter paper patch attached by double-sided adhesive tape to a 4x6 cm piece of thin polythene was saturated with test substance and placed over the shaved site. The patch was held in place by adhexive plaster wrapped around the trunk behind the forelimbs.
- Duration: 13 to 14 days
- Concentrations: intradermal injection 1 %, covered patch application 40 %

B. CHALLENGE EXPOSURE
- No. of exposures: 3
- Day(s) of challenge:
- Exposure period: 24 hrs
- Test groups: 1
- Control group: Treated controls: At first challenge they are treated in exactly the same way as the test animals. Untreated controls: At every challenge in the test are treated in exactly the same way as the test animals.
- Site: flank. For each animal, an 8 mm diameter filter paper patch in am 11 mm aluminium patch test cup was saturated with the test substance at the selected challenge concentration and applied to the site. The patch was held in place by adhesive plaster wound around the trunk.
- Concentrations: 10 %
- Evaluation (hr after challenge): 24 and 48 hrs
Positive control substance(s):
no
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
4 animals were noted to have barely perceptible erythema.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
1 animal was noted to have barely perceptible erythema.
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
1 animal was noted to have spots of erythema.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
other: Challenge 3
Hours after challenge:
24
Group:
test group
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
other: Challenge 3
Hours after challenge:
48
Group:
test group
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: Treated controls
Dose level:
0
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
3 animals were noted to have barely perceptible erythema.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
other: Treated control
Dose level:
0
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
2 animals were noted to have barely perceptible erythema.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: Untreated control
Dose level:
0
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
1 animal was noted to have barely perceptible erythema.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
other: Untreated control
Dose level:
0
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
1 animal was noted to have barely perceptible erythema.
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
other: Untreated control
Dose level:
0
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
1 animal was noted to have barely perceptible erythema.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Untreated control
Dose level:
0
No. with + reactions:
0
Total no. in group:
4
Key result
Reading:
other: Challenge 3
Hours after challenge:
24
Group:
other: Untreated controls
Dose level:
0
No. with + reactions:
0
Total no. in group:
4
Key result
Reading:
other: Challenge 3
Hours after challenge:
48
Group:
other: Untreated control
Dose level:
0
No. with + reactions:
0
Total no. in group:
4
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
0
Clinical observations:
Negative controls were performed as 2 groups: Treated and untreated controls (see further for more details)
Remarks on result:
not measured/tested
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0
No. with + reactions:
0
Total no. in group:
0
Clinical observations:
A positive control was not reported
Remarks on result:
not measured/tested
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was assessed for skin sensitisation according to the method described in Magnusson and Kligman (1970). Sensitisation was induced in guinea pigs by intradermal injections and supplemented 7 days later by an occluded patch. Three challenges were then given by occluded patch. None of the animals sensitised after 3 challenges and so the test substance is not considered to be a sensitiser by this method.
Executive summary:

Male and female Dunkin-Hartley guinea pigs were purchased from Environmental Safety Division. A preliminary, dose range-finding study was conducted on 4 male guinea pigs weighing 360 g- 428 g with the test substance being administered via intradermal injection. A preliminary, dose range-finding study was conducted on 4 male guinea pigs weighing 454 g- 546 g with the test substance being administered via an occluded patch.

The main study was conducted using 1 group of 10 guinea pigs, composed of 6 males and 4 females. Sensitisation was induced by intradermal injections and supplemented 7 days later by an occluded patch at dose levels 1 % and 40 % respectively. Three challenges were then given by occluded patch at the dose level 10 %. None of the animals sensitised after 3 challenges and so the test substance is not considered to be a sensitiser by this method.

All animals were observed at 24 and 48 hours after each challenge; reactions were noted.

There were no positive results for sensitization after 3 challenges. Four animals were noted to have barely perceptible erythema at 24 hours after the first challenge. One animal was noted to have barely perceptible erythema at 48 hours after the first challenge. One animal was noted to have barely perceptible erythema at 24 hours after the second challenge.

None of the animals sensitised after 3 challenges and so the test substance is not considered to be a sensitiser by this method.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In chemico/vitro skin sensitization:

DPRA (OECD 442c):

The Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of test a substance towards peptides. This assay has been validated for a broad range of low-molecular weight chemicals and it was found to detect reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).

The test substance AMYL SALICYLATE was dissolved in acetonitrile and mixed with a Cysteine and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by AMYL SALICYLATE was determined by HPLC-UV.

AMYL SALICYLATE was non-reactive with the Cys-peptide. Co-elution occurred with the Lyspeptide. It was thus rated with the Cysteine 1:10 prediction model according to the guideline and classified into the minimal reactivity class. It is therefore considered a non-sensitizer according to the DPRA.

Keratinosens (OECDD 442d):

The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response. This assay has been validated for a broad range of low-molecular weight chemicals and it was found to respond to skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA). It forms part of OECD guideline 442d.

The test substance Amyl Salicylate was dissolved in DMSO and tested according to the standard operating procedure of the KeratinoSensTM assay at 12 concentrations in three repetitions, each time in three replicates. After a 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.

Amyl Salicylate was moderately toxic to the KeratinoSens™ cells. It did not significantly induce the luciferase gene above a threshold of 1.5 in any of the three repetitions. It is therefore considered a non-sensitizer according to the prediction model of the KeratinoSens™ assay.

h-CLAT (OECD 442e):

The study was conducted to investigate the potential of Amyl Salicylate to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the

purpose of hazard classification and labelling.

The test article was dissolved in dimethyl sulfoxide (DMSO) and a dose finding assay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75).

Eight stock solutions were prepared by 1.2-fold serial dilutions using DMSO to give eight doses ranging from 0.335 x CV75 to 1.2 x CV75. These stock solutions were then diluted 250-fold into the culture medium (working solutions).

Aliquots of 500 μL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 cells per well. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 24±0.5 hours.

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 μL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4°C for 15 minutes.

After blocking, the cells were split into three aliquots of 180 μL into a 96-well plate, centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4°C for 30 minutes.

The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

In both experiments, RFI values >150% were obtained for CD86 and values >200% were obtained for CD54. The test article therefore gave a positive prediction in this assay.

The EC150 for CD86 was 34.79 μg/mL and the EC200 for CD54 was 35.81 μg/mL.

The test article, Amyl Salicylate, was considered to give a positive prediction in the human Cell Line Activation Test.

In vivo skin sensitization:

A Magnusson and Kligman maximisation study in the guinea pig was conducted. The study has been scored a klimisch rating of 2.

Male and female Dunkin-Hartley guinea pigs were purchased from Environmental Safety Division. A preliminary, dose range-finding study was conducted on 4 male guinea pigs weighing 360 g- 428 g with the test substance being administered via intradermal injection. A preliminary, dose range-finding study was conducted on 4 male guinea pigs weighing 454 g- 546 g with the test substance being administered via an occluded patch.

The main study was conducted using 1 group of 10 guinea pigs, composed of 6 males and 4 females. Sensitisation was induced by intradermal injections and supplemented 7 days later by an occluded patch at dose levels 1 % and 40 % respectively. Three challenges were then given by occluded patch at the dose level 10 %. None of the animals sensitised after 3 challenges and so the test substance is not considered to be a sensitiser by this method.

All animals were observed at 24 and 48 hours after each challenge; reactions were noted.

There were no positive results for sensitization after 3 challenges. Four animals were noted to have barely perceptible erythema at 24 hours after the first challenge. One animal was noted to have barely perceptible erythema at 48 hours after the first challenge. One animal was noted to have barely perceptible erythema at 24 hours after the second challenge.

None of the animals sensitised after 3 challenges and so the test substance is not considered to be a sensitiser by this method.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Amyl Salicylate was found not to possess any skin sensitization potential in vitro (DPRA, Keratinosens and h-CLAT weight of evidence) or in vivo (GPMT).

Based on the weight of evidence of the data available on Amyl Salicylate, no classification is necessary according to the (EC) No 1272/2008 Regulation (CLP).