4-phenylbenzophenone

Endpoint summary

Description of key information

The data from a reliable In Chemico Skin Sensitization: Direct Peptide Reactivity Assay (DPRA) undertaken to evaluate the skin sensitisation potential of the substance showed that the substance was not a dermal sensitiser. However precipitate formation was observed in the lysine incubation samples after incubation and thus the % SPCL depletion might be underestimated. Consequently, this SPCL result is uncertain and should be interpreted with due care.

The substance was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway employing the KeratinoSens assay. The substance is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations <1000 µM with a cell viability of >70% compared to the vehicle control.

The substance was evaluated for its potential to induce skin sensitization in mice after three epidermal exposures of the animals in the in vivo Local Lymph Node Assay (LLNA). The SI values calculated for the substance concentrations 5, 10 and 25% were 1.3, 2.3 and 2.9, respectively. The highest mean SI value was 2.9 for the 25% concentration, which is close to the threshold for classification (SI ≥ 3). Based on these results the substance would be regarded as a skin sensitizer and warrants classification as skin sensitizer (Category 1B).

A QSAR analysis ( DEREK NEXUS version 5.0.2) was conducted on the substance. The substance did not yield any alerts for skin sensitization. The QSAR model concluded and predicted the substance to be not sensitizing to the skin.

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:

13 Jun 2017 - 27 Jun 2017

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Sponsor; 20161118
- Expiration date of the batch: 17 November 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Solubility and stability of the test substance in the solvent/vehicle: Yes, maximum temperature: 90°C; Solubility of the test item in an appropriate solvent was assessed before performing the DPRA.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No reactivity with the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
-Preparation of test item: No correction for the purity/composition of the test item was performed.
- Final preparation of a solid: For the cysteine and lysine reactivity assay, respectively, 42.67 mg of and 26.05 mg Omnirad 4 PBZ were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1652 µL and 1009 µL ACN to obtain 100 mM solutions.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Liquid

Positive control results:

Cinnamic aldehyde - The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 78.1% ± 1.8%. 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

Value:

0.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Parameter:

other: SPCL depletion

Value:

2.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Acceptability of the Cysteine Reactivity Assay:
The correlation coefficient (r2) of the SPCC standard calibration curve was 0.996. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
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 Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 2.3%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 18.78. The mean A220/A258 ratio ± 10% range was 16.90-20.65. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 78.1% ± 1.8%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Acceptability of the Lysine Reactivity Assay:
The correlation coefficient (r2) of the SPCL standard calibration curve was 0.991. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
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 CV of the peptide areas for the nine Reference Controls B and C was 2.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 14.32. The mean A220/A258 ratio ± 10% range was 12.89-15.75. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 60.9% ± 1.9%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Test item	SPCC depletion		SPCL depletion						DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD	Mean of SPCC and SPCL depletion				Cysteine 1:10 / Lysine 1:50 prediction model
Omnirad 4 -PBZ	0.6%	1.1%	2.5%	2.7%	1.5%				Negative: No or minimal reactivity

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, this DPRA test is valid. The substance 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. However, since precipitation was observed upon addition of the test item to the SPCL peptide solution,it is not certain how much test item remained in the solution to react with the SPCL peptide. Consequently, this SPCL result is uncertain and should be interpreted with due care.

Executive summary:

An In Chemico Skin Sensitization: Direct Peptide Reactivity Assay (DPRA) was employed to evaluate the skin sensitisation potential of the substance by determining the reactivity of the substance towards model synthetic peptides containing either cysteine or lysine, and to categorize the test item in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers. The substance was used neat as provided and the study met the acceptance criteria. In the cysteine reactivity assay the test item showed 0.6% SPCC depletion while in the lysine reactivity assay the test item showed 2.5% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.5% and as a result Omnirad 4-PBZ was classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitate formation was observed in the lysine incubation samples after incubation, the % SPCL depletion might be underestimated. Consequently, this SPCL result is uncertain and should be interpreted with due care.

Reference 2

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

4th of October 2017

Reliability:

2 (reliable with restrictions)

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:

QSAR Model as weight of evidence for substance.

Qualifier:

no guideline available

Version / remarks:

QSAR analysis ( DEREK NEXUS version 5.0.2)

Principles of method if other than guideline:

Expert derived structural alerts for skin sensitisation (2D SARs), physicochemical properties and associated reasoning. Following alert evaluation, Derek will make a prediction of skin sensitisation potency for alerting query compounds, where possible. This is based on the activity (EC3 values) for nearest neighbours derived from a local lymph node assay data set.

GLP compliance:

no

Positive control results:

N/A

Key result

Remarks on result:

not measured/tested

Key result

Remarks on result:

not measured/tested

Key result

Remarks on result:

not measured/tested

Interpretation of results:

GHS criteria not met

Conclusions:

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. 4-phenylbenzophenone is predicted to be not sensitizing to the skin.

Executive summary:

QSAR analysis ( DEREK NEXUS version 5.0.2) was conducted on the substance. The substance did not yield any alerts for skin sensitization. The QSAR model concluded and predicted the substance to be not sensitizing to the skin.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 July 2017 - 04 August 2017

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:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 20161118
- Expiration date of the lot/batch: 17 November 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature away from light

Positive control results:

EC1.5 (µM) for the positive controls = 23.9µM and 39 in experiment 1 and 2, respectively (acceptance criteria is between 5 and 125 µM).
Imax = 3.7 and 2.5 in experiment 1 and 2, respectively (acceptance criteria is above 1.5).

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5

Value:

0.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5

Value:

18.84

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see table 1

Table 1- Historical Control Data for the KeratinoSens Studies

	Positive Control
	EC1.5	Imax
Range	5.7 -109.0	1.79 -11.13
Mean	43.2	3.51
SD	29.0	1.60
n	46	46

Table 2 - Overview EC1.5, Imax, IC30 and IC50 Values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test Item Experiment 1	<0.06	18.84	37	51.9
Test Item Experiment 2	0.64	1.7	N/A	N/A
Positive Control Experiment 1	23.9	3.7	N/A	N/A
Positive Control Experiment 2	39	2.5	N/A	N/A

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The substance is classified as positive in the KeratinoSens assay (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The substance was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway employing the KeratinoSens assay according to OECD TG 442D. The substance showed toxicity in experiment 1 (IC₃₀value of 37µM and IC₅₀value of 52µM). A biologically relevant, dose-related induction of the luciferase activity (EC_1.5values of < 0.06µM and 0.64µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (I_max) was 18.84-fold and 1.70-fold in experiment 1 and 2, respectively. The substance is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations <1000 µM with a cell viability of >70% compared to the vehicle control.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 December 2017 - 30 January 2018

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:

Section 4, Health Effects (2010).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 20161118
- Expiration date of the lot/batch: 22 November 2018

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: Young adult animals (approximately 10 weeks old) were selected.
- Weight at study initiation: 17.3 to 22.7 g.
- Housing: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
- IN-LIFE DATES: From: 03 January 2018 to 29 January 2018.

Vehicle:

dimethylformamide

Concentration:

10% and 25% concentration.

No. of animals per dose:

5/group

Positive control substance(s):

other: For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available for at least the recent 9 years showing reproducible and consistent positive results.

Positive control results:

For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available for at least the recent 9 years showing reproducible and consistent positive results.

Key result

Parameter:

SI

Value:

2.9

Test group / Remarks:

25% (top concentration)

Key result

Parameter:

SI

Value:

2.3

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

5%

Interpretation of results:

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

Conclusions:

The substance was found to be a skin sensitiser in the in vivo Local Lymph Node Assay.

Executive summary:

The substance was evaluated for its potential to induce skin sensitization in mice after three epidermal exposures of the animals in the in vivo Local Lymph Node Assay (LLNA). Five female CBA/J mice were treated with the substance at concentrations of 5, 10 or 25% 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). Three days after the last exposure, all animals were injected with³H-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. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. 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 the substance concentrations 5, 10 and 25% were 930, 1696 and 2101 DPM, respectively. The mean DPM/animal value for the vehicle control group was 731 DPM. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.3, 2.3 and 2.9, respectively. The highest mean SI value was 2.9 for the 25% concentration, which is close to the threshold for classification (SI ≥ 3).  Based on these results the substance would be regarded as a skin sensitizer and warrants classification as skin sensitizer (Category 1B).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the findings of two reliable in vitro studies (Direct Peptide Reactivity Assay (DPRA) and the KeratinoSens assay) and an in vivo LLNA study conducted on the substance, classification of the substance is justified (Skin Sens. 1B, H317: May cause an allergic skin reaction).