Ammonium 3-nitrobenzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation, other

Remarks:

Defined approach

Type of information:

other: Bayesian Network DIP (BN-ITS-3) for hazard and potency identification of skin sensitizers (P&G)

Adequacy of study:

key study

Study period:

2018-03-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The chemical falls in the applicability domain of this defined approach.

Justification for type of information:

1. SOFTWARE
DC ITS SkinSens Integrated testing strategy and prediction of potency for skin sensitization

2. MODEL (incl. version number)
ChemAxon calculator, Douglas Connect Protein binding model, JSME Editor

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
N.OC(=O)c1cccc(c1)[N+]([O-])=O

4. SCIENTIFIC VALIDITY OF THE MODEL
Please refer to attached 'model report'

5. APPLICABILITY DOMAIN
The substance falls within the applicability domain of the model (please refer to attached 'prediction report')

6. ADEQUACY OF THE RESULT
Results of this model can be used in quantitative risk assessment (as 4 potency categories can be provided), please refer to attached 'model report')

Cross-reference

Reason / purpose for cross-reference:

other: study integrated in the defined approach

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017-12-11 - 2017-12-19 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

defined approach

Qualifier:

according to guideline

Guideline:

other: OECD 442E, In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Part 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT) (adopted 29. July 2016)

Deviations:

yes

Remarks:

see "deviations", but not affecting the validity of the study

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The REACH Regulation requires in vitro tests as a first approach.
The h-CLAT addresses the third key event of the skin sensitisation adverse outcome pathway (AOP) that was defined by the OECD in 2012 and is a part of the AOP-based “two out of three” skin sensitisation integrated testing strategy for hazard identification (Bauch et al., 2012).

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a dry, closed vessel at room temperature (20 ± 5 °C), protected from humidity.

OTHER SPECIFICS: Log Kow = -1.52

Details on the study design:

See free-text field "Any other information on materials and methods incl. tables"

Positive control results:

appropriate, see tables below

Key result

Run / experiment:

other: 1st (actually experiment II)

Parameter:

other: EC200 (for CD54)

Remarks:

in µg/ml

Value:

550.14

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Remarks:

vehicle controls

Positive controls validity:

valid

Key result

Run / experiment:

other: 1st (actually experiment II)

Parameter:

other: EC150 (for CD86)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Remarks:

vehicle controls

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

A calculation of the EC150 (for CD86) was not possible, no dose-response was evident, only one of the RFI values was above 150 at a lower dose, i.e. 334.9 µg/ml.

Key result

Run / experiment:

other: 2nd (actually experiment III)

Parameter:

other: EC200 (for CD54)

Remarks:

in µg/ml

Value:

569.06

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Remarks:

vehicle controls

Positive controls validity:

valid

Key result

Run / experiment:

other: 2nd (actually experiment III)

Parameter:

other: EC150 (for CD86)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Remarks:

vehicle controls

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

A calculation of the EC150 (for CD86) was not possible since none of the RFI values was above 150.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not stated

DEMONSTRATION OF TECHNICAL PROFICIENCY: done

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: no

Interpretation of results:

other: potential skin sensitizer

Conclusions:

The study was performed according to OECD TG 442E under GLP on the registered substance itself. Positive and negative control were valid.
The h-CLAT addresses the third key event of the skin sensitisation adverse outcome pathway (AOP) that was defined by the OECD in 2012 and is a part of the AOP-based “two out of three” skin sensitisation integrated testing strategy for hazard identification (Bauch et al., 2012). The sensitising potential of the test item was assessed by quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells, after incubation with the test item. The measured expression levels were used to distinguish potential skin sensitizers from non-sensitizers.
In both valid experiments (II and III) the RFI of CD54 was > 200 % at four non-cytotoxic test item concentrations (578.7 µg/ mL, 694.4 µg/ mL, 833.3 µg/ mL, 1000.0 µg/ mL).
Experiment II: The RFI of CD86 was > 150 % at one non-cytotoxic test item concentration (334.9 µg/mL).
Experiment III: The RFI of CD86 was not ≥ 150 % in any test item concentration.
In conclusion, it can be stated that under the experimental conditions of this study, the test item, Ammonium-3-nitrobenzoate, was positive in the h-CLAT and is therefore considered to have the potential to activate dendritic cells and is a potential skin sensitizer. Nevertheless it is not considered necessary to classify it in a two out of three approach, as outlined in the defined approach section of this data set.

Executive summary:

The determination of the sensitising potential of Ammonium-3-nitrobenzoate was done with the “In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)” following OECD 442E under GLP.

This in vitro study was performed to assess the sensitising potential of the test item Ammonium-3-nitrobenzoate by quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells.

In total one pre-test and three experiments (experiment I - III) with a treatment period of 24 hours were performed whereby experiment I was invalid and had to be repeated. Therefore, in total two valid experiments (experiment II and III) were performed.

For the experiments, the highest nominal applied concentration (1000 µg/mL) was chosen based on the results obtained in the pre-test. A geometric series (factor 1.2) of 7 dilutions thereof was prepared.

As solvent control for the test item, DMSO was used in a final concentration of 0.2 % in culture medium.

As positive control, 2,4-dinitrochlorobenzene (DNCB, CAS n. 97-00-7, ≥ 99% purity) was used.

In both valid experiments (II and III) the RFI of CD54 was > 200 % at four non-cytotoxic test item concentrations.

Experiment II: The RFI of CD86 was > 150 % at one non-cytotoxic test item concentration.

Experiment III: The RFI of CD86 was not ≥ 150 % in any test item concentration.

Since the majority result of the two individual runs is positive, the test item is considered as “positive”.

Conclusion: Under the experimental conditions of this study, the test item, Ammonium-3-nitrobenzoate, was positive in the h-CLAT and is therefore considered to have the potential to activate dendritic cells and therefore to be a potential skin sensitizer.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Bayesian Integrated Testing Strategy (ITS) for Skin Sensitization Potency Assessment: A Decision Support System for Quantitative Weight of Evidence and Adaptive Testing Strategy

Author:

Jaworska, J. S.; Natsch, A.; Ryan, C.; Strickland, J.; Ashikaga, T.; Miyazawa, M.

Year:

2015

Bibliographic source:

Arch. Toxicol. 2015, 89, 2355–2383.

Reference 2

Reference Type:

other: Application programming interface

Title:

DC ITS SkinSens Integrated testing strategy and prediction of potency for skin sensitization

Author:

Jaworska, J. S.

Year:

2016

Bibliographic source:

https://its.douglasconnect.com/

Materials and methods

Test guideline

Guideline:

other: OECD GD 256

Version / remarks:

Annex I, Case study IX

Principles of method if other than guideline:

- Software tool(s) used including version: DC ITS SkinSens Integrated testing strategy and prediction of potency for skin sensitization
- Model(s) used: ChemAxon calculator, Douglas Connect Protein binding model, JSME Editor
- Model description: The Bayesian Network ITS/DS for hazard and potency identification of skin sensitisers (BN DIP) estimates skin sensitisation potency in the LLNA, TG 429, expressed as probability distribution of LLNA pEC3, among 4 potency classes: nonsensitisers (NS), weak (W), moderate (M), and combined strong and extreme (S) sensitisers. In addition, it guides testing strategy by value of information. Please refer also to 'Attached justification'
- Justification of QSAR prediction: The BN DIP purpose is to provide skin sensitisation hazard and potency information for chemicals considering their mode of action. The information can be used in quantitative risk assessment (as 4 potency categories can be provided)

Type of study:

other: A defined approach consists of a fixed data interpretation procedure (DIP) (e.g. statistical models) applied to data (e.g in silico predictions, in chemico, in vitro data) generated with a defined set of information sources to derive a prediction.

Justification for non-LLNA method:

data from in silico models and the h-CLAT test were integrated in a defined approach

Test material

Test material information

Constituent 1

Reference substance name:

3-nitrobenzoic acid

EC Number:

204-508-5

EC Name:

3-nitrobenzoic acid

Cas Number:

121-92-6

Molecular formula:

C7H5NO4

IUPAC Name:

3-nitrobenzoic acid

Specific details on test material used for the study:

N.OC(=O)c1cccc(c1)[N+]([O-])=O

Results and discussion

In vitro / in chemico

Results

Key result

Parameter:

other: EC3

Remarks on result:

other: non-sensitiser

Remarks:

Bayes factor: 4.6313

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Combining the EC200 value of 299700 μM derived in the h-CLAT assay with calculated values indicating bioavailability and protein-binding in this defined approach, the substance is predicted to be 'not a sensitiser'. The reliability of the result is substantial (Bayes Factor 4.6313).

Executive summary:

Data indicating bioavailability from in silico models and the EC200 from the CLAT test were integrated in a defined approach ( The Bayesian Network ITS/DS for hazard and potency identification of skin sensitisers (BN DIP)). This DIP (BN ITS-3) is based on Bayesian Network methodology. Combining the EC200 value of 299700 μM derived in the h-CLAT assay with calculated values indicating bioavailability and protein-binding in this defined approach, the substance is predicted to be 'not a sensitiser'. The reliability of the result is substantial (Bayes Factor 4.6313).