2-methylpentyl 2-hydroxybenzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05-03-2020 to 19-03-2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed under GLP. Relevant validity criteria were met with acceptable restrictions. Test method considered to cover Key Event-1 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017). The result will be considered within a weight of evidence assessment for C&L purposes.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

yes

Remarks:

All acceptance criteria were met, with the exception of the SD value of the SPCL depletion which was above the acceptability criteria for the DPRA. See 'other effects/acceptance of results' and 'Applicant's summary and conclusions' for more information.

GLP compliance:

yes

Remarks:

Guideline study performed under GLP. Relevant validity criteria were met with acceptable restrictions. Test method considered to cover Key Event-1 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017).

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

2-methylpentyl 2-hydroxybenzoate

EC Number:

850-366-8

Cas Number:

98969-19-8

Molecular formula:

C13H18O3

IUPAC Name:

2-methylpentyl 2-hydroxybenzoate

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light container flushed with nitrogen
- Other: colourless liquid

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422C – In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (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 at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.
- HPLC-PDA (UV) methodology are reported in the full study report.

PREPARATION OF TEST SOLUTIONS
- Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol and ethanol. At a concentration of 100 mM, the test item was soluble in ACN. Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in the study. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The dissolution of the test item in the SPCC and SPCL assay buffers was also evaluated by diluting the test item stock solution in the buffer-based incubation mixtures. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Preparation of the peptide/derivative stock solutions: [Synthetic Peptide Containing Cysteine (SPCC) and Synthetic Peptide Containing Lysine (SPCL)]
1. Cysteine: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.0 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
2. Lysine: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.2 mg of SPCL in 19.69 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes (and/or sonication if necessary).
- Preparation of the test chemical solutions: For both the cysteine and lysine reactivity assay 45.2 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 2.03 mL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the a clear solution being formed was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Preparation of the positive controls, reference controls and co-elution controls:
1. Cysteine: SPCC Reference Control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN. The SPCC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report). The mean peptide concentration of Reference Controls A was 0.516 ± 0.002 mM while the mean peptide concentration of Reference Controls C was 0.507 ± 0.002 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
2. Lysine: SPCL Reference Control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN. The SPLC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report). The mean peptide concentration of Reference Controls A was
0.482 ± 0.011 mM while the mean peptide concentration of Reference Controls C was 0.496 ± 0.009 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
3. Positive controls: PC samples were prepared from 750 μL ’stock solution’ of 0.667 mM SPCC or SPCL (as applicable), 250 μL Cinnamic aldehyde solution (100 mM in ACN) and (in case of Cysteine) further 200 μL ACN.

INCUBATION
- Incubation conditions: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC analysis the samples were visually inspected for precipitation. Samples that showed phase separation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.
- Precipitation noted: (i) In the Cysteine reactivity assay : upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation was observed in the co-elution control (CC) and the test item samples ; (ii) In the Lysine reactivity assay: upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation was observed in the co-elution control (CC) and the test item samples.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Yes. HPLC-PDA (UV) methodology are reported in the full study report. The concentration of SPCC or SPCL was spectrophotometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards. The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C using standard formula (see: OECD TG 442C).
- Verification of the suitability of the HPLC for test chemical and control substances: In addition to the above, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
DATA EVALUATION
- Cys and Lys peptide detection wavelength: See above. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm for both the Cysteine Reactivity Assay and Lysine Reactivity Assay, are presented in the full study report.

- ACCEPTABILITY CRITERIA:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 0.9994 and SPLC r2 = 1.0000)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 72.8% ± 0.2% and SPCL 65.0% ± 0.9%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.2% and SPCL PC : SD = 0.9%)
(iv) mean peptide concentration of Reference Controls A, C is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C reference controls: 0.516 ± 0.05 mM, and 0.507 ± 0.002 mM ; Lysine A, C reference controls: 0.482 ± 0.011 mM and 0.496 ± 0.009 mM, respectively).
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 1.3% ; Lysine: Reference Controls B and C was 2.9%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm and 258 nm was observed at the retention time of SPCC and SPLL. For the test item/A-cys samples, the mean SPCC A220/A258 area ratio was 37.88. For the test item/A-lys samples, the mean SPCL A220/A258 area ratio was 30.69. For each sample, a ratio in the range of 90% < mean area ratio of control samples < 110% gives a good indication that co-elution has not occurred.
All acceptance criteria were fulfilled for the PC (cinnamic aldehyde).
All relevant acceptability criteria were met.
- Synthetic peptides:
Cysteine- containing peptide: Ac-RFAACAA-COOH (MW=750.9) – full details on source provided in full study report.
Lysine-containing peptide: Ac-RFAAKAA-COOH (MW=775.9) – full details on source provided in full study report.
- Controls:
Positive control (PC): Cinnamic aldehyde (CAS 104-55-2; 99.1%) – full details on source provided in full study report.
Negative control (NC): Vehicle = Acetonitrile (ACN)
Evaluation of results: In accordance with OECD TG 442C – Table 1.
Test item reactivity was determined by mean peptide depletion and was rated as high, moderate, low, or minimal:
Mean peptide depletion [%] Reactivity
> 42.47 high reactivity (Positive)
> 22.62 < 42.47 moderate reactivity (Positive)
> 6.38 < 22.62 low reactivity (Positive)
< 6.38 minimal reactivity (Negative)

Results and discussion

Positive control results:

- All PC acceptability criteria were met.
- PC CYS-peptide depletion (mean): 72.8% ± 0.2% (high reactivity)
- PC LYS-peptide depletion (mean): 65.0% ± 0.9% (high reactivity)

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

mean

Parameter:

mean cystein depletion

Remarks:

Mean (%) Cys-peptide depletion

Value:

1.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: (mean ; n=3):1.1% ± 1.2% ; negative : No or minimal reactivity

Remarks:

n = 3 ; See 'any other information on results incl. tables' for further information

Results 2

Key result

Run / experiment:

mean

Parameter:

mean lysine depletion

Remarks:

Mean (%) Lys-peptide depletion

Value:

21.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: (mean ; n=2): 24.4% ± 23.0% ; positive ; moderate reactivity

Remarks:

n = 2 ; one sample excluded. See 'any other information on results incl. tables' for further information

Outcome of the prediction model:

other: See comments provided below in 'Any other information on results incl. tables' and/or 'Applicant's summary and conclusion'

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.
- Other:
(i) In the Cysteine reactivity assay : upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation was observed in the co-elution control (CC) and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.
(ii) In the Lysine reactivity assay: upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation was observed in the co-elution control (CC) and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

DEMONSTRATION OF TECHNICAL PROFICIENCY: 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 at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met , with the exception of the mean Percent SPCL Depletion variability, see comments below.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 0.9994 and SPLC r2 = 1.0000)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 72.8% ± 0.2% and SPCL 65.0% ± 0.9%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.2% and SPCL PC : SD = 0.9%)
(iv) mean peptide concentration of Reference Controls A, C is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C reference controls: 0.516 ± 0.05 mM, and 0.507 ± 0.002 mM ; Lysine A, C reference controls: 0.482 ± 0.011 mM and 0.496 ± 0.009 mM, respectively).
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 1.3% ; Lysine: Reference Controls B and C was 2.9%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm and 258 nm was observed at the retention time of SPCC and SPLL. For the test item/A-cys samples, the mean SPCC A220/A258 area ratio was 37.88. For the test item/A-lys samples, the mean SPCL A220/A258 area ratio was 30.69. For each sample, a ratio in the range of 90% < mean area ratio of control samples < 110% gives a good indication that co-elution has not occurred.
All acceptance criteria were fulfilled for the PC (cinnamic aldehyde).
All relevant acceptability criteria were met with the exception of: the mean Percent SPCL Depletion for the test item was 21.4% ± 23.0% (estimated value). The standard deviation value of the SPCL depletion was above the acceptability criterium for the DPRA as stated in the OECD 442C guideline. As this was caused by the occurrence of a phase separation in the test item samples in combination with interference of a test item related peak with SPCL, DPRA results were accepted.

Any other information on results incl. tables

Table 1.0 – Acceptability of the DPRA

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r2) standard calibration curve	>0.99	0.9994	>0.99	1.0000
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.516 ± 0.002	0.50 ± 0.05	0.482 ± 0.011
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.507 ± 0.002	0.50 ± 0.05	0.496 ± 0.009
CV (%) for RC samples B and C	<15.0	1.3	<15.0	2.9
Mean peptide depletion PC (cinnamic aldehyde) (%)	60.8-100	72.8	40.2-69.0	65.0
SD of peptide depletion PC (cinnamic aldehyde) (%)	<14.9	0.2	<11.6	0.9
SD of peptide depletion for the test item (%)	<14.9	1.2	<11.6	23.0 #

Where: RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation

 

Table 2.0 – Results of the DPRA with the test item

	SPCC depletion (CYSTEINE)		SPCL depletion (LYSINE)		Mean of SPCC and SPCL depletion
	Mean	± SD	Mean	± SD
Test item	1.1%	±1.2%	21.4% #1	±23.0%	11.3%

#1 : based on only two replicates.

 

Note:

Within the results of the lysine assay:

(1) In the CC sample no peak was observed at the retention time of SPCL. This indicated that there was no co-elution of the test item with SPCL. For the test item/A-lys samples, the mean SPCL A220/A258 area ratio was 29.42. Since this was within the 27.62-33.76 range, this again indicated that there was no co-elution of the test item with SPCL, however, in the test item/A-lys-2 and the test item/A-lys-3 samples it appeared that a test item related peak interfered with the SPCL peak at both wavelengths.

(2) As the overlap in retention time between the test item related peak and SPCL at the 220 nm wavelength was incomplete, an estimation of the Percent SPCL Depletion was made. For this estimation, the data of the test item/A-lys-3 sample was excluded from calculations as the SPCL peak area (1767491) was much higher than that of the mean of the peptide areas for the nine Reference Controls B and C (1581310). Therefore the Lysine reactivity (positive) prediction was based on only two replicates.

 

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a phase separation was observed in the test item samples. An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion were presented. In the cysteine reactivity assay the test item showed 1.1% SPCC depletion while in the lysine reactivity assay the test item showed 21.4% SPCL depletion (estimated value). The mean of the SPCC and SPCL depletion was 11.3% (estimated value) and as a result the test item was considered to be positive in the DPRA. As the overlap in retention time between the test item and SPCL was incomplete, the SPCL depletion was estimated and used in the Cysteine 1:10 / Lysine 1:50 prediction model, however, assignment to a reactivity class was not (formally) made (within the study).

 

Conclusion:

The test item gave a positive in the DPRA and was not classified into a reactivity class (formally, within the study) due phase separation being observed in the incubation period in SPCC and SPCL test item-samples.

However applicant assessment indicates using the Cysteine 1:10 / Lysine 1:50 prediction model :

(i) the Mean of SPCC and SPCL depletion was in the > 6.38 < 22.62 - predicted low reactivity (Positive) class

(ii) the estimated result for lysine is suggested as possible: > 22.62 < 42.47 - predicted moderate reactivity (Positive) class

The result will be considered within a weight of evidence assessment for Classification and Labelling purposes

Applicant's summary and conclusion

Interpretation of results:

other: The test item gave a positive in the DPRA and was not classified into a reactivity class (formally, within the study) due phase separation being observed. The result will be considered within a weight of evidence assessment for C&L purposes

Conclusions:

The test item gave a positive in the DPRA and was not classified into a reactivity class (formally, within the study) due phase separation being observed in the incubation period in SPCC and SPCL test item-samples.
However applicant assessment indicates using the Cysteine 1:10 / Lysine 1:50 prediction model :
(i) the Mean of SPCC and SPCL depletion was in the > 6.38 < 22.62 - predicted low reactivity (Positive) class
(ii) the estimated result for lysine is suggested as possible: > 22.62 < 42.47 - predicted moderate reactivity (Positive) class
The result will be considered within a weight of evidence assessment for Classification and Labelling purposes

Executive summary:

The study was performed to the OECD TG 442C in chemico Direct Peptide Reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item. The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the SPCC depletion for the test item, were all within the acceptability criteria for the DPRA. the standard deviation value of the SPCL depletion was above the acceptability criterium for the DPRA as stated in the OECD 442C guideline. As this was caused by the occurrence of a phase separation in the test item samples in combination with interference of a test item related peak with SPCL, DPRA results were accepted. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a phase separation was observed in the test item samples. An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion were presented. In the cysteine reactivity assay the test item showed 1.1% SPCC depletion while in the lysine reactivity assay the test item showed 21.4% SPCL depletion (estimated value). The mean of the SPCC and SPCL depletion was 11.3% (estimated value) and as a result the test item was considered to be positive in the DPRA. As the overlap in retention time between the test item and SPCL was incomplete, the SPCL depletion was estimated and used in the Cysteine 1:10 / Lysine 1:50 prediction model, however, assignment to a reactivity class was not made.

 

Applicant assessment indicates: the test item gave a positive in the DPRA and was not classified into a reactivity class (formally, within the study) due phase separation being observed in the incubation period in SPCC and SPCL test item-samples.

However applicant assessment indicates using the Cysteine 1:10 / Lysine 1:50 prediction model :

(i) the Mean of SPCC and SPCL depletion was in the > 6.38 < 22.62 - predicted low reactivity (Positive) class

(ii) the estimated result for lysine is suggested as possible: > 22.62 < 42.47 - predicted moderate reactivity (Positive) class

The result will be considered within a weight of evidence assessment for Classification and Labelling purposes