4-hydroxybenzophenone

Endpoint summary

Description of key information

Studies carried out according to recognised testing guidelines under GLP certification.

The objective of these studies is to reach an overall conclusion on the endpoint skin sensitization based on all available relevant information, including in silico, in chemico and in vitro data.

A DEREK assessment, a DPRA assay, a KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

A DEREK NEXUS assessment yielded an alert for a substituted phenol related to skin sensitization and predicted and EC3 of 1.5%. In the DPRA, the substance did not significantly react with lysine- or cysteine moieties; therefore, interaction with proteins via binding is not expected to take place. The substance gave a positive response in a KeratinoSensTM assay, indicating that 4-Hydroxy-benzophenone can activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes. The results of the assays do not provide an unanimous conclusion on skin sensitization. Therefore additional information is required to reach a conclusion on skin sensitization. Performance of a U-SENSTM assay is considered to give no additional information, as cytotoxicity is expected resulting in an ambiguous test outcome. Since testing further in vitro would not lead to an overall conclusion and the current data-set is not adequate for classification and risk assessment on the endpoint skin sensitization it is considered justified to perform an in vivo study to assess the skin sensitizing properties of 4-Hydroxy-benzophenone.

The weight of evidence approach is based on in silico/in chemico/in vitro data, addressing each of the following key events of skin sensitization on its own or together:

1. Key event 1: Covalent binding of the electrophilic substance to proteins; tested by OECD 442C: Direct Peptide Reactivity Assay (DPRA)

2. Key event 2: Release of pro-inflammatory cytokines and induction of cyto-protective pathways in keratinocytes; tested by OECD 442D: ARE-Nrf2 Luciferase Test Method or KeratinoSensTM assay

3. Key event 3: Activation and maturation of dendritic cells; OECD 442E Myeloid U937 Skin Sensitization Test (U-SENSTM)

4. Key event 4: Presentation of the chemical allergen by dendritic cells to naïve T-cells, which leads to their differentiation and proliferation into allergen-specific memory T-cells; not generally accepted in vitro test available yet.

If information from test method(s) addressing one or two of these key events allows classification and risk assessment according to point 8.3 of Annex VII of the REACH Regulation, studies addressing the other key event(s) need not be conducted. According to the Guidance on information requirements and chemical safety assessment R7a (most recent version), to reach a conclusion on (non-)classification, the following questions should be addressed:

Does the evidence enable to conclude that the substance is not a skin sensitizer? If so, conclude on no classification.

Does the evidence enable to conclude that the substance is presumed to produce significant sensitization in humans i.e. Cat. 1A? If so, classify accordingly.

Does the evidence enable to conclude that the substance is a skin sensitizer and significant sensitization in humans i.e. Cat. 1A can be excluded? If so, it is presumed that the substance would be a moderate skin sensitizer i.e. Cat. 1B and it is recommended to classify accordingly.

In case none of these conditions are met, e.g. when Cat. 1A cannot be excluded, further testing needs to be performed, in vivo testing being the last resort. At the moment no accepted in vitro studies are available to discern between Cat. 1A and 1B.

In case of positive in chemico/in vitro skin sensitization tests and absence of reliable indication for potency by DEREK, for the time being performance of an in vivo study is the only option to determine the degree of potency (see CLP Regulation 3.4 Respiratory or skin sensitisation).

No data were available that would preclude performance of the studies to determine the potential for skin sensitization. Therefore, STEP 1 studies were performed, i.e. a DEREK assessment (project no 20151987), a DPRA assay (project no 20151989) and a KeratinoSensTM assay (project no 20151990) were performed.

Results of studies performed

DEREK NEXUS version 6.0.1 yielded an alert for 4-hydroxy-benzophenone for skin sensitization based on the presence of a substituted phenol group. 4-Hydroxy-benzophenone is predicted to be sensitizing to the skin (equivocal). DEREK NEXUS predicted an EC3 of 1.5% (moderate sensitizer) based on LLNA data from eleven structurally related substances.

A valid DPRA test was performed according to OECD 442C and GLP. For the DPRA assay, 4-hydroxy-benzophenone was dissolved completely in acetonitrile at 100 mM. Upon preparation as well as after incubation of the synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) with test item no precipitate or phase separation was observed in any of the samples. No co-elution of the test item with SPCC or SPCL was observed. In the cysteine reactivity assay the test item showed 0.1% SPCC depletion while in the lysine reactivity assay the test item showed 1.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.8% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

A valid KeratinoSens^TMassay was performed according to OECD 442D and GLP. For the KeratinoSens^TMassay 4-Hydroxy-benzophenone was dissolved in dimethyl sulfoxide to a final concentration of 40 mg/mL in experiment 1 and 10 mg/mL in experiment 2 and 3. Test concentrations of 0.20 – 400 μg/mL were used in experiment 1 (2-fold dilution series) and 8.6–100 μg/mL(1.25-fold dilution series) were used in experiment 2 and 3. No precipitate was observed at any dose level tested. Three independent experiments were performed. In the first experiment the test item showed toxicity (calculated IC₃₀values of 35 μg/mL and IC₅₀values of 63 μg/mL). A dose-related induction of the luciferase activity (EC1.5 value of 19 μg/mL) was measured and the maximum luciferase activity induction (I_max) was 1.89-fold. In the second experiment the test item showed toxicity (calculated IC30 values of 90 μg/mL and IC50 values of 97 μg/mL). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 71 μg/mL) was measured and the Imax was 1.65-fold. Due to the big difference in IC₃₀, IC₅₀and EC1.5 between experiment 1 and 2 an additional third experiment was performed to confirm the positive results observed in experiment 1 and 2. In the third experiment, the test item showed no toxicity. A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 7.5 μg/mL) was measured and the I_maxwas 4.38-fold. The test item is classified as positive in the KeratinoSensTM assay since clear positive results (>1.5-fold induction) were observed in all 3 experiment at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control.

As the above studies; DPRA, Keratinosens and DEREK together yield an inconclusive result, an LLNA study (OECD 429) is currently being conducted as a conclusive test.

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:

11 Oct 2018 to 16 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

Adopted June, 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™

Version / remarks:

Adopted March, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Appearance: White powder
Purity/Composition: 99.72%, assumed 100% for testing
Test item storage: At room temperature desiccated

Details on the study design:

Test System:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Rationale:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

Cell Culture:

Basic medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).

Maintenance medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).

Exposure medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 76 – 101%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 to 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Subculturing:
Cells were subcultured upon reaching 80 to 90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

Experimental Design:
Plating of Cells:
For testing, cells were 80 to 90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+9 in experiment 1, P+13 in experiment 2 and P+3 in experiment 3.

Treatment of Cells:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0°C in the presence of 5% CO2. Initially, experiment 1 and 2 did not pass all the acceptability criteria and therefore these experiments wererepeated. In total 3 valid experiments were performed.

Luciferase Activity Measurement:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 to 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.55 and the EC1.5 43 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.31 and the EC1.5 50 µM.
Experiment 3: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.92 and the EC1.5 26 µM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5 (µM)

Value:

19

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

EC1.5 = 43µM

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5 (µM)

Value:

71

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

EC1.5 = 50 µM

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: EC1.5 (µM)

Value:

7.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

EC1.5 = 26 µM

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.89

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Imax = 3.55

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.65

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Imax = 3.31

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: Imax

Value:

4.38

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Imax = 2.92

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

In conclusion, 4-Hydroxy-benzophenone is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of 4-Hydroxy-benzophenone to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens^TM assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 1803004 of 4-Hydroxy-benzophenone was a white powder.  The test item was dissolved in dimethyl sulfoxide up to 40 mg/mL.  From this stock 11 spike solutions in DMSO were prepared.  The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.20 – 400 µg/mL (2-fold dilution series).  The highest test concentration was the highest dose required in the current guideline.  A narrower dose-response analysis was performed in experiment 2 and 3 using a lower dilution factor of 1.25-fold to investigate the induction observed in experiment 1 in more detail.  No precipitate was observed at any dose level tested.  Three independent experiments were performed.

All experiments passed the acceptance criteria:

•       The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.  

•       The EC1.5 of the positive control was within two standard deviations of the historical mean (43 µM, 50 µM and 26 µM in experiment 1, 2 and 3, respectively).  A dose response was observed and the induction at 250 µM was higher than 2-fold (3.55-fold, 3.31-fold and 2.92-fold in experiment 1, 2 and 3, respectively).

•       Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (12%, 4.3% and 8.8% in experiment 1, 2 and 3, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

In the first experiment the test item showed toxicity (IC30 values of 35 µg/mL and IC50 values of 63 µg/mL).  A biologically relevant, dose-related induction of the luciferase activity (EC_1.5 values of 19 µg/mL) was measured and the maximum luciferase activity induction (Imax) was 1.89-fold.  In the second experiment the test item showed toxicity (IC₃₀ values of 90 µg/mL and IC₅₀ values of 97 µg/mL).  A biologically relevant, dose-related induction of the luciferase activity (EC_1.5 values of 71 µg/mL) was measured and the maximum luciferase activity induction (I_max) was 1.65-fold.  Due to the big difference in IC₃₀, IC₅₀ and  EC_1.5 between experiment 1 and 2 an additional third experiment was performed to confirm the positive results observed in experiment 1 and 2.  In the third experiment the test item showed no toxicity (No IC₃₀ or IC₅₀ value).  A biologically relevant, dose-related induction of the luciferase activity (EC_1.5 values of 7.5 µg/mL) was measured and the maximum luciferase activity induction (Imax) was 4.38-fold.

The test item is classified as positive in the KeratinoSens^TM assay since clear positive results (>1.5-fold induction) were observed in all 3 experiment at test concentrations < 200 µg/mL with a cell viability of >70% compared to the vehicle control.

In conclusion, 4-Hydroxy-benzophenone is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Reference 2

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

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:

1. SOFTWARE
DEREK NEXUS v 6.0.1.

2. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL

Chemical name: 4-hydroxy-benzophenone
CAS Number 1137-42-4
Molecular weight: 198.22
Molecular formula C13H10O2

3. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization based on the presence of molecular substructures. LHASA (see Appendix I) has inserted validation comments for the skin sensitization alerts.

4. APPLICABILITY DOMAIN
See QMRF

5. ADEQUACY OF THE RESULT
See QMRF

Qualifier:

no guideline required

Principles of method if other than guideline:

DEREK NEXUS contains 90 alerts for skin sensitisation, together with reasoning rules encoding physicochemical descriptors. In addition to a prediction of skin sensitisation potency for alerting query compounds, Derek evaluates potentially misclassified and unclassified features in cpompounds that do not activate skin sensitsation alerts or examples.

GLP compliance:

no

Remarks:

QSAR data

Justification for non-LLNA method:

QSAR data used as part of an in-vitro testing strategy.

Key result

Run / experiment:

other: Structural Alert

Parameter:

other:

Remarks on result:

other: 439 Substituted Phenol

Key result

Run / experiment:

other: Predicted LLNA EC3

Parameter:

other:

Remarks on result:

other: 1.5% modertate sensitiser

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

DEREK NEXUS version 6.0.1 yielded an alert for 4-hydroxy-benzophenone for skin sensitization based on the presence of a substituted phenol group. 4-Hydroxy-benzophenone is predicted to be sensitizing to the skin (equivocal). DEREK NEXUS predicted an EC3 of 1.5% (moderate sensitizer) based on data on closest structurally-related substances.

Executive summary:

DEREK NEXUS version 6.0.1 yielded an alert for 4-hydroxy-benzophenone for skin sensitization based on the presence of a substituted phenol group. 4-Hydroxy-benzophenone is predicted to be sensitizing to the skin (equivocal). DEREK NEXUS predicted an EC3 of 1.5% (moderate sensitizer) based on data on closest structurally-related substances.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 Jan 2019 to 16 Jan 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 (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Appearance: White powder
Purity/Composition: 99.72%, assumed 100% for testing
Test item storage: At room temperature desiccated

Details on the study design:

Test Item (4-Hydroxy-benzophenone)
Identification: 4-Hydroxy-benzophenone
Test Facility test item number: 209445/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC, synonym or trade name: 4-Hydroxy-benzophenone
CAS number: 1137-42-4
Molecular formula: C13H10O2
Molecular weight: 198.22 g/mol

Reference Item (Positive Control Cinnamic Aldehyde)
Identification: Cinnamic aldehyde
Test Facility Test Item Number: RS473/C
Appearance: Yellow liquid
CAS Number: 104-55-2
Molecular Formula: C9H8O
Molecular Weight: 132.16 g/mol
Batch: MKCB9907
Purity: 99.1%
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2021 (expiry date)
Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
Purity/composition correction factor: No correction factor required

The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. The characterization of the test item was conducted under a sponsor or sponsor subcontractor quality environment. A Certificate of Analysis was provided to the Test Facility.

Reserve Samples:
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility. The sample will be destroyed after the expiry date.

Test Item Inventory and Disposition:
Records of the receipt, distribution, and storage of test item were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

Dose Formulation and Analysis:

Preparation of Test Item:
No correction for the purity/composition of the test item was performed.
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN).

Test item stock solutions were prepared freshly for each reactivity assay.

For both the cysteine and lysine reactivity assay 34.09 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1720 µL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

Any residual volumes were discarded.
 

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.
Rationale: Recommended test system in the international OECD guideline for DPRA studies.
Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Batch: See Appendix 7 for detailed information.
Storage: The peptides were stored in the freezer (≤ 15°C) for a maximum of 6 months.

Reagents:
Acetonitrile (ACN): HPLC grade, Fisher Chemicals, Loughborough, England
Ammonium acetate: Fractopur, Merck, Darmstadt, Germany
Ammonium hydroxide: 25%, Merck
Disodium hydrogen phosphate (Na2HPO4·12H2O): Emsure, Merck
Milli-Q water (MQ): Tap water purified by reversed osmosis and subsequently passed over activated carbon and ion exchange cartridges; Millipore, Bedford, MA, USA
Sodium dihydrogenphosphate dehydrate(NaH2PO4·H2O): Lab-Honeywell, Germany
Trifluoroacetic acid (TFA): >99%, Sigma Aldrich, Zwijndrecht, The Netherlands

Experimental Design:
Preparation of Solutions for Cysteine Reactivity Assay:
Synthetic Peptide Containing Cysteine (SPCC) Stock Solution:
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 11.2 mg of SPCC in 22.36 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

SPCC Reference Control Solutions:
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.

Positive control results:

The results of the positive control cinnamic aldehyde are presented in Table 7 (Appendix 3). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.4% ± 0.4%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Key result

Parameter:

other: SPCC depletion (percentage)

Value:

0.1

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

SPCC depletion 72.4%

Remarks on result:

no indication of skin sensitisation

Key result

Parameter:

other: SPCL depletion (percentage)

Value:

1.4

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

SPCL depletion = 59.3%

Remarks on result:

no indication of skin sensitisation

Solubility Assessment of the Test Item

At a concentration of 100 mM, 4-Hydroxy-benzophenone was soluble in ACN.  Therefore this solvent was used to dissolve the test item in this DPRA study.

Cysteine Reactivity Assay

The reactivity of 4-Hydroxy-benzophenone towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC analysis, following 23.5 hours of incubation at 25±2.5°C.  Representative chromatograms of CCcys-209445/A and 209445/A-cys samples are presented in Appendix 4.  An overview of the retention time at

220 nm and peak areas at 220 nm and 258 nm are presented in Table 3 (Appendix 3).

Acceptability of the Cysteine Reactivity Assay

The SPCC standard calibration curve is presented in Figure 1 (Appendix 2).  The correlation coefficient (r2) of the SPCC standard calibration curve was 0.9999.  Since the r2 was >0.99, the SPCC standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 4 (Appendix 3).  The mean peptide concentration of Reference Controls A was 0.503 ± 0.001 mM while the mean peptide concentration of Reference Controls C was 0.497 ± 0.003 mM.  The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM.  This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.

The SPCC peak areas for Reference controls B and C are presented in Table 5 (Appendix 3).  The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 0.7%.  This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCC A220/A258 area ratios of Reference controls A, B and C are presented in Table 6 (Appendix 3).  The mean area ratio (A220/A258) of the Reference Control samples was 37.45.  The mean A220/A258 ratio ± 10% range was 33.70-41.19.  Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Results: Cysteine Reactivity Assay for the Test Item

Preparation of a 100 mM 4-Hydroxy-benzophenone stock solution in ACN showed that the test item was dissolved completely.  Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected.  No precipitate or phase separation was observed in any of the samples.

The results of the cysteine reactivity assay for the test item are presented in Table 8 (Appendix 3).  In the CC sample no peak was observed at the retention time of SPCC (see chromatogram in Appendix 4).  This demonstrated that there was no co-elution of the test item with SPCC.  

For the 209445/A-cys samples, the mean SPCC A220/A258 area ratio was 37.14.  Since this was within the 33.70-41.19 range, this again indicated that there was no co elution of the test item with SPCC.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C.  The mean Percent SPCC Depletion for the test item was 0.1% ± 0.1%.

Lysine Reactivity Assay

The reactivity of 4-Hydroxy-benzophenone towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC analysis, following 23.5 hours of incubation at 25±2.5°C.  Representative chromatograms of CClys-209445/A and 209445/A-lys samples are presented in Appendix 4.  An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 9 (Appendix 3).

Acceptability of the Lysine Reactivity Assay

The SPCL standard calibration curve is presented in Figure 2 (Appendix 2).  The correlation coefficient (r2) of the SPCL standard calibration curve was 0.9998.  Since the r2 was >0.99, the SPCL standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 10 (Appendix 3).  The mean peptide concentration of Reference Controls A was 0.508 ± 0.013 mM while the mean peptide concentration of Reference Controls C was 0.476 ± 0.028 mM.  The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM.  This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

The SPCL peak areas for Reference controls B and C are presented in Table 11 (Appendix 3).  The CV of the peptide areas for the nine Reference Controls B and C was 4.5%.  This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCL A220/A258 area ratios of Reference controls A, B and C are presented in Table 12 (Appendix 3).  The mean area ratio (A220/A258) of the Reference Control samples was 31.76.  The mean A220/A258 ratio ± 10% range was 28.59-34.94.  Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 13 (Appendix 3).  The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C.  The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 59.3% ± 0.5%.  This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Results Lysine Reactivity Assay for the Test Item

Preparation of a 100 mM 4-Hydroxy-benzophenone stock solution in ACN showed that the test item was dissolved completely.  Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected.  No precipitate or phase separation was observed in any of the samples.

The results of the lysine reactivity assay for the test item are presented in Table 14 (Appendix 3).  In the CC sample no peak was observed at the retention time of SPCL (see chromatogram in Appendix 4).  This demonstrated that there was no co-elution of the test item with SPCL.  

For the 209445/A-lys samples, the mean SPCL A220/A258 area ratio was 31.81.  Since this was within the 28.59-34.94 range, this again indicated that there was no co elution of the test item with SPCL.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C.  The mean Percent SPCL Depletion for the Test Item was 1.4% ± 1.2%.

DPRA Prediction and Reactivity Classification

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below.  In the cysteine reactivity assay the test item showed 0.1% SPCC depletion while in the lysine reactivity assay the test item showed 1.4% SPCL depletion.  The mean of the SPCC and SPCL depletion was 0.8% and as a result the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Text Table 9       

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
4-Hydroxy-benzophenone	0.10%	±0.1%	1.40%	±1.2%	0.80%	Negative: No or minimal reactivity

SD = Standard Deviation.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, this DPRA test is valid. 4-Hydroxy-benzophenone was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The objective of this study was to determine the reactivity of 4-Hydroxy-benzophenone towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL).  After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm.  SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The study procedures described in this report were based on the most recent OECD guideline.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.  An overview of the obtained assay validation parameters is presented in the table below.

Text Table 1       

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.9999	>0.99	0.9998
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.503 ± 0.001	0.50 ± 0.05	0.508 ± 0.013
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.497 ± 0.003	0.50 ± 0.05	0.476 ± 0.028
CV (%) for RC samples	<15.0	0.7	<15.0	4.5
B and C
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	72.4	40.2-69.0	59.3
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.4	<11.6	0.5
SD of peptide depletion for the test item (%)	<14.9	0.1	<11.6	1.2

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below.  In the cysteine reactivity assay the test item showed 0.1% SPCC depletion while in the lysine reactivity assay the test item showed 1.4% SPCL depletion.  The mean of the SPCC and SPCL depletion was 0.8% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Text Table 2       

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
4-Hydroxy-benzophenone	0.10%	±0.1%	1.40%	±1.2%	0.80%	Negative: No or minimal reactivity

SD = Standard Deviation.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.  4-Hydroxy-benzophenone was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 May 2019 to 19 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

EC No 640/2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Appearance: White powder
Purity/Composition: 99.72%
Test item storage: At room temperature desiccated

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST SYSTEM
Condition: Inbred, SPF-Quality

Source: Janvier, Le Genest-Saint-Isle, France

Number of Animals: 20 Females (nulliparous and non-pregnant). Five females per group.

Age at the Initiation of Dosing: Young adult animals (approximately 11 weeks old) were selected.

Weight at the Initiation of Dosing: 19.6 to 24.2 g.

JUSTIFICATION FOR TEST SYSTEM AND NUMBER OF ANIMALS
The CBA/J mouse was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW). The test method and number of animals were based on the test guidelines.

The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures are summarized in Appendix 3 of this report. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.

The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

ANIMAL IDENTIFICATION
At study assignment, each animal was identified using a tail mark with indelible ink.

ENVIRONMENTAL ACCLIMATION
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

SELECTION, ASSIGNMENT, REPLACEMENT AND DISPOSITION OF ANIMALS
Animals were assigned to the study at the discretion of the coordinating bio technician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study. Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. The disposition of all animals was documented in the study records.

HUSBANDRY
Housing:
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records. Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Environmental Conditions:
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 42 to 57%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food:
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.

It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Municipal tap-water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment:
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

Veterinary Care:
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Vehicle:

dimethylformamide

Concentration:

0, 10, 25 and 60 % w/w test item

No. of animals per dose:

5 females

Details on study design:

Justification of Route and Dose Levels:
Dose route and dose concentrations used are in compliance with the OECD test guidelines for LLNA studies.

PRE-SCREEN TEST
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.

Two test item concentrations were tested; a 25% and 60% concentration. The highest concentration was the highest concentration that could be prepared homogeneously.

The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

Allocation:
Text table 1
Allocation
Group[1] animal numbers induction (test item; % w/w)
1 01 - 05 Vehicle control 0 (N,N-dimethylformamide (DMF))
2 06 - 10 Experimental low concentration 10
3 11 - 15 Experimental Intermediate concentration 25
4 16 - 20 Experimental high concentration 60
[1] five females per group

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the Nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

Tissue Processing for Radioactivity - Day 6:
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements - Day 7:
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

In-life Procedures, Observations, and Measurements:
Mortality/Moribundity Checks:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations:
Postdose Observations:
Postdose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.
Body Weights:
Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy).

Irritation:
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing), according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Erythema and eschar formation:
No erythema ..........................................................................................................................................................0
Very slight erythema (barely perceptible) .............................................................................................................1
Well-defined erythema ...........................................................................................................................................2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) ................................3
Severe erythema (beet redness) to eschar formation preventing grading of erythema ......................................4

Terminal procedures:
No necropsy was performed, since all animals survived until the end of the observation period.

Key result

Parameter:

SI

Value:

1

Variability:

± 0.1

Test group / Remarks:

Group 1, 0% w/w Test substance

Key result

Parameter:

SI

Value:

1.1

Variability:

± 0.3

Test group / Remarks:

Group 2, 10% w/w Test substance

Key result

Parameter:

SI

Value:

1.4

Variability:

± 0.2

Test group / Remarks:

Group 2, 25% w/w Test substance

Key result

Parameter:

SI

Value:

0.9

Variability:

± 0.3

Test group / Remarks:

Group 4, 60% w/w Test substance

Cellular proliferation data / Observations:

PRE-SCREEN TEST
At a 25 and 60% test item concentration, no signs of systemic toxicity were noted and up to very slight irritation was observed. Therefore, a 60% concentration was selected as highest concentration for the main study.

MAIN STUDY
Skin Reactions / Irritation
The very slight erythema of the ears as shown by animals treated at 60% on Days 2 and/or 3 was considered not to have a toxicologically significant effect on the activity of the nodes. White test item remnants were present on the dorsal surface of the ears of test item treated animals on Days 2 and/or 3 which did not hamper scoring of the skin reactions.

Systemic Toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic Examination of the Lymph Nodes and Surrounding Area
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 60% were 656, 854 and 559 DPM, respectively. The mean DPM/animal value for the vehicle control group was 624 DPM. The SI values calculated for the test item concentrations 10, 25 and 60% were 1.1, 1.4 and 0.9, respectively.

RESULTS

For detailed results see Appendix 1.

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 60%, 4-Hydroxy-benzophenone was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 60%.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity (see Appendix 3).

Based on these results, 4-Hydroxy-benzophenone would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this study was to evaluate whether 4-Hydroxy-benzophenone induces skin sensitization in CBA/J mice after three epidermal exposures of the animals under the conditions described in this report.

The study was carried out based on the guidelines described in:

• OECD, Section 4, Health Effects, No.429 (2010).

• EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".

• EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 25 and 60% test item concentration, no signs of systemic toxicity were noted and up to very slight irritation was observed. Therefore, a 60% concentration was selected as highest concentration for the main study.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 60% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-dimethylformamide (DMF)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes of the animals of the experimental and control groups were

considered normal in size. No macroscopic abnormalities of the surrounding area were noted

for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 60% were 656, 854 and 559 DPM, respectively. The mean DPM/animal value for the vehicle control group was 624 DPM. The SI values calculated for the test item concentrations 10, 25 and 60% were 1.1, 1.4 and 0.9, respectively.

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 60%, 4-Hydroxy-benzophenone was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 60%. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, 4-Hydroxy-benzophenone would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

4-Hydroxy-benzophenone yielded an alert for skin sensitization based on the presence of a substituted phenol group and predicted an EC3 of 1.5% based on LLNA data. Both positive and negative results in the LLNA and GPMT were observed in compounds containing a substituted phenol, therefore the alert is equivocal for skin sensitization. Based on the absence of SPCC and SPCL depletion observed in the DPRA the substance is not expected to bind with endogenous proteins. The KeratinoSensTM assay showed clearly activation of keratinocytes before cytotoxic concentrations were reached in all three experiments. This indicates that the cells may have become sensitized after exposure to the test item. The KeratinoSensTM assay and the DPRA result differ in their outcome for the skin sensitizing properties of 4-Hydroxy-benzophenone and thus additional data is required. Further in vitro testing with an U-SENSTM assay is considered not to give additional information. The cytotoxic properties of 4-Hydroxy-benzophenone are expected to negatively affect the dendritic cells resulting in an unreliable test outcome. The additional LLNA study has shown that this substance is not classified as a skin sensitiser.