Registration Dossier

Administrative data

Description of key information

In vitro skin sensitization studies, performed on Nitrosodiphenylamine, showed that the substance is a skin sensitizer. To determine the potency of skin sensitization, no reliable read-across with a structural analogue was found, so a default classification in the sub-category 1A is proposed to avoid new animal testing.

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:
key study
Study period:
16 June 2017 - 04 July 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:
04 February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.
Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

Cysteine peptide (batch 111016HS_MHeW0117, supplier JPT Peptide Technologies GmbH)
Lysine peptide (batch 220114HSDWW0117, supplier JPT Peptide Technologies GmbH)

Vehicle used: Based on solubility results, the retained vehicle was acetonitrile (without sonication step).

Positive control: cinnamaldehyde (CAS No. 104-55-2), batch No. MKBV4784V, supplied by Sigma Aldrich. Its molecular weight was 132.16 g/mol and the purity of the batch used was 98.9%. The positive control was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in acetonitrile at 100 mM. The formulation was a colorless limpid solution and was used just after its preparation.

Criteria: Interpretation of results
The run was considered valid if the following criteria were fully met:
- the calibration curve should have a coefficient of determination (r2) >= 0.99,
- the mean peptide concentrations of the reference control A samples should be within ± 10% of the nominal concentration,
- the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
- for the cysteine peptide, the mean percent depletion value should be between 60.8 and 100% with a SD < 14.9%,
- for the lysine peptide, the mean percent depletion value should be between 40.2 and 69.0% with a SD < 11.6%,
- the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0%.
The test item’s results were considered valid if the following criteria were fully met:
- the mean peptide concentrations of the reference control C samples prepared in the appropriate solvent should be within ± 10% of the nominal concentration,
- the maximum SD for the test item replicates should be < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.
Parameter:
other: % depletion
Remarks:
lysine peptide
Value:
0.02
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Parameter:
other: % depletion
Remarks:
cysteine peptide
Value:
91.34
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: % mean depletion
Remarks:
Cysteine + Lysine
Value:
45.68
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: The mean of cysteine and lysine %depletion = 45.68%. Accordingly, the test item was considered have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples (Figures 1 and 4) indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:
- for the cysteine peptide, the mean depletion value was 91.34%,
- for the lysine peptide, the mean depletion value was 0.02%.

It is to be noted that since precipitate and micelles were observed at the end of the incubation with the lysine peptide, the corresponding peptide depletion may be underestimated. Therefore, the mean depletion value of 0.02% cannot be drawn with sufficient confidence.

The mean of the percent cysteine and percent lysine depletions was equal to 45.68%. Accordingly, the test item was considered have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

Conclusion: Under the experimental conditions of this study, the test item N-Nitrosodiphenylamine was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.
Interpretation of results:
other: positive in the DPRA assay (high peptide reactivity)
Conclusions:
Under the experimental conditions of this study, the test item N-Nitrosodiphenylamine was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.
Executive summary:

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.

The design of this study was based on the OECD guideline No. 442C and the study was performed in compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

 

Methods

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violetdetection at 220 nm.

Peptide reactivity was reported as percent depletion basedon the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

 

Results

The test item was dissolved at 100 mM inacetonitrile.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide:

.            for the cysteine peptide, the mean depletion value was 91.34%,

.            for the lysine peptide, the mean depletion value was 0.02%.

 

The mean of the percent cysteine and percent lysine depletions was equal to 45.68%. Accordingly, the test item was considered to have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

 

Conclusion

Under the experimental conditions of this study, the test item N-Nitrosodiphenylamine was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 June 2017 - 11 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:
Februray 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details on the study design:
IN VITRO TEST SYSTEM
KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.
Supplier: this cell line was provided by Givaudan.
Batch: C1.
Storage conditions: at -80°C.
Mycoplasm: absence of mycoplasm was confirmed.

MATERIALS AND METHODS
Vehicle and negative control: DMSO.
Positive control: Cinnamic aldehyde (CA).

Test item formulations: On the basis of solubility results, the test item was dissolved in DMSO at 200 mM.
One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 to obtain a total of 12 concentrations in a 96-well plate; this 96 well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96 well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

STUDY DESIGN:
Solubility assay:
The test item was found soluble in DMSO at 200 mM. Then, this stock formulation was diluted in treatment culture medium to the final concentration of 2000 µM. Aa visual inspection of the sample was performed to evaluate the presence or absence of precipitate/emulsion.

Treatment:
The test item was tested in two independent runs using cells from a different passage number.
After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 µL of treatment medium.
From the Master plate 4x, a volume of 50 µL was added to each well of the three white assay plates and 50 µL to the transparent plate for the cytotoxicity evaluation. All plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells. The plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.

Endpoint measurements:
Microscopic observation to evaluate the presence or absence of precipitate - transparent plate.
Luminescence flash signal to evaluate induction signal - white plates.
Absorbance signal to evaluate the cytotoxicity - transparent plate.

RESULTS ANALYSIS
Data evaluation was performed using a validated Excel sheet. The generated raw data (luminescence data for the luciferase activity and absorbance data for the MTT test) were pasted into an Excel template, and all data processing was performed automatically.

For the MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative control wells.
For the luciferase data, the average value of the six negative control wells was set to 1, and for each well in the plate, the fold induction was calculated in relation to this value.

For wells in which a statistically significant gene-induction (using a student test, also called T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data:
- Imax: maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured,
- EC1.5: concentration at which a 1.5-fold luciferase gene induction is obtained,
- IC50 and IC30: concentrations effecting a reduction of cellular viability by 50% and 30%,
- Indication whether significant 1.5-fold gene induction occurred below the IC30.

The data were plotted in graphs and the Imax and the EC1.5 values were visually checked since uneven dose response curves or large variation may lead to wrong extrapolations.
Also, the individual and overall geometric means IC50 and IC30 were calculated, when applicable.

ACCEPTANCE AND EVALUATION CRITERIA
Acceptance criteria
Each run was considered valid if the following criteria were met:
- the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
- the average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of Cinnamic Aldehyde was carefully checked, and the run was accepted if there was a clear dose response with increasing luciferase activity at increasing concentrations for the positive control,
- the average Coefficient of Variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%.

Evaluation criteria of the test item:
The results of each run are analyzed individually and if the test item is classified as positive in two runs, the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.

The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
- the EC1.5 value is < 1000 µM (or < 200 µg/mL for test item without MW),
- there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).
Run / experiment:
other: Experiments 1 & 2
Parameter:
other: I max value
Value:
6.33
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
other: Experiments 1 & 2
Parameter:
other: EC1,5 (µM)
Value:
37.41
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
All acceptance criteria were fulfilled for the positive and negative controls in each run; both runs were therefore considered as validated.

Both runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:
- slight to strong precipitate was observed in test item-treated wells at the end of the 48-hour treatment period at concentrations = 1000 µM in either run,
- during the first run, no noteworthy decrease in cell viability was noted and thus no IC30 or IC50 was calculated since the cell viability was > 70%. During the second run, a decrease in cell viability (i.e. cell viability < 70%) was noted at dose-levels = 1000 µM, and the corresponding IC30 and IC50 were 526.06 and 736.53 µM, respectively,
- statistically significant gene-fold inductions above the threshold of 1.5 was noted in comparison to the negative control at concentrations = 62.5 µM in the first run, and at concentrations = 125 µM in the second run. An apparent dose response relationship was also noted during both runs,
- the Imax values were 6.33 and 7.55 and the calculated EC1.5 were 37.41 and 86.92 µM in the first and second run, respectively.

The evaluation criteria for a positive response were met in both runs, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.
Interpretation of results:
other: positive in the KeratinoSens assay
Conclusions:
Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.
Executive summary:

The objective of this study was to evaluate the potential of the test ite, to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

The design of this study was based on the guideline No. 442D and the study was performed in compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Principle

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

 

Methods

 The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed.

 

Results

 

All acceptance criteria were fulfilled for the positive and negative controls in each run; both runs were therefore considered as validated.

 

Bothruns were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

 

At these tested concentrations:

slight to strong precipitate was observed in test item-treated wells at the end of the 48-hour treatment period at concentrations =1000 µM in either run,

during the first run, no noteworthy decrease in cell viability was noted and thus no IC30 or IC50 was calculated since the cell viability was > 70%. During the second run, a decrease in cell viability (i.e. cell viability < 70%) was noted at dose levels = 1000 µM, and the corresponding IC30 and IC50 were 526.06 and 736.53 µM, respectively,

statistically significant gene-fold inductions above the threshold of 1.5 was noted in comparison to the negative control at concentrations = 62.5 µM in the first run, and at concentrations = 125 µM in the second run. An apparent dose-response relationship was also noted during both runs,

the I max values were 6.33 and 7.55 and the calculated EC1.5 were 37.41 and 86.92 µM in the first and second run, respectively.

 

The evaluation criteria for a positive response were met in both runs, the final outcome is therefore positive.This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

Conclusion

 

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered toactivate the Nrf2 transcription factor.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

DPRA / OECD 442C (Chevallier 2017):

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.

The reactivity of the test item was evaluated in chemicoby monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.

Peptide reactivity was reported as percent depletion basedon the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

The test item was dissolved at 100 mM inacetonitrile.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide:

.            for the cysteine peptide, the mean depletion value was 91.34%,

.            for the lysine peptide, the mean depletion value was 0.02%.

The mean of the percent cysteine and percent lysine depletions was equal to 45.68%. Accordingly, the test item was considered to have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

Under the experimental conditions of this study, the test item N-Nitrosodiphenylamine was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.

KeratinoSens / OECD 442D (Richez 2017):

The objective of this study was to evaluate the potential of the test ite, toactivate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed.

Both runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

slight to strong precipitate was observed in test item-treated wells at the end of the 48-hour treatment period at concentrations =1000 µM in either run,

during the first run, no noteworthy decrease in cell viability was noted and thus no IC30 or IC50 was calculated since the cell viability was > 70%. During the second run,a decrease in cell viability(i.e.cell viability < 70%)was noted at dose levels = 1000 µM, andthe corresponding IC30 and IC50 were 526.06 and 736.53 µM, respectively,

statistically significant gene-fold inductions above the threshold of 1.5 was noted in comparison to the negative control at concentrations = 62.5 µM in the first run, and at concentrations = 125 µM in the second run. An apparent dose-response relationship was also noted during both runs,

the I max values were 6.33 and 7.55 and the calculated EC1.5 were 37.41 and 86.92 µM in the first and second run, respectively.

The evaluation criteria for a positive response were met in both runs, the final outcome is therefore positive.This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered toactivate the Nrf2 transcription factor.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Registered substance: Nitrosodiphenylamine (CAS number 86-30-6)

 

Reporting format for defined approaches to testing and assessment based on multiple information sources

 

1 Summary

In vitro skin sensitization studies, performed on Nitrosodiphenylamine, showed that the substance is a skin sensitizer. To determine the potency of skin sensitization, no reliable read-across with a structural analogue was found, so a default classification in the sub-category 1A is proposed to avoid new animal testing.

 

2 General information

2.1 Identifier: In order to evaluate the potential skin sensitizer properties and to not use animal tests, in vitro skin sensitization studies were performed.

 

2.2 Reference to main scientific papers:

-       ECHA.Guidance on Information Requirements and Chemical Safety Assessment.ChapterR.7a: Endpoint specific guidance. Version 6.0 – July 2017

-       ECHA.Guidance on Information Requirements and Chemical Safety Assessment.ChapterR.7c: Endpoint specific guidance. Version 2.0 – November 2014

-       OECD (2012) The Adverse Outcome Pathway for Skin Sensitization Initiated by Covalent Binding to Proteins, Part 1: Scientific Evidence, Series on Testing and Assessment No.168 (ENV/JM/MONO(2012)10/PART1), available at:http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2012)10/part1&doclanguage=en

-       OECD (2016) Guidance document on the reporting of defined approaches and individual information sources to be used within integrated approaches to testing and assessment (IATA) for skin sensitization. Series on testing & assessment No.256.

 

3 Endpoint addressed

Skin sensitization

 

4 Definition of the purpose

Meeting the REACH information requirement for skin sensitization (Annex VII, 8.3) and the relevant classification and/or risk assessment obligations.

 

5 Rationale underlying the construction of the defined approach

 “Due to the complexity of the skin sensitization endpoint, a combination of alternative test methods (e.g.in silico, in chemico and in vitro) in a Weight-of-Evidence approach needs to be considered to increase confidence in the final assessment of skin sensitization, e.g. a combination of read-across and non-animal test methods can be useful in concluding on the assessment of skin sensitization. “ (ECHA Guidance R7a, 2017, page 302).

The rationale of the defined approach is based on the strategy described in the ECHA Guidance R7a (2017, Pages 304-307,Figure R.7.3–2 Testing and assessment strategy for evaluating the skin sensitization potential of substances).

 

Element

Information

Conclusion for the registered substance

Existing data on physico-chemical properties

1

Is the substance a strong acid (pH= 2.0) or base (pH= 11.5), corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature?

 

NO

Existing human data

2

Are there adequate existing human data, which provide evidence that the substance is a skin sensitizer?

 

NO

Existing animal data from sensitisation studies

3

Are there data from existing studies on skin sensitisation in laboratory animals (LLNA, GPMT, or Buehler test, EU B.42, B.50, B.51 and B.6/OECD TGs 429, 442A, 442B and 406), which provide sound conclusive evidence that the substance is a sensitizer, or non-sensitizer?

 

NO

Existing (Q)SAR data and read-across

4

Do “read-across” from structurally and mechanistically related substances and/or do suitable (Q)SAR predictions reliably indicate skin sensitization potential or the absence thereof of the substance?

 

NO

Existing in chemico and in vitro data

5a

Is there evidence/hypothesis of dermal bioavailability based on physico-chemical, in silico, in vitro or in vivo data?

 

YES

 

5b

Has the substance demonstrated peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. OECD TG 442c)? (Key event 1 of the AOP), and/or

Has the substance demonstrated activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. OECD TG 442d)?(Key event 2 of the AOP), and/or

Has the substance demonstrated induction of the cell surface markers(CD54 and/or CD86) on monocytic cells in a validated in vitro test, e.g. h-CLAT?(Key event 3 of the AOP).

 

NO

5c

Are there data from (a)non-validatedin vitrotest(s), which provide evidence that the substance may be a skin sensitizer?

 

NO

Weight-of-Evidence analysis

6

The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-5) into account, is there sufficient information to meet the information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?

 

NO

Generation of new non-animal data

7a

Does the substance demonstrate peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. B. 59/OECD TG 442c)?(Key event 1 of the AOP)

 

YES

The test OECD 442c was performed and showed positive results.

7b

Does the substance demonstrate activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. B.60/OECD TG 442d)?(Key event 2 of the AOP)

 

YES

The test OECD 442d was performed and showed positive results.

7c

Does the substance demonstrate induction of the cell surface markers (CD54 and/or CD86) of monocytic cells in a validated in vitro test (e.g. h-CLAT)?(Key event 3 of the AOP)

 

n/a

study not performed

7d

Is any additional testing/generation of data considered necessary in order to conclude on classification, or e.g. to explain the inconsistent data obtained in previous elements or to address the Key event 4 of the AOP (T-cell proliferation) with an in vitro test?

 

NO

Weight-of-Evidence analysis

8

The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-7) into account, is there sufficient information to meet the respective information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?

 

YES

Based on the in vitro studies, the registered substance is considered to be skin sensitizer without data on potency.

The category 1A is proposed by default to avoid new animal testing.

The registered substance is considered to be a strong skin sensitizer (Skin sens.1A).

 

Generation of new in vivo data for sensitization as a last resort (Annex VII to the REACH Regulation)

9

Does the substance demonstrate sensitizing properties in an EU/OECD adoptedin vivotest, the LLNA (EU B.42/OECD TG 429)?

 

n/a

study not performed

 

 

6 Description of the individual information sources used within the approach

·        Dermal bioavailability :

No experimental toxicokinetic study is available on Nitrosodiphenylamine.

However, as per REACH guidance document R7.c (2014, pages 172-174), information on dermal absorption may be deduced from the physicochemical properties, including: molecular weight (198 g/mol), water solubility (35 mg/L) and the partition coefficient (Log Kow of 3,13).

NItrosodiphenylamine is a liquid having low molecular weight (< 500 g/mol), favourable to the dermal absorption. With a water solubility of 35 mg/L, the substance is considered to be moderately soluble, allowing a good dermal absorption. Log Kow value between 1 and 4 is favourable to a dermal absorption particularly if water solubility is high.To sum up, the physical-chemical properties of Nitrosodiphenylamine concluded that the substance is well absorbed after dermal exposure.That’s is confirmed by the carcinogenicity study performed by dermal route.

 

·        In vitro skin sensitization tests:

First of all,an in Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) was performed according to the OECD test guideline (N°442c).The objective of this study was to determine the reactivity of Nitrosodiphenylamine 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 photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated. In the cysteine reactivity assay, the test item showed 91,34% SPCC depletion while in the lysine reactivity assay the test item showed 0,02% SPCL depletion.The mean of the cysteine and lysine depletion was 45,68% and as a result Nitrosodiphenylamine was considered to be positive in the DPRA with high peptide reactivity.

 

Then, anIn Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method was performed according to the OECD test guideline (N°442D).The objective of this study was to evaluate the ability of Nitrosodiphenylamine to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensÔassay.

Nitrosodiphenylamine showed no toxicity in experiment 1. However,during the second run, a decrease in cell viability (i.e. cell viability < 70%) was noted at dose level of 1000 µM, and the corresponding IC30 and IC50 were 526.06 and 736.53 µM, respectively. A statistically significant dose-related induction of the luciferase activity (EC1.5 values of 37,41µMand 86,92µMin experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (I max) was 6,33-fold and 7,55-fold in experiment 1 and 2 respectively.Nitrosodiphenylamine is classified as positive in the KeratinoSens assaysince positive results (I max >1.5).

 

 

7 Data interpretation procedure applied

The data interpretation procedure (DIP) is currently not designed to provide information on the potency of a sensitiser. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E or the modified Myeloid U937 Skin Sensitisation Test (mMUSST)]. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification.

 

8 Substances used to develop and test the DIP

The DIP and the training set are described in the OECD documents (2012, 2016).

Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.

 

9 Predictive capacity of the approach

The DIP can be applicable to Nitrosodiphenylamine.

The training set used to define the DIP includes one amine substance.

 

10 Discussion and conclusion

The physical-chemical properties of Nitrosodiphenylamine concluded that the substance is well absorbed after dermal exposure. So, the skin sensitization potential of this substance should be evaluated as required by REACh regulation.

In absence of reliable skin sensitization data on Nitrosodiphenylamine,an in Chemico Skin Sensitization: Direct Peptide Reactivity Assay (DPRA) was performed according to the OECD test guideline (N°442c).The mean of the cysteine and lysine depletion was 45,68% and as a result Nitrosodiphenylamine was considered to be positive in the DPRA.

Then, anIn Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method was performed.Nitrosodiphenylamine is classified as positive in the KeratinoSens assay.

In summary, Nitrosodiphenylamine showed positive results for the Key Events 1 and 2 of the skin sensitization AOP developed by OECD. So, the substance is considered to be a skin sensitizer according to the “2 out of 3 - Sens ITS” rules.

However, no information is available on the potency of skin sensitization allowing to choose a sub-category of classification according to the Regulation EC n°1272/2008. As no information on the potency was found for Nitrosodiphenylamine, the subcategory 1A is proposed by default in order to avoid new animal testing.

Based on the weight-of-evidence approach, Nitrosodiphenylamine is classified as skin sensitizer, subcategory 1A according to the Regulation EC n°1272 (CLP).