Bis(4-(1,1,3,3-tetramethylbutyl)phenyl) ether

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date 16 Jun. 2020, Experimental Completion Date 19 Jun. 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Peptide Reactivity Assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(4-(1,1,3,3-tetramethylbutyl)phenyl) ether

EC Number:

202-956-6

EC Name:

Bis(4-(1,1,3,3-tetramethylbutyl)phenyl) ether

Cas Number:

101-58-6

Molecular formula:

C28H42O

IUPAC Name:

1-(2,4,4-trimethylpentan-2-yl)-4-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]benzene

Test material form:

liquid

Specific details on test material used for the study:

Purity: 90.2 % Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)ether

In chemico test system

Details on the study design:

The OECD AOP (The adverse outcome pathway for skin sensitisation) is initiated by key event 1 (Covalent interaction with skin proteins), which is followed sequentially by three key events (KE): (KE2) keratinocyte activation, (KE3) dendritic cell activation, and (KE4) proliferation of antigen-specific T cells. However, none of the assays addressing the different KEs is currently accepted as stand-alone test method and may not be sufficient to conclude on the presence or absence of skin sensitisation potential of chemicals. But supports the discrimination between skin sensitisers and non sensitisers in combination with other complementary data.
The direct peptide reactivity assay (DPRA) addresses the molecular initiating event (KE1) of the AOP. It is an in chemico assay to quantify the depletion of synthetic model peptides caused by known amounts of the test item measured by HPLC.
This study is performed in order to evaluate the reactivity of Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)ether towards cysteine and lysine containing peptides. The peptide depletion compared to the solvent controls is calculated and leads to a DPRA prediction (reactive/positive or non-reactive/negative) that could be used to support the discrimination between skin sensitisers and non-sensitisers. Additionally, an assignment to one of four reactivity classes could be made in order to possibly support a potency assessment.
The DPRA is part of a tiered strategy for the evaluation of the skin sensitization potential. Thus, all data generated with the present Test Guideline OECD 442C and EU-Method B.59 should be used in the context of an integrated approach to testing and assessment (IATA).
The test item was incubated for 24 h at 25 °C together with Cys- and Lys-peptides, respectively. The peptide concentration after the incubation period was measured using HPLC-UV.
Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptides, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

Results and discussion

Positive control results:

One valid experiment was performed.
Experiment 1 was valid for both peptide assays. In the Lys-peptide assay, the percent mean area ratio 220/258 nm for the positive control was slightly out of the range 90-110%. The mean area ratio is an indicator for peak purity and sign for co-elution. As the chromatogram of the co-elution control shows no sign of co-elution this might be due to really small peaks at 258 nm. The mean area ratio is no acceptance criterion and has no influence on the result of the test item peptide depletion or the result of the study.

In vitro / in chemico

Results

Run / experiment:

other: One valid experiment was performed. Experiment 1 was valid for both peptide assays. In the Lys-peptide assay, the percent mean area ratio 220/258 nm for the positive control was slightly out of the range 90-110%.

Parameter:

other: reactivity towards the Cys- and Lys-peptides.

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not soluable neither in the media used for the h-CLAT nor in the one for the LuSens. Therefore it is not possible to perform the studies according tom OECD 422E and OECD 422D.
The test item is soluable in Isopropanol so it is possible to perform the Direct Peptide Reactivity Assay: DPRA according to OECD 422C.

The DPRA prediction is “negative” according to the Cysteine 1:10/Lysine 1:50 prediction model. Thus, under the experimental conditions reported, the test item Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)ether shows no reactivity towards the two model synthetic peptides.
This assignment supports the discrimination between skin sensitisers and non-sensitisers in the framework of an integrated approach (IATA).
For sensitising potency assessment within an IATA, the test item Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)ether could be assigned to the reactivity class that covers no or minimal reactivity under the conditions of this study.
As the calculated peptide depletion is minimal (i.e. much smaller than the limit required for a positive classification) the classification as “negative” is reasonable despite the slight precipitation.
As the result is unambiguos there is no reason to perform a further study with animals.

Executive summary:

This in chemico study was performed in order to evaluate the reactivity of the test item Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)ether towards cysteine (Cys-) and lysine (Lys-) containing peptides.

The calculated peptide depletion could be used to support the discrimination between skin sensitisers and non-sensitisers. The DPRA is part of a tiered strategy for the evaluation of skin sensitisation potential in the context of an integrated approach to testing and assessment (IATA).

The test item was incubated for 24 h at 25 °C together with Cys- and Lys-peptides, respectively. The peptide concentration after the incubation period was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptides, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

 

One valid experiment was performed.

Experiment 1 was valid for both peptide assays. In the Lys-peptide assay, the percent mean area ratio 220/258 nm for the positive control was slightly out of the range 90-110%. The mean area ratio is an indicator for peak purity and sign for co-elution. As the chromatogram of the co-elution control shows no sign of co-elution this might be due to really small peaks at 258 nm. The mean area ratio is no acceptance criterion and has no influence on the result of the test item peptide depletion or the result of the study.

The test item “Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)ether” shows no reactivity towards the Cys- and Lys-peptides.

Therefore, the DPRA prediction is “negative” with “no or minimal” reactivity.

According to the guideline OECD 442C, in samples with observed precipitation or phase separation the peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result and it should be interpreted with due care.

In the test item replicates of the Cys- and Lys-peptide assay, slight precipitation was observed immidiatelly upon addition of the test item and after the incubation period.

As the calculated peptide depletion is minimal (i.e. much smaller than the limit required for a positive classification) the classification as “negative” is reasonable despite the slight precipitation.

The peptide depletion values after incubation are shown in Table 3a:

Table 3a Results

	Cys-peptide depletion [%]	Lys-peptide depletion [%]	Mean peptide depletion [%]
Experiment 1	0.00	0.00	0.00