4,4'-(4-methylpentane-2,2-diyl)bis((heptyloxy)benzene)

Endpoint summary

Description of key information

An in chemico skin sensitisation assay was conducted on the substance employing synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). Precipitation was noted following incubation for both SPCC and SPCL. Therefore, it was not possible to conclude whether the sufficient levels of the substance had reacted with the peptides. For this reason it is not possible to conclude on the skin sensitisation potential of this substance using Cystine 1:10 or Lysine 1:50 prediction model.

An in silico approach employing DEREK NEXUS was used to obtain a prediction on the potential for skin sensitisation. The result did not yield any alerts for skin sensitization.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28th January 2019 - 6th February 2019

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))

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Recommended test system in the international OECD guideline for DPRA studies.

Specific details on test material used for the study:

Batch (Lot) Number: AS455433
Expiry date: 01 November 2020 (retest date) (taken from label)
Physical Description: Colourless to light yellow viscous liquid
Purity/Composition: 98.5%
Storage Conditions: At room temperature protected from light

Additional information
Test Facility test item number: 209996/A
Purity/Composition correction factor: No correction factor required

Details on the study design:

Test system.

Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.

Experimental Design.

A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.4 mg of SPCC in 20.76 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

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. In addition, a RCcysCIPA sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCIPA sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL IPA.

Sample Incubations:

After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and substance 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 23.6 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. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.

HPLC Analysis.

SPCC and SPCL peak areas in the samples were measured by HPLC. Sample analysis was performed using the following systems:

System 1 (used for Cysteine Reactivity Assay):
Alliance separations module 2695 (Waters, Milford, MA, USA)
Dual λ absorbance detector 2487 (Waters)

System 2 (used for Lysine Reactivity Assay):
Alliance separations module 2695 (Waters, Milford, MA, USA)
Dual λ absorbance detector 2487 (Waters)

Positive control results:

SPCL mean deplition for Cinnamic Aldehyde is 57.0% ± 0.7%. SPCC mean deplition for Cinnamic Aldehyde is 70.7% ± 0.3%.

Key result

Run / experiment:

other: 1

Parameter:

other: SPCC mean depletion (%)

Value:

1.8

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Precipitation observed following the incubation

Key result

Run / experiment:

other: 1

Parameter:

other: SPCL mean deplition (%)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Precipitation observed following the incubation

SPCC Peak Area, Concentration, Depletion and Area Ratio (A₂₂₀/A₂₅₈) of the Substance Samples.

Sample code	Peak area at 220 nm (µAU)	Concentration (mM)	SPCC Depletion	Peak area at 258 nm (µAU)	Area ratio (A220/A258)
209996/A-cys-1	1866636	0.509	1.6%	49473	37.73
209996/A-cys-2	1870198	0.510	1.4%	49090	38.10
209996/A-cys-3	1852693	0.505	2.3%	49417	37.49
		Mean	1.8%	NA	37.77
		SD	0.5%	NA	0.31

SPCL Peak Area, Concentration, Depletion and Area Ratio (A₂₂₀/A₂₅₈) of the Substance Samples.

Sample code	Peak area at 220 nm (µAU)	Concentration (mM)	SPCL Depletion	Peak area at 258 nm (µAU)	Area ratio (A220/A258)
209996/A-lys-1	1587711	0.483	0.0%	50991	31.14
209996/A-lys-2	1605124	0.488	0.0%	51014	31.46
209996/A-lys-3	1592852	0.484	0.0%	50692	31.42
		Mean	0.0%	NA	31.34
		SD	0.0%	NA	0.18

SD = Standard Deviation, NA = Not Applicable.

Interpretation of results:

GHS criteria not met

Remarks:

Precipitation of the substance was observed after the incubation period for both SPCC and SPCL, is not known how much of the substance remained in the solution to react with the peptides. Consequently, this negative result is uncertain.

Conclusions:

In the in chemico skin sensitisation assay the substance was negative and classified in the “no or minimal reactivity class”. However, since precipitation was observed before and after the incubation period for both SPCC and SPCL, this negative result is uncertain and should be interpreted with due care.

Executive summary:

The in chemico skin sensitisation study conducted in accordance with OECD TG 442C, 'In Chemico Skin Sensitization: Direct Peptide Reactivity Assay'. The substance was incubated with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) for 23.6 h at 25 °C. The SPCC and SPCL Percent Depletion Values were calculated. It was shown that after the incubation of substance with peptides there was a precipitate present for both SPCC and SPCL. The substance is classified in the “no or minimal reactivity class”. However, as the precipitation was observed it was not possible to conclude whether the sufficient levels of the substance had reacted with the peptides, therefore this negative result is uncertain and should be interpreted with due care.

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

In silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

10th December 2018 - 10th December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Preliminary study conducted using in silico (DEREK NEXUS) prior to the in vitro OECD TG preferred studies.

Principles of method if other than guideline:

The objective of this study was to obtain a prediction on the potential for skin sensitization of the substance with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.
DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitization alerts.The level of likelihood of a structure being sensitizing to skin is expressed in terms of:
Certain: There is proof that the proposition is true.
Probable: There is at least one strong argument that the proposition is true and there are no arguments against it.
Plausible: The weight of evidence supports the proposition.
Equivocal: There is an equal weight of evidence for and against the proposition.
The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment.

DEREK NEXUS contains an expert-derived functionality that can provide negative predictions for skin sensitization. This functionality further evaluates those compounds which do not fire any skin sensitization alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of skin sensitization data, producing the following outcomes:
In compounds where all features in the molecule are found in accurately classified compounds from the reference set, a negative prediction is displayed: inactive.
For those query compounds where features in the molecule are found in non-alerting skin sensitizers in the Lhasa reference set, the prediction remains negative and the misclassified (1) features are highlighted to enable the negative prediction to be verified by expert assessment.
In cases where features in the molecule are not found in the Lhasa reference set, the prediction remains negative and the unclassified (2) features are highlighted to enable the negative prediction to be verified by expert assessment.
If a substance is predicted to be a skin sensitizer, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental data from the closest structurally-related substances (at least 3 substances should be present) using the following equation:

EC3Q = MWQ/ (sum of omegaNN/sum of TNN)

MW = molecular weight
T = Tanimoto similarity score
Omega=weghting factor=(MWNN/EC3) * TNN
Q = query compound
NN = nearest neighbour
The EC3 is the estimated concentration needed to produce a stimulation index of 3.

(1) Misclassified features are those that have been derived from non-alerting substances in the Lhasa test reference set;
(2) Unclassified features are those that have not been found in the Lhasa test reference set.

Specific details on test material used for the study:

CAS Number 1951440-04-2
Molecular weight: 466.74
Molecular formula C32H50O2

Key result

Run / experiment:

other: 1

Parameter:

other: Skin Sensitisation alert

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no indication of skin sensitisation

Remarks:

Assessment with DEREK NEXUS did not yield any skin sensitization alerts for this structure.

Interpretation of results:

study cannot be used for classification

Conclusions:

The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitization.

Executive summary:

An in silico approach employing DEREK NEXUS used to obtain a prediction on the potential for skin sensitisation. The result did not yield any alerts for skin sensitization. This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitization.

Substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available