Diethyl [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate

Endpoint summary

Description of key information

Sensitization involves a number of key steps in order to take place and can be described in terms of an adverse outcome pathway (AOP). These include reactivity with skin proteins (peptide reactivity), activation of skin cells (keratinocyte activation) and immune cells (dendritic cell activation). The studies carried out for this substance address these key steps. The results are then used in a predefined evaluation scheme to determine hazard classification as a sensitizer by a weight-of-evidence approach.

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:

weight of evidence

Study period:

19 Sep 2014 - 21 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, in-house validated procedure, pre-OECD

Qualifier:

according to guideline

Guideline:

other: Direct peptide binding assay (DPRA) performed as described in Bauch C. et al. (2011), Toxicology in Vitro 25, 1162 – 1168.

Qualifier:

according to guideline

Guideline:

other: OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: Direct Peptide Reactivity Assay (DPRA), assessed on 13 Nov 2013 at http://www.oecd.org

Principles of method if other than guideline:

In the DPRA the reactivity of a test item (=test substance) towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated. For this purpose the test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm. The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Details on the study design:

synthetic peptides (Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH; Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH)

Positive control results:

The peptide depletion by the positive control was 38.86%.

Parameter:

other: mean peptide depletion in %

Value:

0

The mean C-peptide depletion, caused by the test substance was determined to be -1.34%. The mean K-peptide depletion, caused by the test substance was determined to be -1.24%. Negative depletions were considered to be “zero” for calculation of the mean peptide

depletion, which was thus calculated to be 0.00%. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that the test article shows a minimal

chemical reactivity in the DPRA under the test conditions chosen.

Interpretation of results:

other: minimal chemical reactivity

Executive summary:

The reactivity of the test article towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was prepared at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity. The following results were obtained in the DPRA: The test substance was not soluble in one of the vehicles used for the assay. Thus a homogeneous suspension in acetonitrile was used for sample preparation. The test substance was soluble in acetonitrile. The samples with the test substance and the peptide stock solutions were suspensions. After 24 hours suspensions were noticed. Thus all samples were centrifuged prior to HPLC analysis. The mean C-peptide depletion, caused by the test substance was determined to be -1.34%. The mean K-peptide depletion, caused by the test substance was determined to be -1.24%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that the test article shows a minimal chemical reactivity in the DPRA under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 Sep 2014 - 21 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant and in-house validated study

Qualifier:

according to guideline

Guideline:

other: Keratinocyte activation assay (LuSens) as described in Bauch et al. (2012) Regul Toxicol Pharmacal, 63(3):489-504.

Principles of method if other than guideline:

The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using the genetically modified keratinocytes (LuSens, Bauch et al. 2012). It employs the reporter gene for luciferase under the control of an antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. The endpoint measurement is the up-regulation of the luciferase activity after 48 hours incubation with test substances. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signaling pathway (Ade et al. 2009, Natsch 2012, Natsch & Emter 2008, Vandebriel et al. 2010).

GLP compliance:

yes (incl. QA statement)

Type of study:

other: in-vitro keratinocyte activation assay (LuSens)

Details on the study design:

cells used: LuSens cells (transgenic keratinocyte cell line derived from HaCaT cells)
The reporter gene cell line LuSens was prepared in collaboration with the RWTH Aachen University. This keratinocyte cell line derived from HaCaT cells carries a reporter gene for luciferase under the control of an antioxidant-response-element (ARE) and hence monitors Nrf2 transcription factor activity.
LuSens cells are routinely cultured in D-MEM (Cat. No. Biochrom FG 0445)+ 10 % FBS + 1 % Penicillin / Streptomycin, Puromycin dihydrochloride 25 μL.

Run / experiment:

other: 1

Parameter:

other: fold luciferase induction

Value:

0.87

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

no statistical increase at concentrations affording at least 70% survivability

Run / experiment:

other: 2

Parameter:

other: fold luciferase induction

Value:

0.73

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

no statistical increase at concentrations affording at least 70% survivability

After 48 hours of exposure to test substance luciferase activity in LuSens cells was not statistical significantly induced affording at least 70% viability in at least two consecutive concentrations of two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential.

Summary of LuSens Main Experiments:

Concentration (test substance) µg/mL	1st experiment						2nd experiment
	fold induction		rel. viability [%]		t-test		fold induction		rel. viability [%]		t-test
	mean	SD	mean	SD	p-value	markers	mean	SD	mean	SD	p-value	markers
3.4	1.10	0.74	101.8	6.1	0.422	n.s.	0.77	0.44	88.6	2.8	0.231	n.s.
4.1	1.21	0.33	98.7	6.5	0.193	n.s.	0.82	0.27	80.2	7.8	0.187	n.s.
5.0	0.87	0.09	87.5	6.8	0.091	n.s.	1.00	0.39	86.3	13.7	0.493	n.s.
5.9	0.92	0.15	89.0	4.7	0.266	n.s.	0.73	0.41	78.9	12.7	0.188	n.s.
7.1	1.31	0.43	79.9	3.8	0.172	n.s.	1.05	0.26	72.9	5.6	0.397	n.s.
8.6	1.60	0.45	66.1	1.2	0.068	n.s.	0.98	0.31	75.0	11.8	0.459	n.s.
10.3	1.46	0.15	53.8	1.4	0.004	**	0.94	0.35	66.1	5.7	0.405	n.s.
12.3	1.52	0.26	45.0	3.8	0.026	*	1.03	0.37	46.6	7.0	0.451	n.s.
VC	1.00	0.37	100.0	6.5	-	-	1.00	0.29	100.0	7.0	-	-
EGDMA (18 µg/mL)	5.37	1.03	110.1	9.6	0.000	**	6.60	0.76	106.5	7.4	0.000	**
LA (450 µg/mL)	0.80	0.17	105.4	4.0	0.057	n.s.	0.95	0.36	118.6	4.8	0.395	n.s.

n.s. = no statistical significance

* = p < 0.05

** = p < 0.01

Interpretation of results:

other: no induction of antioxidant response genes in a keratinocyte cell line

Executive summary:

The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined thereafter by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. The following results were observed: At concentrations used in the main test the test substance was soluble in DMSO (100 x stock solutions) and soluble in 1% DMSO in medium 3 (final concentrations). After 48 hours precipitates were not noticed in any concentration. Luciferase activity in LuSens cells was not statistical significantly induced affording at least 70% viability in at least two consecutive concentrations of two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Due to the complexity of the skin sensitization process a single in vitro assay is not sufficient to adequately assess this toxicological endpoint. Therefore, a combination of several methods addressing key steps of the sensitization process: protein reactivity (DPRA), activation of keratinocytes (LuSens or KeratinoSens) and activation of dendritic cells (MUSST or h-CLAT) were used (test battery).

These in vitro studies cover three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10). These study types have undergone in-house validation using 54 substances (Bauch et al., 2012, Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015, Regul Toxicol Pharmacol. 71: 337 -351).

Based on the results of the in house validation (Bauch et al., 2012, Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. This was confirmed in the validation study with more substances (Urbisch 2015): When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from theDPRA, LuSens and mMUSST had a sensitivity of 90%, a specificity of 90% and an accuracy of 90%.

 

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauchet al. 2012; Table 1). If two assays (DPRA, LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

Table 1: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.

DPRA	LuSens/ KeratinoSens^TM	MUSST/ h-CLAT	Test battery evaluation
positive	positive	positive	sensitizer
positive	positive	negative	sensitizer
positive	negative	positive	sensitizer
positive	negative	negative	non-sensitizer
negative	positive	positive	sensitizer
negative	positive	negative	non-sensitizer
negative	negative	positive	non-sensitizer
negative	negative	negative	non-sensitizer

The in vitro sensitization test battery was initiated with the DPRA and the LuSens. As an unambiguous result was obtained after these two tests, no further test was performed. Each individual assay was performed under GLP and the cell-based assay LuSens consisted of at least two independent experiments. Positive and negative controls were included in each experiment and confirmed the functionality and validity of the individual assays.

 

The test battery applicability is limited when testing substances insoluble in the commonly used vehicles and highly volatile substances. Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods. The substance under evaluation did not have any of the above mentioned limitations and the outcome of this assay is therefore considered adequate for hazard identification for the endpoint of skin sensitization.

The test substance is not peptide reactive and does not activate keratinocytes. In accordance with the published evaluation scheme (Bauch et al., 2012, Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, the test article is judged to be not a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification,Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be not classified for skin sensitization under Regulation (EC) No. 1272/2008.