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Description of key information

Results obtained from the in chemico Direct Peptide Reactivity Assay (OECD 442C), with the test item p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) indicated that the test item is not a potential skin sensitiser. Results obtained from the experimental human Cell Line Activatioin Test (OECD 442E), with the test item p-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) demonstrated an in vitro sensitizing potential with miT of 1869 ug/ml (in active substance).

Study OECD 442D in progress (05/2019), preliminary results negative. Based on available information and preliminary results the substance is considered not a sensitizing substance according to the CLP regulation 1272/2008/EC.

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:
20 October 2017 - 24 January, 2018
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
Qualifier:
according to guideline
Guideline:
other: DB-ALM (INVITTOX) Protocol 154: Direct Peptide Reactivity assay (DPRA) for skin sensitisation testing
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SANCO Guideline 3030/99 rev.4, Technical Material and Preparations: Guidance for generating and reporting methods of analysis in support of pre- and post-registration data requirements for Annex II and Annex III of Directive 91/414
Version / remarks:
July 11, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
other: Direct Peptide Reactivity Assay – DPRA
Details on the study design:
Principle of the DPRA Method

The reactivity of a test chemical and synthetic cysteine or lysine containing peptides was evaluated by combining the test chemical with a solution of the peptide (reaction samples) and monitoring the remaining concentration of the peptide following 24 ± 2 hours of interaction time at room temperature (25 ± 2.5°C). The peptide is a custom material containing phenylalanine to aid in detection and either cysteine or lysine as the reactive centre. Relative concentrations of the peptide following the 24 hour reaction time were determined by HPLC with gradient elution and UV detection at 220 nm. Samples were prepared and analysed in triplicates in batches to keep the total HPLC analysis time less than 30 hours.

Steps of the DPRA Method done in chronological order

- Solubility assessment of the test chemical – ultrapure water was used as a solvent
- Preparation of buffer solutions
- Pre-weighting of test chemicals and positive control
- Pre-weighting of cysteine or lysine peptide for the stock solution
- Test chemical and positive control solution preparation
- Peptide stock solution preparation
- Serial dilution of standards
- Assembling of standards, reaction samples, positive controls, reference controls (A, B and C) and co-elution controls. For each set of control/sample replicates, the triplicate vials are prepared individually but from the same solutions.
- Preparation of HPLC system (column equilibration)
- HPLC analysis
- Data evaluation

The vials were capped, vortexed to mix and placed to the HPLC autosampler for 24 ± 2 h incubation at 25± 2.5 ° C in the dark. HPLC analysis of the batch of reaction samples started 24 ± 2 h hours after the test chemical was added to the peptide solution. The batches were consisted of 2 parts: one part with the A reference controls, the calibration standards and the co-elution controls. These samples could be run before the 24 ± 2 h incubation time ends and right before the other part started or right after the other part. The other part contained the B and C reference controls, the positive controls and the reaction samples and these samples were run right after the 24 ± 2 h incubation time ended.

Demonstration of Proficiency

Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

Rejected Runs and Failure to Meet Acceptance Criteria

Individual chromatograms or runs could be rejected if the failure could be attributed to an assignable cause (e.g. error in reagent preparation, pipetting error, instrument failure). Reasons for rejections are indicated and discussed in the raw data. Rejected runs were repeated accordingly. Rejected chromatograms are also printed and reported in the raw data.

Percent peptide depletion

The concentration of the peptide was determined in each reaction sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of the peptide using the linear calibration curves derived from the standards.

The percent peptide depletion was determined in each reaction sample measuring the quotient of the peak area and the mean reference control C peak area, according to the formula described below.

peptide percent depletion =[1-( (peak area of the reaction sample)/(mean peak area of reference controls C ) )]× 100

Co-elution

The test chemical did not absorbed at 220 nm significantly at the same retention time as the peptides. Thus, there was no co-elution of the test chemical observed with either of the peptides (see representative chromatograms in Appendix I). The range of retention times for cysteine peptide was between 8.396 and 8.488. The range of retention times for lysine peptide was between 6.064 and 6.183.

Apparatus

HPLC System Conditions

HPLC system: SHIMADZU LC2030 (Prominence-i LC-2030C)
Serial number: L21445402951AE

Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 µm)
Serial number: USRY003976

Column temperature: 30°C
Sample temperature: 25°C
Detector: 220 nm (258 nm)
Injection volume: 7µL
System equilibration: 50% phase A and 50% phase B for 2 hours at 30°C and running the gradient twice before injecting the first sample
Run time: 20 min
Flow conditions: gradient flow

Table 2. Gradient flow conditions
Time Flow A phase (%) B phase (%)
0 min 0.35 mL / min 90 10
10 min 75 25
11 min 10 90
13 min 10 90
13.5 min 90 10
20 min gradient ends

Buffer Solutions

Phosphate buffer pH 7.5 ± 0.05
18 (v/v) % 0.1 M sodium phosphate monobasic
(of Sodium Phosphate Monobasic Monohydrate (NaH2PO4 · H2O) in purified water)
82 (v/v) % 0.1 M sodium phosphate dibasic
(of Sodium Phosphate Dibasic Heptahydrate (Na2HPO4 · 7H2O) in purified water)
pH was adjusted with either the monobasic or dibasic solution

Ammonium acetate buffer pH 10.2 ± 0.05
- 0.1 M Ammonium Acetate (CH3CO2NH4) in purified water
- pH was adjusted with Ammonium Hydroxide (NH4OH) drop by drop

Peptide Stock Solutions

Cysteine peptide, 0.667 mM, 0.501 mg/mL stock solution:
The needed amount of peptide was calculated based on the equation below (0.01312 g ± 10%). The previously calculated amount of the peptide (0.01352 g) was pre-weighted and 25 mL of pH 7.5 phosphate buffer was added in the volumetric glass right before beginning the assay.

Lysine peptide, 0.667 mM, 0.518 mg/mL stock solution:
The needed amount of peptide was calculated based on the equation below (0.01385 g ± 10%). The previously calculated amount of the peptide (0.01391 g) was pre-weighted and 25 mL of pH 10.2 acetate buffer was added in a volumetric glass right before beginning of the assay.

In a 25 mL volumetric glass:

(molecular weight)/(% purity) ×250=calculated amount of the peptide (mg)

Calibration Solutions

Six calibration standard points were prepared by serial dilution of the peptide stock solutions with the following nominal molarities: STD 1 = 0.534 mM, STD 2 = 0.267 mM, STD 3 = 0.1335 mM, STD 4 = 0.0667 mM, STD 5 = 0.0334 mM and STD 6 = 0.0167 mM. As dilution buffer a 20% acetonitrile:buffer solution (phosphate or ammonium acetate) was used. For the zero standard point (STD 7 = 0 mM) dilution buffer was used.

Positive Control Stock Solutions

100 mM solutions of the positive control chemical in acetonitrile were prepared just before use. The needed amount of test chemical was calculated (0.0668 g ± 10%) based on the molecular weight and purity of the substance with the equation below. 0.0665 g Cinnamaldehyde was weighted for the positive stock solution used for the cysteine peptide depletion determination and 0.0665 g Cinnamaldehyde was weighted for the stock solution used for lysine peptide depletion determination.

In a 5 mL volumetric glass:

(molecular weight)/(% purity) ×50=calculated amount of the peptide (mg)

Test Chemical Stock Solutions

100 mM solutions of the test chemical in the appropriate solvent (Section 5.1.3) were prepared just before use. The needed amount of test chemical was calculated (0.1614 g ± 10 %) and weighted based on the single average molecular weight. 0.1647 g test chemical was weighted for the stock solution used for the cysteine peptide depletion determination and 0.1645 g test chemical was weighted for the stock solution used for lysine peptide depletion determination.

Phases for HPLC

Mobile Phase A – 0.1 % (v/v) trifluoroacetic acid in ultra-pure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile
Mobile Phase C – 50 - 50 % (v/v) acetonitrile and ultrapure water
Positive control results:
The acceptance criteria were met in case of the positive control with the cysteine peptide depletion value of 73.08 % and a mean lysine peptide depletion value of 52.80 %. The SD of the percent peptide depletions of the positive control was 0.76 % and 0.42 % for the cysteine and lysine depletion respectively.
Key result
Parameter:
other: Mean % obtained peptide depletion
Value:
5.83
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
The chromatograms were evaluated with the help of “LabSolutions” software and the calculations were carried out using “Microsoft Office Excel”.

System suitability

Reference control A replicates were included in HPLC run the sequence to verify the HPLC system suitability prior the analysis. The mean peptide concentration of A reference control sample replicates was 0.51 mM for the cysteine peptide and 0.49 mM for the lysine peptide.

A standard calibration curve was generated for both cysteine and lysine peptides using serial dilutions standards from the peptide stock solutions. Calibration standard points were analysed by linear regression. Means of the peak areas versus the concentrations of both peptides showed good linearity with r2 = 0.9993 for the cysteine peptide and r2 = 1.0000 for the lysine peptide, covering the concentration range from 0.534 mM to 0.0167 mM. The back-calculated values for all the nominal concentrations of both peptides were within the acceptance criteria, not more than 16 % for the LOQs and not more than 12 % for the other calibration standards. Thus, all standards were accepted. All system suitability criteria were within acceptable limits and therefore the runs can be considered valid.

Analysis sequences

Reference control B replicates were included in the sequence to verify the stability of the peptide over time and reference control C replicates were used to verify that the solvent of the test item did not impact the percent peptide depletion. The mean cysteine peptide concentration of the reference control C replicates was 0.49 mM and the mean lysine peptide concentration of the reference control C replicates were 0.49 mM, which were within the acceptable 0.50 ± 0.05 mM range. Moreover the CV % for the nine reference control B and C replicates in acetonitrile and water respectively were much smaller than the acceptable 15 % for both peptides, since it was 0 % and 1 % for cysteine and lysine peptides. All validity criteria were within acceptable limits and therefore the study can be considered valid.

Cysteine and lysine depletion and mean peptide depletion of the test item

The acceptance criteria were met in case of the positive control with the cysteine peptide depletion value of 73.08 % and a mean lysine peptide depletion value of 52.80 %. The SD of the percent peptide depletions of the positive control was 0.76 % and 0.42 % for the cysteine and lysine depletion respectively.

The percent cysteine peptide depletion with the test item was 7.85 % while the percent lysine depletion was 3.80 %. The maximum standard deviation for the test chemical replicates was 4.31 % for the percent cysteine depletion and 0.70 % for the percent lysine depletion.
In Appendix II for the test chemical, the peptide peak areas of each replicate, their mean and CV %, the peptide depletion values for each replicate, their mean and SD and description of all relevant observations (solubility, precipitate, co-elution) are shown.

Assigning the test chemical to a reactivity class and category

The mean percent cysteine and percent lysine depletion values were calculated for the test chemical and the positive control. By using the cysteine 1:10 / lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of Integrated Approaches to Testing and Assesment (IATA). Application of the prediction models assigned the test chemical to a reactivity class (minimal, low, moderate or high reactivity).
On the basis of the cysteine 1:10 / lysine 1:50 prediction model, chemicals assigned to the minimal reactivity class should be classified as non-sensitisers whereas chemicals assigned to the low, moderate or high reactivity class should be classified as sensitisers.

The percent cysteine peptide depletion was 7.85 % while the percent lysine depletion was 3.80 %. The mean of percent cysteine and lysine peptide depletion of p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) was 5.83 % which is a minimal reactivity class classifying the test chemical into the non-sensitizer reactivity category.

Reference control A replicates for cysteine peptide

 

Peptide

peak area

at 220 nm

Peptide conc. calculated (mM)

Peptide peak area

Peptide concentration

Mean

CV

%

Mean (mM)

SD

CV %

ref A I

2312357

0.51

2298160

1%

0.51

0.0054

1%

ref A II

2270274

0.50

ref A III

2311848

0.51

 

Reference control A replicates for lysine peptide

 

Peptide

peak area

at 220 nm

Peptide conc. calculated (mM)

Peptide peak area

Peptide concentration

Mean

CV

%

Mean (mM)

SD

CV %

ref A I /1

2325482

0.49

2342526

1%

0.49

0.0052

1%

ref A I / 2

2323877

0.49

ref A I / 3

2325266

0.49

ref A II

2361455

0.50

ref A III

2376552

0.50

Reference control B and C replicates for cysteine peptide

Name, replicate number

Peptide

peak area

at 220 nm

Peptide conc. calculated (mM)

Peptide peak area

Peptide concentration

Mean

CV

%

Mean (mM)

SD

CV %

ref B I

2277206

0.51

2229905

3%

0.50

0.0132

0%

ref B II

2314043

0.51

ref B III

2273305

0.50

ref B I / 2

2183832

0.49

ref B II / 2

2241259

0.50

ref B III / 2

2186396

0.49

ref C I

2269044

0.50

ref C II

2196368

0.49

ref C III

2127689

0.47

Reference control B and C replicates for lysine peptide

Name, replicate number

Peptide

peak area

at 220 nm

Peptide conc. calculated (mM)

Peptide peak area

Peptide concentration

Mean

CV

%

Mean (mM)

SD

CV %

ref B I

2358218

0.49

2350478

0%

0.49

0.0051

1%

ref B II

2367146

0.50

ref B III

2372111

0.50

ref B I / 2

2361731

0.50

ref B II / 2

2364654

0.50

ref B III / 2

2372738

0.50

ref C I

2331096

0.49

ref C II

2310946

0.48

ref C III

2315666

0.49

Cysteine peptide depletion values for the positive control and the test item

Name, replicate number

Peptide

peak area

at 220 nm

Peptide conc. calculated (mM)

Peptide depletion

%

SD

(%)

ref C, rep I

2269044

0.50

-

-

ref C, rep II

2196368

0.49

-

ref C, rep III

2127689

0.47

-

p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), rep I

2026171

0.45

10.70 %

4.31

p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), rep II

1977588

0.44

9.96 %

p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), rep III

2066181

0.46

2.89 %

CINNAMALDEHYDE, rep I

604294

0.13

73.27 %

0.76

CINNAMALDEHYDE, rep II

609531

0.14

72.24 %

CINNAMALDEHYDE, rep III

586063

0.13

73.72 %

Lysine peptide depletion values for the positive control and the test item

Name, replicate number

Peptide

peak area

at 220 nm

Peptide conc. calculated (mM)

Peptide depletion

%

SD

(%)

ref C, rep I

2331096

0.49

-

-

ref C, rep II

2310946

0.48

-

ref C, rep III

2315666

0.49

-

p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), rep I

2224712

0.47

4.56%

0.70

p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), rep II

2226839

0.47

3.64%

p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), rep III

2241845

0.47

3.19%

CINNAMALDEHYDE, rep I

1124094

0.24

52.33%

0.42

CINNAMALDEHYDE, rep II

1109162

0.23

53.14%

CINNAMALDEHYDE, rep III

1116488

0.23

52.93%

Mean peptide depletion values for the positive control and the test chemical

Name, replicate number

Obtained mean % cysteine peptide depletion

Obtained mean % lysine peptide depletion

Mean % obtained peptide depletion

p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

7.85 %

3.80 %

5.83 %

CINNAMALDEHYDE

73.08 %

52.80 %

62.94 %

Prediction model Cysteine 1:10 / Lysine 1:50

Mean depletion values

Reactivity class

Reactivity category

Less than 6.38 %

Minimal reactivity

NON-SENSITISER

Between 6.38 % and 22.62 %

Low reactivity

SENSITISER

Between 22.62 % and 42.47 %

Moderate reactivity

More than 42.14 %

High reactivity

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was formulated with ultrapure water to obtain a homogenous solution at a concentration of 100 mM. The back calculated values of the reference control replicates were within the expected molarity concentration range and the linearity of standard calibration points showed good linearity.

The positive control replicates showed the expected percent peptide depletion values within acceptable limits. The experiment was considered to be valid.


Results obtained from this in chemico Direct Peptide Reactivity Assay, with the test item p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) indicated that the test item is not a potential skin sensitiser. The mean percent peptide depletion value of p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) was 5.83 %, which assigned the test chemical to the minimal reactivity class and a non-sensitiser category.

The DPRA result has been considered scientifically valid for the evaluation of the skin sensitisation potential. Consequently, information generated by the DPRA can already be used in a weight-of-evidence approach to support regulatory decision making, e.g to characterise equivocal responses in in vivo studies (e.g. conflicting results from multiple studies). Moreover, for the purposes of some regulations (for example REACH in the EU) a positive DPRA result should be considered sufficient to classify a test material as a skin sensitizer. However correct prediction of skin sensitisation potential of substances requires integration of information from multiple sources. Additionally combination of non-animal methods (in silico, in chemico, in vitro) within IATA will be needed to fully substitute for the animal tests currently in use, given the restricted AOP mechanistic coverage of each of the currently available non-animal test methods.
Executive summary:

This study was undertaken to evaluatethe skin sensitization potential of the test itemp-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)in chemico. The Direct Peptide Reactivity Assay (DPRA) proposed the molecular initiating event of the skin sensitisation Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of the test chemical towards cysteine and lysine model synthetic peptides.

 

Before beginning the assay thesolubility of the test chemical was assessed and ultrapure water was chosen as the appropriate solvent. Test chemical stock solutions were prepared in ultrapure water at the concentration of 100 mM. Cysteine and lysine peptide stock solutions were prepared at the concentrations of 0.501 mg/mL and 0.518 mg/mL with sodium phosphate buffer (pH=7.5) and ammonium acetate buffer (pH=10.2) respectively. Calibration standards were made by serial dilutions from the peptide stock solutions. Positive control stock solutions were prepared in acetonitrile at the concentration of 100 mM.The test chemical stock solutions were combined with the peptide stock solutions (1:10 and 1:50 ratio with cysteine and lysine peptides respectively) asreaction samples. Positive controls, reference controls and co-elution controls were assembled with the peptide stocks.The vials were placed to the HPLC autosampler for 24 ± 2 h incubation at 25 ± 2.5 °C in the dark.High performance liquid chromatography (HPLC) analysis of the batch of reaction samples started after the incubation peiod. Concentrations of the peptides following reaction time were determined by HPLC with gradient elution and UV detection at 220 nm.

 

Cysteine and lysine depletion values were used to categorize the test chemical in one of four classes of reactivity. By using the cysteine 1:10 / lysine 1:50 prediction model, the threshold of 6.38 % mean percent peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of Integrated Approaches to Testing and Assesment (IATA).

 

The positive control replicates showed the expected percent peptide depletion values within acceptable limits. The back-calculated values of the reference control replicates were within the expected molarity concentration range. The experiment was considered to be valid.

The mean percent peptide depletion value ofp-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)was 5.83 %, which is under the 6.38 % threshold of discrimination between sensitisers and non sensitisers.Results obtained from this
in chemicoDirect Peptide Reactivity Assay, with the test item p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) indicated that the test item is not a potential skin sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018 - study in progress
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)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Key result
Parameter:
other: Imax
Value:
0
Remarks on result:
other: preliminary results, study in progress
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16, October, 2017- 15, December, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 442E
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
other: Human Cell Line Activation test (h-CLAT)
Details on the study design:
Objective of the study
To evaluate the in vitro intrinsic senzitizing potential of the test item Revatol NS, using the human cell line activation test (h-CLAT).

Cytotoxicity was induced on THP-1 cells by Revatol NS annd was observed from the 1st activation test. In the followings the dose ranges were between 1594-3417ug/ml to cover cytotoxic profile until 50% of dead cells.

test system:ThP-1 cells
Preliminary study: cytotoxicity assay
Cell toxicity assessment was performed by determining cell viability on THP-1 cells using the7-AAD inclusion method.Eight concentration of the test item was prepared from the max conc of 1000ug/ml.

Main study: activation test
Based on the preliminary testthe max conc selected for activation test did not exceed 1000ug/ml when the test item was disslved or stably dispersed in Ethanol or DMSO and 5000ug/ml when the test item was dissolved in saline vehicle.
THP1 cells were stained FITC conjugated monoclonal antibodies as: anti-human CD54, anti-human CD86, FITC labeled mouse Ig G1. Cells were also stained with 7-AAD;
Fluorescence intensity was measured by flow cytometry.
Positive control results:
Positive controls:
DCNB (CV: crit:>50%; result: 83%)
NiSo4(CV: crit:>50%; result: 82%)
According to the results the testsystem was validated.
Key result
Run / experiment:
other: 1-3
Parameter:
other: Activation test (ug/ml)
Value:
3 417
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: 1-3
Parameter:
other: MIT ug/ml
Value:
1 869
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

in the 2nd exp dose response realtionship was observed for CD54 expression compared with the negative control: increasing 2.23 -3.01fold.

in the 3nd exp dose response realtionship was observed for CD54 expression compared with the negative control: increasing 2.06 -4.04fold.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on the results of the test item p-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) demonstrated an in vitro sensitizing potential with miT of 1869 ug/ml (in active substance) in condition of the experimental human Cell Line Activatioin Test during the study.
Executive summary:

Objective of the study

To evaluate the in vitro intrinsic senzitizing potential of the test item Revatol NS, using the human cell line activation test (h-CLAT).

Cytotoxicity was induced on THP-1 cells by Revatol NS annd was observed from the 1st activation test. In the followings the dose ranges were between 1594-3417ug/ml to cover cytotoxic profile until 50% of dead cells.

test system:ThP-1 cells

Preliminary study: cytotoxicity assay

Cell toxicity assessment was performed by determining cell viability on THP-1 cells using the7-AAD inclusion method.Eight concentration of the test item was prepared from the max conc of 1000ug/ml.

Main study: activation test

Based on the preliminary testthe max conc selected for activation test did not exceed 1000ug/ml when the test item was disslved or stably dispersed in Ethanol or DMSO and 5000ug/ml when the test item was dissolved in saline vehicle.

THP1 cells were stained FITC conjugated monoclonal antibodies as: anti-human CD54, anti-human CD86,  FITC labeled mouse Ig G1. Cells were also stained with 7-AAD;

Fluorescence intensity was measured by flow cytometry.

In the assay conditiions reproducible increase of CD54 expression compared to the negative controll at least for five levels of p-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) was noticed. No increase was observed in case of CD 86 expression.

Based on the results of the test item p-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) demonstrated an in vitro sensitizing potential with miT of 1869 ug/ml (in active substance) in condition of the experimental human Cell Line Activatioin Test during the study.

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