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Description of key information

The substance did not yield any alerts for skin sensitization using DEREK NEXUS version 6.0.1. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The substance is predicted to be not sensitizing to the skin.

The substance was negative in a reliable in chemico assay (DPRA) and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.   However, the substance was positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) in a reliable in vitro study. The substance was also found to be positive in the OECD 442E, U-SENS assay in which an increase in the expression levels of CD86 cell surface marker in the U937 cell line was observed.

Consequently, an in vivo murine LLNA was performed to evaluate the sensitisation potential of the substance in female mice (five animals/concentration). The SI of 2.2 (at the highest concentration of 50%) indicates that the substance is not a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 February 2018 - 15 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
yes
Remarks:
The SPCL depletion for the positive control was outside the acceptance criteria. Since this was only slightly above the upper limit while all other acceptability criteria were met, the result was accepted.
GLP compliance:
yes
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 171116
- Expiration date of the lot/batch: 16 November 2019

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: Solubility of the test item in an appropriate solvent was assessed before performing the DPRA.

Positive control results:
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 74.2% ± 1.6%. 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: Mean of SPCC and SPCL depletion
Value:
0.9
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
All validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent SPCC value for the positive control, the standard deviation value of the peptide depletion for the positive control and the standard deviation value of the peptide depletion for the test item, were within the acceptability criteria for the DPRA.

 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
1.5%   ±2.6% 0.2%   ±0.4%  0.9%  Negative: No or minimal reactivity
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, the substance was negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

The reactivity of the substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was evaluated in the Direct Peptide Reactivity Assay (DPRA). The aim was to categorize the substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers. In the cysteine reactivity assay the substance showed 1.5% SPCC depletion while in the lysine reactivity assay the substance showed 0.2% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.9% and as a result the substance 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.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March 2018 - 30 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted 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: 171116

STABILITY AND 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: Dissolved in DMSO

Positive control results:
EC1.5 of the positive control was between 5 and 125 µM (61 µM and 98 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.75-fold and 2.72-fold in experiment 1 and 2, respectively).
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
5.98
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
5.05
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
The EC1.5 of the positive control was between 5 and 125 µM (61 µM and 98 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.75-fold and 2.72-fold in experiment 1 and 2, respectively).
The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (8.8% and 14% in experiment 1 and 2, respectively).

Overview EC1.5, Imax, IC30and IC50Values

 EC1.5(µM)  Imax  IC30(µM)  IC50(µM)
Test item Experiment 1   <0.98  5.98  3.6  5.4
 Test item Experiment 2  <0.49  5.05  4.1  6.8
 Pos Control Experiment 1  61  2.75  N/A   N/A
 Pos Control Experiment 2  98  2.72   N/A  N/A 
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, the substance is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described.
Executive summary:

The ability of the substance to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway was evaluated in the in vitro KeratinoSens assay. Two independent experiments were performed. 4,4’-Bis(diethylamino)benzophenone showed toxicity (IC30values of 3.6µM and 4.1µM and IC50values of 5.4µM and 6.8 µM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5values of < 0.98µM and < 0.49µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 5.98-fold and 5.05-fold in experiment 1 and 2 respectively. The substance is positive in the KeratinoSensTMassay since positive results (>1.5-fold induction) were observed at test concentrations < 1000µM with a cell viability of >70% compared to the vehicle control.

Endpoint:
skin sensitisation, other
Remarks:
QSAR
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
9th Febuary
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
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Principles of method if other than guideline:
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.

The level of likelihood of a structure being sensitizing to skin is expressed in terms of:
Certain There is proof that the proposition is true.
Probable There is at least one strong argument that the proposition is true and there are no arguments against it.
Plausible The weight of evidence supports the proposition.
Equivocal There is an equal weight of evidence for and against the proposition.

The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment.
If a substance is predicted to be no skin sensitizer, DEREK NEXUS contains an expert-derived functionality to provide negative predictions for skin sensitization. This functionality further evaluates those compounds which do not fire any skin sensitization alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of Ames test or skin sensitization data, producing the following outcomes:
• In compounds where all features in the molecule are found in accurately classified compounds from the reference set, a negative prediction is displayed: inactive.
• For those query compounds where features in the molecule are found in non-alerting skin sensitizers in the Lhasa reference set, the prediction remains negative and the misclassified features are highlighted to enable the negative prediction to be verified by expert assessment.
• In cases where features in the molecule are not found in the Lhasa reference set, the prediction remains negative and the unclassified features are highlighted to enable the negative prediction to be verified by expert assessment.

If a substance is predicted to be a skin sensitizer, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental data from the closest structurally-related substances (at least 3 substances should be present) using the following equation:
GLP compliance:
not specified
Justification for non-LLNA method:
QSAR
Specific details on test material used for the study:
Identification: 4,4’ Bis(diethylamino) benzophenone
Trade name: Omnirad EMK
CAS Number: 90-93-7
Molecular weight: 324.46
Molecular formula: C21H28N2O
Species:
other: N/A
Strain:
other: N/A
Sex:
not specified
Details on test animals and environmental conditions:
N/A
Positive control results:
N/A due to the nature of QSAR
Key result
Remarks on result:
not measured/tested
Other effects / acceptance of results:
N/A
Key result
Remarks on result:
not measured/tested
Key result
Remarks on result:
not measured/tested
Cellular proliferation data / Observations:
N/A
Interpretation of results:
study cannot be used for classification
Conclusions:
DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the substance. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. 4,4’ Bis(diethylamino) benzophenone is predicted to be not sensitizing to the skin.
Executive summary:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the substance. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The substance is predicted to be not sensitizing to the skin. Due to the nature of QSAR, the substance endpoint and assay cannot be used as a primary source for classification however will be considered in a weight of evidence context.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2018 - 28 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
yes
Remarks:
A deviation from the maximum level of daily mean target humidity (71%) occurred on one day. This study plan deviation is considered not to have affected the integrity of the study.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Appearance: White to pale yellow green powder
Batch: 171116
Substance storage: At room temperature protected from light
Stable under storage conditions until: 16 November 2019 (expiry date)
Substance handling: Use amber glassware or wrap container in aluminum foil
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Age at the Initiation of Dosing: Young adult animals (approximately 10 weeks old) were selected.
Weight at the Initiation of Dosing: 20.0 to 23.3 g.
Acclimatisation period: 5 days.
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 44 to 71%. 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.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 or 50% w/w
No. of animals per dose:
Five per concentration.
Details on study design:
Substance concentrations selected for the main study were based on the results of a pre-screen test. At 50% and 25% concentrations, no signs of systemic toxicity were noted and no irritation was observed and therefore the 50% 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 the substance at concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
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.
Key result
Parameter:
SI
Value:
2.7
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
2.2
Test group / Remarks:
50%

During the pre-test, no signs of systemic toxicity were noted and no irritation was observed at 50% and 25% concentrations, and therefore the 50% concentration was selected as highest concentration for the main study.

For one animal treated at 10% (Animal No. 8), scabs of the eyelids in combination with closed eyes were noted on Days 5 and 6. In addition, the right lymph node of this animal was considered to be significantly enlarged compared to normal and the animal showed a DPM value of 8539, which was found to be a statistical outlier based on the Dixon’s Q test. Based on the above, the animal was excluded from interpretation and is therefore not included in the results below. 

No irritation was observed in any of the animals.

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.

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not a skin sensitizer according to the recommendations made in the test guidelines and as such does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact.
Executive summary:

A study was undertaken to evaluate the potential of the substance to induce delayed contact hypersensitivity using the murine LLNA according to OECD Test Guideline 429. Evaluation of irritation was also conducted in parallel by measurement of ear thickness. A preliminary test was conducted in order to define the concentration of the substance to be used in the main test. The concentrations of the substance selected for the main study were based on the results of a pre-screen test. At 50% and 25% concentrations, no signs of systemic toxicity were noted and no irritation was observed and therefore the 50% 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 the substance at concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). 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. 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. No irritation was observed in any of the animals. 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 at concentrations of 10, 25 and 50% were 1114, 996 and 926 DPM, respectively. The mean DPM/animal value for the vehicle control group was 412 DPM. The SI values calculated for the substance at concentrations of 10, 25 and 50% were 2.7, 2.4 and 2.2, respectively. Since there was no indication that the substance elicits a SI3 when tested up to 50%, the substance was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated substance concentration that will give a SI =3) (if any) exceeds 50%. Based on these results, the substance would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 May 2018 - 13 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-SENS™)’ (9 October 2017).
Version / remarks:
9 October 2017
Deviations:
no
GLP compliance:
yes
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
Appearance: White to pale yellow green powder
Batch: 171116
Test item storage: At room temperature protected from light
Stable under storage conditions until: 16 November 2019 (expiry date)
Details on study design:
Three experiments were conducted per concentration to demonstrate reproducibility of the results and conclusion.
Positive control results:
Experiment 1: The positive control (TNBS) showed a S.I. ≥ 195% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2: The positive control (TNBS) showed a S.I. ≥ 300% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 3: The positive control (TNBS) showed a S.I. ≥ 212% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Key result
Parameter:
other: EC150
Run / experiment:
Experiment 1
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
Based on the observed cytotoxicity, colour and solubility interference.
Key result
Parameter:
other: EC150
Run / experiment:
Experimnent 2
Value:
2.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC150
Run / experiment:
Experiment 3
Value:
> 0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Experiment 1: CV70 value < 1.0 µg/mL
Experiment 2: No CV70 value
Experiment 3: CV70 value 0.41
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The substance is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line).
Executive summary:

The ability of the substance to increase the expression levels of CD86 cell surface marker was evaluated in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay. The experiment was conducted according to the most recent OECD guideline. In all experiments the positive (TNBS) and negative (LA) control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly. In the first experiment the substance showed toxicity at all tested concentration (CV70value < 1.0 µg/mL). No biologically relevant induction of the CD86 activity (No EC150value) was measured at any of the concentrations. The individual run conclusion was Positive based on the observed cytotoxicity, colour and solubility interference. In the second experiment the substance showed no toxicity (No CV70value). A biologically relevant, induction of the CD86 activity (EC150value 2.5 µg/mL) was measured. The individual run conclusion was Positive based on the CD86 activity observed. Because of the difference in results of cell viability and CD86 activity between the first and second experiment an additional third experiment was performed. In the third experiment the substance showed toxicity (CV70value 0.41). A biologically relevant induction of the CD86 activity (EC150value< 0.1 µg/mL) was measured. The individual run conclusion was Positive based on CD86 activity, cytotoxicity interference and colour interference.  

Overall, the substance is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at concentrations with a cell viability of >70% compared to the vehicle control in 2 out of 3 experiments. Moreover interference (cytotoxicity, colour and solubility) was observed in all experiments.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

A structure activity assessment and three in vitro assays gave mixed results. The structure activity predicted negative and the DPRA assay gave a negative response. However an in vitro Keratinosens and an in vitro U-SENS both gave a positive result. Given the mixed pattern of results an in vivo LLNA assay was undertaken to give a definitive result and this study was negative. Taking into account all the results obtained, in particular the LLNA result, the balance of evidence is against classification.