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

Endpoint summary

Description of key information

 

Summary

This assessment addresses the requirements of conducting skin sensitization assessment for the substance according to Annex VII section 8.3 of the REACH Regulation (Regulation (EC) No 1907/2006 and Commission Regulation (EU) 2016/1688).

The assessment of physico-chemical properties of the substance showed that substance cannot readily undergo dermal penetration. However, none of specified conditions in column 2 of Annex VII section 8.3 of the REACH Regulation was fulfilled to exclude the substance from the skin sensitization testing. A weight of evidence of the assessed (Q)SAR models suggested that the substance does not show the skin sensitization.

Following the Guidance for skin sensitization (IATA No 256, 2016), this assessment discussed in some extent chemical structure/properties and molecular initiating event (MIE). However, to confirm the assessed result and to cover the remaining key events at the cellular level, further in chemico and in vitro testing of the substance using OECD test methods 442C, 442D and 442E was considered.

From the (Q)SAR and in chemico assessments the result was consistent and showed that the substance was not sensitizer. In addition, the absence of the possible skin metabolites in the OECD QSAR Toolbox confirmed that the in chemico assessment (DPRA, OECD 441C) supplied reliable data on the test item, which was likely exempted from the pre- and pro-hapten sensitizing properties. Therefore, it can be concluded that the substance is likely not a skin sensitizer and no further testing will be required.

 

 

Introduction

2-(benzotriazol-2-yl)-4-tert-butylpheno (IUPAC name)l, CAS: 3147-76-0, EC: 221-57-4) is a chemical currently preregistered by the European Chemicals Agency (ECHA).

According to Annex VII (Regulation (EC) No 1907/2006) and the testing strategies for the 2018 registration deadline (ECHA Guidance, Chapter R.7a, 2016), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step.

Before to start any new testing an assessment of all available in vitro data, in vivo data, historical human data, data from valid (Q)SARs and data from structurally related substances (read-across approach) is requested. The screened PROQUEST database gateway services are related with the substance identification, physicochemical and toxicological properties.

Testing does not need to be conducted if the conditions specified in column 2 of Annex VII section 8.3 of the REACH Regulation and include:

a) the available information indicate the substance should be classified for skin corrosion or skin sensitisation, or

b) substance is a strong acid (pH< 2.0) or base (pH> 11.5), or

c) substance is (spontaneously) flammable in air at room temperature.

It is important to ensure that the chosen non-animal test methods (e.g.in vitro,in chemicoorin silico) are suitable for the substance in order to obtain adequate information. For example there may be limitations such as low solubility or log K_owof the test substance, that would hinder the use of a particularin vitromethod. The main limitations of thein chemico or in vitro methods are related to the absence of, or limited, metabolic capacity of the test system and hence pre- and pro-haptens (chemicals activated by auto oxidation or chemicals requiring enzymatic activation to exert their sensitisation activity, respectively) may not be correctly identified and therefore, in the case of a negative outcome the prediction may be a false negative. When the non-animal testing methods are used to fulfil the Annex VII information requirement for skin sensitisation, a case-specific justification should be provided on why and how thein vitrodata, taken within a weight of evidence approach, can be used to cover the REACH information requirement. In that weight of evidence justification, e.g. coverage of the key events (see above), quality and reliability of data (positive evidence has more weight that the negative), limitations and the scope of each test methods used, and consistency of the results need to be considered (ECHA document 10162).

In vivo methods can only be used if the in chemico or in vitro test methods are not adequate for the substance or cannot be used for classification and risk assessment. On the other hand, ECHA reminds that with the amended requirements, if a substance is predicted to be a skin sensitizer based on the available data, skin sensitisation potency should also be assessed. There is currently no standardised way to assess potency with the in vitro methods and therefore thein vivotest may still be necessary (ECHA/NI/16/32). The potency estimation may be exempted if an existing in vivo study does not allow potency estimation and the study has been performed according to internationally-adopted test methods and good laboratory practice.

Based on ECHA approach (ECHA Guidance, Chapter R.7a, 2016) the testing strategy was discussed. This includes structural considerations, physico-chemical properties, check for exclusion conditions specified in Annex VII section 8.3, (Q)SAR ((Quantitative) Structure-Activity Relationship), information from structurally similar substances, in vitro/in chemico data, animal studies and human data.

 

 

Identification                CAS: 3147-76-0, EC: 221-57-4

Molecular weight:        267.33 g/mol

pH:                              6

Log P (o/w):                6.15

Water solubility:           2.63 mg/L

Flammability:              not spontaneously flammable

Acute oral toxicity         > 2 000 mg/kg

Skin corrosion:           non-corrosive

Skin irritation:             not irritant (client and SDS) but irritant Category 2 (CLP inventory)

Eye irritation:               irritant Category 2 (CLP inventory)

 

 

Appraisal of available data

The substance is a phenolic benzotriazole with low molecular weight (267.33 g/mol) solid compound at room temperature poorly soluble in water with a lipophilicity (log P = 6.15). Although the substance is small, it is expected that it would be poorly absorbed through the skin since its log P value is high and its solubility in water poor.

It is not corrosive to the skin and not spontaneously flammable liquid; therefore, the compound may not be exempted from the skin sensitization testing based on the listed conditions in the column 2 of Annex VII section 8.3 of the REACH Regulation.

From ECHA compiled inventory of substances likely to meet the criteria of Annex III to the REACH Regulation, it is a suspected respiratory sensitizer but not a suspected skin sensitizer (ECHA website).

The substance is a member of the phenolic benzotriazole family (ECCC Monograph, 2016).

 

There is sensitization data on two of the category members. However, one was found as negative and the other as positive in guinea pig maximization tests.

From the databases searched via PROQUEST database gateway services no additional relevant information on the skin sensitization properties of the substance have been found. Therefore, as the next step, the in silico assessment was performed.

 

Appraisal of (Q)SAR Modelling

The prediction of the skin sensitisation potential of 2-(2H-benzotriazol-2-yl)-tert-butylphenol (CAS 3147-76-0) was performed with BIOVIA Discovery Studio (TOPKAT) 4.5, VEGA NIC 1.1.4 (CAESAR), OECD QSAR Toolbox 4.0, Toxtree 2.6.13 and DEREK Nexus 5.0.2. The TOPKAT model for skin sensitisation was used in the extended modus including data from the Envigo database. In addition, the two analogues with sensitization data were also included in the training set. The results are summarised below.

 

Skin sensitization modelling of 2-(2H-benzotriazol-2-yl)-tert-butylphenol (CAS: 3147-76-0)

Model	Prediction result	Reliability (model statistics)
DEREK	No alert	Not applicable
CAESAR (VEGA)	Sensitising	Not reliable, out of domain
TOPKAT	Not sensitising	Reliable
OECD QSAR Toolbox	Not sensitising	Reliable (confidence p=0.0313)
Toxtree	No alert	Not applicable
Danish QSAR Database	Inconclusive	Not reliable (inconclusive, out of domain)

The query structure did not trigger an alert for skin sensitisation in DEREK. The absence of an alert in DEREK should however not be equated with the absence of any skin sensitising properties of the query structure. It only shows that not any of the expert rules implemented were triggered by the structure.

The positive prediction with CAESAR is characterised by several uncertainties (see, QPRF) and therefore considered to be not reliable.

TOPKAT predicted the query structure to be not sensitising. Significant prediction statistics and considerations on similar structures indicate high confidence and the prediction is therefore considered to be reliable.

Read-across with OECD QSAR Toolbox is based on five category members representing all functional groups of the query structure, triggering no alert for skin sensitising properties, and all having the same experimental value as predicted for the query structure. The prediction is therefore considered to be reliable. In addition, the skin metabolism simulator of OECD QSAR Toolbox did not create any metabolite, indicating the absence of potentially relevant metabolites.

No alert for skin sensitisation reactivity domains was triggered in Toxtree.

The predictions from the Danish QSAR Database taking into account the results from three models are considered to be inconclusive.

With two reliable predictions, the absence of any alert in Toxtree and DEREK and no indication on skin sensitising metabolites there is a strong argument for a weight of evidence that the substance can be considered to be not sensitizing.

 

 

In chemico/vitroassessment

In order to confirm the assessedin silicoresult and to cover the remaining key events at the cellular level, furtherin chemicoandin vitrotesting of 2-(2H-benzotriazol-2-yl)-tert-butylphenol using OECD test methods 442C, 442D and 442E was considered.

The performed DPRA showed minimal reactivity of 2-(2H-benzotriazol-2-yl)-tert-butylphenol in the peptides (OECD 441C), therefore the test item was concluded from this assay to be not sensitizer (Envigo Ref. VD51KX).

The substance was of limited use in respect to the OECD 442D KeratinoSens™ and OECD 442E h-CLAT assays as the log P value 6.15 of the substance was out of applicability domain of these in vitro tests and therefore they were not recommended.

 

 

 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

According to Annex VII (Regulation (EC) No 1907/2006) and the testing strategies for the 2018 registration deadline (ECHA Guidance, Chapter R.7a, 2016), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step.

Qualifier:

according to guideline

Guideline:

other: ECHA Guidance, Chapter R.7a, 2016.

GLP compliance:

no

Specific details on test material used for the study:

SMILES
CC(C)(C)c1ccc(O)c(c1)-n1nc2ccccc2n1
InChI
1S/C16H17N3O/c1-16(2,3)11-8-9-15(20)14(10-11)19-17-12-6-4-5-7-13(12)18-19/h4-10,20H,1-3H3

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Predicted values (model result): Sensitiser

Predicted values (comments): According to VEGA's evaluation scheme the prediction may be not reliable.

Input for prediction: Smiles

Descriptor values: Not provided

Applicability domain (OECD Principle 3)

Domains:

i. Predicted compound is out of the applicability domain of the model.

ii. Three unknown fragments and two infrequent fragments found.

iii. Considerations on the mechanism domain are not applicable since statistical model.

Structural analogues

i. CAS: 1675-54-3

ii. SMILES: O=S(=O)(NCCN(c1ccc(N)c(c1)C)CC)C

iii. SMILES: O=C(c1cc(ccc1C)C)CC(=O)c2cc(ccc2C)C

iv. CAS: 55302-96-0

Consideration on structural analogues

With 72% the average similarity of the four most similar analogues to the query structure is considered high. One out of four structures is a non-sensitiser thus indicating moderate concordance with the prediction for the query structure. Accuracy between predicted and actual result is moderate as one was predicted false-positively. Not all functional groups of the query structure are represented by the analogues.

The uncertainty of the prediction (OECD principle 4)

Uncertainty may be indicated due to moderate concordance and accuracy with regard to the four most similar structures and several fragments of the query structure not found or found too infrequently in the structures of the training set. Taking further into account that three out of six similar structures provided by CAESAR are non-sensitiser, concordance with the predicted result for the query structure is even worse (i.e., low).

The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5)

Not applicable since statistical model

Adequacy (Optional)

Regulatory purpose

Skin sensitisation endpoint for assessing the skin sensitisation potential with in vitro and in silico methods according to ECHA Guidance, Chapter R.7a, 2016.

Approach for regulatory interpretation of the model result

Result is directly applicable since no conversion of the result is required.

Outcome

Sensitiser. Due to many uncertainties there is no confidence in the prediction.

Interpretation of results:

other: predicted sensitiser

Conclusions:

Outcome
Sensitiser. Due to many uncertainties there is no confidence in the prediction.

Conclusion
The prediction is considered to be not reliable.

Reference 2

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

According to Annex VII (Regulation (EC) No 1907/2006) and the testing strategies for the 2018 registration deadline (ECHA Guidance, Chapter R.7a, 2016), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step.

Qualifier:

according to guideline

Guideline:

other: ECHA Guidance, Chapter R.7a, 2016

GLP compliance:

no

Specific details on test material used for the study:

SMILES CC(C)(C)c1ccc(O)c(c1)-n1nc2ccccc2n1
InChI 1S/C16H17N3O/c1-16(2,3)11-8-9-15(20)14(10-11)19-17-12-6-4-5-7-13(12)18-19/h4-10,20H,1-3H3

Remarks on result:

other: Inconclusive prediction

See attachment

Interpretation of results:

other: Inconclusive

Conclusions:

The prediction from the Danish QSAR Database was inconclusive

Reference 3

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

According to Annex VII (Regulation (EC) No 1907/2006) and the testing strategies for the 2018 registration deadline (ECHA Guidance, Chapter R.7a, 2016), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step.

Qualifier:

according to guideline

Guideline:

other: ECHA Guidance, Chapter R.7a, 2016

GLP compliance:

no

Specific details on test material used for the study:

SMILES CC(C)(C)c1ccc(O)c(c1)-n1nc2ccccc2n1
InChI 1S/C16H17N3O/c1-16(2,3)11-8-9-15(20)14(10-11)19-17-12-6-4-5-7-13(12)18-19/h4-10,20H,1-3H3

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

See attachment

Interpretation of results:

other: predicted non-sensitiser

Conclusions:

No alert for protein binding for skin sensitisation

Reference 4

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

According to Annex VII (Regulation (EC) No 1907/2006) and the testing strategies for the 2018 registration deadline (ECHA Guidance, Chapter R.7a, 2016), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step.

Qualifier:

according to guideline

Guideline:

other: ECHA Guidance, Chapter R.7a, 2016

GLP compliance:

no

Specific details on test material used for the study:

SMILES CC(C)(C)c1ccc(O)c(c1)-n1nc2ccccc2n1
InChI 1S/C16H17N3O/c1-16(2,3)11-8-9-15(20)14(10-11)19-17-12-6-4-5-7-13(12)18-19/h4-10,20H,1-3H3

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

See attachment

Interpretation of results:

other: predicted non-sensitiser

Conclusions:

The query structure did not trigger an alert for skin sensitisation in DEREK.

Reference 5

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

According to Annex VII (Regulation (EC) No 1907/2006) and the testing strategies for the 2018 registration deadline (ECHA Guidance, Chapter R.7a, 2016), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step.

Qualifier:

according to guideline

Guideline:

other: ECHA Guidance, Chapter R.7a, 2016

GLP compliance:

no

Specific details on test material used for the study:

SMILES CC(C)(C)c1ccc(O)c(c1)-n1nc2ccccc2n1
InChI 1S/C16H17N3O/c1-16(2,3)11-8-9-15(20)14(10-11)19-17-12-6-4-5-7-13(12)18-19/h4-10,20H,1-3H3

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Predicted values (model result): Non-Sensitiser

Predicted values (comments): A Bayesian score of -17.31 being well below the best split value of -1.054 and a probability of <0.01 suggest high confidence in the prediction.

Input for prediction: Smiles

 

Descriptor values

Descriptor Value LogP 4.33 Molecular weight (g/mol) 267.326 Number of hydrogen bond donors 1 Number of hydrogen bond acceptors 3 Number of rotatable bonds in the molecule 2 The fraction of polar surface area over the total molecular surface area 0.176 FCFP_12: Unit functional class extended-connectivity atom type fingerprint with a maximum length of 12 bonds Not applicable

Applicability domain (OECD Principle 3)

Domains:

i. All properties and OPS components are within expected ranges

ii. All fingerprint features of the query molecule are found in the training set.

iii. Considerations on the mechanism domain are not applicable since the contributing FCFP_12 features of the model are selected purely on their Bayesian score (statistical model)

Structural analogues:

i. Drometizole

ii. Homosalate

iii. Sudan I

iv. CAS 25973-55-1

Consideration on structural analogues:

With 52% the average similarity of the four analogues to the query structure is considered moderate. All four similar structures are non-sensitiser thus indicating high concordance with the prediction for the query structure. Accuracy between predicted and actual result is high as all four structures were predicted correctly.

The uncertainty of the prediction (OECD principle 4):

Significant prediction statistics, high concordance between the experimental results of similar structures and the prediction for the query structure and high accuracy in the prediction of these structures suggest high confidence in the prediction.

The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5)

Not applicable since statistical model

Adequacy (Optional)

Regulatory purpose

Skin sensitisation endpoint for assessing the skin sensitisation potential with in vitro and in silico methods according to ECHA Guidance, Chapter R.7a, 2016.

Approach for regulatory interpretation of the model result

Result is directly applicable since no conversion of the result is required.

Interpretation of results:

other: predicted non-sensitiser

Conclusions:

Outcome
There is no evidence of a skin sensitising potential of the query structure. The prediction is characterised by significant statistics (p value, Bayesian score, enrichment), high concordance between the experimental results of similar structures and the prediction for the query structure, high accuracy in the prediction of these structures, and therefore considered reliable.

Conclusion
The prediction will be used together with predictions from other model in a weight of evidence conclusion.

Reference 6

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

According to Annex VII (Regulation (EC) No 1907/2006) and the testing strategies for the 2018 registration deadline (ECHA Guidance, Chapter R.7a, 2016), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step.

Qualifier:

according to guideline

Guideline:

other: ECHA Guidance, Chapter R.7a, 2016

GLP compliance:

no

Specific details on test material used for the study:

SMILES CC(C)(C)c1ccc(O)c(c1)-n1nc2ccccc2n1
InChI 1S/C16H17N3O/c1-16(2,3)11-8-9-15(20)14(10-11)19-17-12-6-4-5-7-13(12)18-19/h4-10,20H,1-3H3

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Predicted values (model result)

No alert skin sensitisation reactivity domains

Predicted values (comments)

Not applicable

Input for prediction

Smiles

Descriptor values

Not applicable

Applicability domain (OECD Principle 3)

Domains

Not applicable. The applicability domain of each alert is defined by its modulating factors.

Structural analogues

Not applicable

Consideration on structural analogues

Not applicable

3.4

The uncertainty of the prediction (OECD principle 4)

Not applicable

The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5)

The alerts were derived from existing mechanistic knowledge, and not through data mining algorithms. No alerts were fired.

Adequacy (Optional)

Regulatory purpose

Skin sensitisation endpoint for assessing the skin sensitisation potential with in vitro and in silico methods according to ECHA Guidance, Chapter R.7a, 2016. 4.2 Approach for regulatory interpretation of the model result Result is directly applicable since no conversion of the result is required.

Interpretation of results:

other: predicted non-sensitiser

Conclusions:

Outcome
No alert for skin sensitisation reactivity domains was triggered by the query structure.

Conclusion
The prediction will be used together with predictions from other model in a weight of evidence conclusion.

Reference 7

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

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)

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%

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

Positive control results:

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

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

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.

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)

 

 

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.

Reference 8

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Remarks:

n silico sensitisation assessment

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

According to Annex VII (Regulation (EC) No 1907/2006) and the testing strategies for the 2018 registration deadline (ECHA Guidance, Chapter R.7a, 2016), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step.

Qualifier:

according to guideline

Guideline:

other: Annex VII section 8.3 of the REACH Regulation (Regulation (EC) No 1907/2006 and Commission Regulation (EU) 2016/1688).

Qualifier:

according to guideline

Guideline:

other: Guidance for skin sensitization

Version / remarks:

IATA No 256, 2016

Key result

Parameter:

other: depletion

Value:

0

Remarks on result:

no indication of skin sensitisation

Remarks:

no indication of skin sensitisation based on QSAR/QSPR prediction

Other effects / acceptance of results:

From the (Q)SAR and in chemico assessments the result was consistent and showed that the substance was not sensitizer. In addition, the absence of the possible skin metabolites in the OECD QSAR Toolbox confirmed that the in chemico assessment (DPRA, OECD 441C) supplied reliable data on the test item, which was likely exempted from the pre- and pro-hapten sensitizing properties. Therefore, it can be concluded that the substance is likely not a skin sensitizer and no further testing will be required.

Results for Skin sensitization modelling of the substance (CAS: 3147-76-0):

Model: Prediction result: Reliability (model statistics)

DEREK No alert Not applicable

CAESAR (VEGA) Sensitising Not reliable, out of domain

TOPKAT Not sensitising Reliable

OECD QSAR Toolbox Not sensitising Reliable (confidence p=0.0313)

Toxtree No alert Not applicable

Danish QSAR Database Inconclusive Not reliable (inconclusive, out of

domain)

Interpretation of results:

GHS criteria not met

Conclusions:

It has been concluded that the substance is likely not a skin sensitizer and no further testing will be required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Conclusion

From the (Q)SAR and in chemico assessments the result was consistent and showed that 2-(2H-benzotriazol-2-yl)-tert-butylphenol was not sensitizer. In addition, the absence of the possible skin metabolites in the OECD QSAR Toolbox confirmed that thein chemico assessment (DPRA, OECD 441C) supplied reliable data on the test item, which was likely exempted from the pre- and pro-hapten sensitizing properties. Therefore, it can be concluded that 2-(2H-benzotriazol-2-yl)-tert-butylphenol is likely not a skin sensitizer and no further testing will be required.