2-(2H-benzotriazol-2-yl)-4-tert-butylphenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 02 January 2018 Experimental completion date: 05 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

2-(2H-benzotriazol-2-yl)-4-tert-butylphenol

EC Number:

221-574-0

EC Name:

2-(2H-benzotriazol-2-yl)-4-tert-butylphenol

Cas Number:

3147-76-0

Molecular formula:

C16H17N3O

IUPAC Name:

2-(2H-1,2,3-benzotriazol-2-yl)-4-tert-butylphenol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 02002IX3
- Expiration date of the lot/batch: 25 February 2018
- Purity: 99.9%

In chemico test system

Details on the study design:

Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Stability Controls and Precision Controls
Stability controls (Reference Control B) and precision controls (Reference control A) of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution in acetonitrile of the test item was prepared.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Acetonitrile solutions of test item and solutions of the positive control were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of either test item or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co elution Controls
Acetonitrile solutions of test item and solutions of the positive control were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either the test item or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of test item and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

Positive control depletion (%):
Cysteine - 70.5 (SD, 0.31%, n=3)
Lysine - 57.4 (SD, 1.10%, n=3)

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

-1.26 %

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

-1.07 %

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All analytical acceptance criteria for each peptide run were met.

Any other information on results incl. tables

Solubility Assessment

The solubility of the test item in acetonitrile at a nominal concentration of 100 mM was confirmed.

 

Reactivity Assessment

All analytical acceptance criteria for each peptide run were met:

 

		Peptide		Standard Linearity		Positive control depletion (%)		Reference controls		Test item
Acceptance criteria		Cysteine		r^2>0.99		60.8-100 (SD <14.9%)		0.45-0.55 mM (CV <15%)		SD <14.9%
		Lysine		r^2>0.99		40.2-69.0 (SD <11.6%)		0.45-0.55 mM (CV <15%)		SD <11.6%
Achieved results		Cysteine		r^2>0.999		70.5 (SD, 0.31%, n=3)		B: 0.501 mM (CV 0.38%, n=6)		SD 0.77% (n=3)
		Lysine		r^2>0.999		57.4 (SD, 1.10%, n=3)		B: 0.503 mM (CV 0.24%, n=6)		SD 0.53% (n=3)

CV         Coefficient of Variation

SD         Standard deviation

 

The depletion of peptide in the presence of the test item was:

	Mean peak area of reference control(µV.sec)	Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by test item (%)
Cysteine	Control B: 826900 (n=6)	837310 (n=3)	-1.26
Lysine	Control B: 782410 (n=6)	790810 (n=3)	-1.07

 

And applying the following depletion model (below), reactivity is classed as no to minimal and the DPRA prediction is positive and the test item is therefore a potential non-sensitizer. 

Mean of Cysteine and Lysine% depletion	Reactivity Class	DPRA Prediction
0%≤ mean% depletion ≤6.38%	No or minimal reactivity	Negative
6.38%< mean% depletion ≤22.62%	Low reactivity	Positive
22.62%< mean% depletion ≤42.47%	Moderate reactivity
42.47%< mean% depletion ≤100%	High reactivity

 

There was no co-elution peaks in either of the Lysine or Cysteine assays.   

 

Overall Achieved Depletion Values

Test item		Cysteine peptide depletion (%)		Lysine peptide depletion (%)		Overall mean depletion (%)		Reactivity class		DPRA prediction
2-(2H-benzotriazol-2-yl)-4-tert-butylphenol		0.00^1		0.00^1		0.00		No to minimal		Negative

¹          Negative results count as zero

 

Individual Achieved Depletion Values

Cysteine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion^2(%)	Mean Depletion (%)	SD  (%)
Positive control	240703	107.98	70.9	70.5	0.31
	245298	110.08	70.3
	245038	109.96	70.4
test item	835526	380.40	-1.04	-1.26	0.77
	844419	384.47	-2.12
	831985	378.78	-0.615

SD      Standard Deviation

¹                Samples prepared at a concentration of 376 µg/mL (0.5 mM)

²                Calculated against a mean Reference Control area of 826900 µV.sec(n=6)

 

Lysine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion^2(%)	Mean Depletion (%)	SD  (%)
Positive control	325235	162.29	58.4	57.4	1.10
	342386	170.87	56.2
	333203	166.28	57.4
test item	793690	396.63	-1.44	-1.07	0.53
	792700	396.14	-1.32
	786045	392.81	-0.465

SD      Standard Deviation

¹                Samples prepared at a concentration of 388 µg/mL (0.5 mM)

²          Calculated against a mean Reference Control area of 782410 µV.sec(n=6)

 

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Solutions of the test item were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. No depletion in either peptide places the test item in the reactivity class of “no to minimal” and hence it is predicted by DPRA to be a potential non-skin sensitizer.