Registration Dossier

Administrative data

Description of key information

A valid DPRA assay was performed according to OECD 442C and GLP principles. The test item was dissolved in acetonitrile. The percent cysteine peptide depletion was 3.72 % while the percent lysine depletion was 23.37 %. The mean percent peptide depletion value of Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide was 13.54 %, which is within the range of low reactivity class values (6.38 % - 22.62 %). Results obtained from this in chemico Direct Peptide Reactivity Assay, with the test item Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide indicated that the test item is a potential skin sensitiser.

 

A valid KeratinosensTMassay was performed according to OECD 442D and GLP principles. Test item was tested at 12 concentrations from 0.2µg/ml-400µg/ml. Two independent experiments were performed. Imax were lower than 1.5 no EC 1.5 was determined. Under the described conditions Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide the KeratinoSens is considered negative (<1.5-fold induction).

 

A valid h-CLAT assay was performed according to OECD 442E and GLP principles. Based on the solubility and the cell toxicity assays the max dose levels selected for the main study was 200ug/ml. In the 1st experiment a dose response relationship was observed for CD54 at any of the tested test item concentration, but without of increase of the CD86 expression. In the 2nd experiment a dose response relationship was observed for CD54 expression compared with the negative control: increasing 2.24 -4.25 fold,but without of increase of the CD86 expression. In the 3rd experiment a dose response relationship was observed for CD54 expression compared with the negative control: increasing 2.28 -4.09 fold, with slight increase of the CD86 expression.

Based on the results of the test item Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide demonstrated an in vitro sensitizing potential with Minimum induction threshold (MIT) of 44 ug/ml under the experimental conditions described in the experimental human Cell Line Activatioin Test.

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:
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
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: DB-ALM (INVITTOX) Protocol 154: Direct Peptide Reactivity assay (DPRA) for skin sensitisation testing
Version / remarks:
Protocol 154
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
other: Direct Peptide Reactivity Assay – DPRA
Specific details on test material used for the study:
Expiring date: 16 May 2018
Details on study design:
Formulation of the Test Item

The test item was formulated and examined in the test as follows: solubility of the test item was evaluated in acetonitrile and water at the concentration of 100 mM. The formulation with acetonitrile was homogenous, however limited solubility of the test item was observed in ultrapure water as the formulation separated into two phases. Since acetonitrile is the preferred vehicle according to the guideline [4], solubility in other accepted vehicles was not evaluated.

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

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 – acetonitrile 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 ± 2h incubation at 25± 2.5°C in the dark. HPLC analysis of the
batch of reaction samples started 24 ± 2h 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 . 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 6.106 and 6.241.
Positive control results:
The positive control replicates showed the expected percent peptide depletion values within acceptable limits.
Key result
Parameter:
other: peptide depletion value
Remarks:
mean %
Run / experiment:
1-3
Value:
13.54
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
low reactivity class
Other effects / acceptance of results:
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 were 0.51 mM for both cysteine and lysine peptides. Data for the reference control A sample replicates are presented in Table 5 and 6.

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 = 0.9998 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. The data and regression analysis of the calibration curves are shown in Appendix II. The graphical representation of cysteine and lysine peptide calibration lines are presented in Figure 1. 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. Both the mean cysteine peptide concentration of the reference control C replicates and lysine peptide concentration of the reference control C replicates were 0.50 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 were much smaller than the acceptable 15 % for both peptides, since it was 2 % and 1 % respectively 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 57.66 %. The SD of the percent peptide depletions of the positive control was 0.76 % and 0.79 % for the cysteine and lysine depletion respectively. With the cysteine peptide the mean peptide concentration of the three reference controls C was 0.50 mM and it was 0.50 mM with the lysine peptide, as well. The calculated concentrations of reference control C met the acceptance criteria.

The percent cysteine peptide depletion with the test item was 3.72 % while the percent lysine depletion was 23.37 %. The maximum standard deviation for the test chemical replicates was 2.81 % for the percent cysteine depletion and 0.49 % 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 3.72 % while the percent lysine depletion was 23.37 %. The mean of percent cysteine and lysine peptide depletion of Reaction mass of N butylphthalimide and n propylphthalimide and N-sec-butylphthalimide was 13.54 % which is a low reactivity class classifying the test chemical into the 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

2358554

0.50

2383511

2%

0.51

0.0091

2%

ref A I / 2

2350028

0.50

ref A I / 3

2349359

0.50

ref A II

2430201

0.52

ref A III

2429415

0.52

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

2240283

0%

0.50

0.0101

2%

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

2260866

0.50

ref C II

2195960

0.49

ref C III

2229676

0.50

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

2319924

0.50

2343538

0%

0.50

0.0065

1%

ref B II

2332923

0.50

ref B III

2368026

0.51

ref B I / 2

2317839

0.50

ref B II / 2

2336105

0.50

ref B III / 2

2413067

0.52

ref C I

2324881

0.50

ref C II

2346847

0.50

ref C III

2332226

0.50

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

2260866

0.50

-

-

ref C, rep II

2195960

0.49

-

ref C, rep III

2229676

0.50

-

CINNAMALDEHYDE, rep I

604294

0.134

73.27%

0.76

CINNAMALDEHYDE, rep II

609531

0.136

72.24%

CINNAMALDEHYDE, rep III

586063

0.130

73.72%

Reaction mass of N-butylphthalimide and n-propylphthalimide

 and N-sec-butylphthalimide, rep I

2205719

0.490

2.44%

2.81

Reaction mass of N-butylphthalimide and n-propylphthalimide

 and N-sec-butylphthalimide, rep II

2043423

0.454

6.95%

Reaction mass of N-butylphthalimide and n-propylphthalimide

 and N-sec-butylphthalimide, rep III

2189829

0.486

1.79%

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

2324881

0.50

-

-

ref C, rep II

2346847

0.50

-

ref C, rep III

2332226

0.50

-

CINNAMALDEHYDE, rep I

965360

0.204

58.48%

0.79

CINNAMALDEHYDE, rep II

1011241

0.214

56.91%

CINNAMALDEHYDE, rep III

989280

0.209

57.58%

Reaction mass of N-butylphthalimide and n-propylphthalimide

 and N-sec-butylphthalimide, rep I

1794618

0.383

22.81%

0.49

Reaction mass of N-butylphthalimide and n-propylphthalimide

 and N-sec-butylphthalimide, rep II

1791359

0.382

23.67%

Reaction mass of N-butylphthalimide and n-propylphthalimide

 and N-sec-butylphthalimide, rep III

1780735

0.380

23.65%

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

CINNAMALDEHYDE

73.08 %

57.66 %

65.37 %

Reaction mass of N-butylphthalimide and

n-propylphthalimide and N-sec-butylphthalimide

3.72 %

23.37 %

13.54 %

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:
other: Reactivity class: Low reactivity
Remarks:
Reactivity category: SENSITISER
Conclusions:
Results obtained from this in chemico Direct Peptide Reactivity Assay, with the test item Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide indicated that the test item is a potential skin sensitiser. The mean percent peptide depletion value of Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide was 13.54 %, which assigned the test chemical to the low reactivity class.
Executive summary:

This study was undertaken to evaluatethe skin sensitization potential of the test itemReaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimidein 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.

 

At the beginning of the assay thesolubility of the test chemical was assessed and acetonitrile was chosen as

the appropriate solvent. Test chemical stock solutions were prepared in acetonitrile 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, as well.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 of Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide was 13.54 %, which is within the range of low reactivity class values

(6.38 % - 22.62 %).Results obtained from this
in chemicoDirect Peptide Reactivity Assay, with the test item Reaction mass of
N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide indicated that the test item is a

potential skin sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16, October, 2017-12, February, 2018
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
4th, 2015
Deviations:
yes
Remarks:
study plan deviation: The IC 70 formula shown in the Study Plan was wrong. The IC70 was calculated with the good one shown page11/17 of this report.
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
One of the biological key takes place in keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling pathway such as ARE dependent pathway.
The genes under ARE control , including AKR1C2 gene identified as a target for detecting skin senzitizers in keratinocytes, are induced by the protein called Nrf2 via Keap1.
The test was done by: KeratinoSens; it consists in evaluating the activation of AKR1C2 in transformed keartinocytes by monitoring the induction of the luciferase gene fused to AKR1C2. The luciferase produced by the cells complexes with luciferin which, in the presence of ATP, produces light , measured in relative llight units (RLU). In parallel the cytotoxicity is measured.

Test item was tested at 12 concentrations from 0.2µg/ml-400µg/ml

Testsystem-.KeartinoSens

Test protocol
Day 1st: cell seeding
Day 2nd: preparation of test item dilutiions
Day2nd: contact between the cells and the test and the reference items
Day 4th: Luciferase activity measurement: after 48 h the medium was aspired, cells were washed with PBS, then 100uM luciferase substrate was added. Plates were incubated for 15 minutes. Luciferase activity was measured by luminometer.

Day4th: cell viability assessment with MTT method
Cells were washed, then staining solution were added to each well; after 4:30 hours incubation cells were treated with SDS; after overnight incubation abs were measured at 540nm.
Positive control results:
Positive control: Cinnamaldehyde

Imax EC1.5 IC70
Mean value 5.09 12.74µM >64µM
Standard dev 2.91 4.91 -
Mean EC1.5 value +/- - 2.9µM= EC1.5 = 22.6µM -

Study is considered valid
Key result
Parameter:
other: viability (geom mean)
Remarks:
CI 70 ug/ml
Run / experiment:
Rep 1, 2
Value:
> 400
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: induction Imax (mean)
Run / experiment:
Rep 1,2
Value:
1.01
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Under the retained conditions Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide code ID-17/08402 may be classified as not skin sensitizer.
Executive summary:

One of the biological key takes place in keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling pathway such as ARE dependent pathway.

The genes under ARE control , including AKR1C2 gene identified as a target for detecting skin senzitizers in keratinocytes, are induced by the protein called Nrf2 via Keap1.

The test was done by: KeratinoSens; it consists in evaluating the activation of AKR1C2 in transformed keartinocytes by monitoring the induction of the luciferase gene fused to AKR1C2. The luciferase produced by the cells complexes with luciferin which, in the presence of ATP, produces light ,  measured in relative llight units (RLU). In parallel the cytotoxicity is measured.

 

Test item was tested at 12 concentrations from 0.2µg/ml-400µg/ml.

 

Testsystem-.KeartinoSens

 

Test protocol

Day 1st: cell seeding

Day 2nd: preparation of test item dilutiions

Day2nd: contact between the cells and the test and the reference items

Day 4th: Luciferase activity measurement: after 48 h the medium was aspired, cells were washed with PBS, then 100uM luciferase substrate  was added. Plates were incubated for 15 minutes. Luciferase activity was measured by luminometer.

 

Day4th: cell viability assessment with MTT method

Cells were washed, then staining solution were added to each well; after 4:30 hours incubation cells were treated with SDS; after overnight incubation abs were measured at 540nm.

 

VIABILITY                                   INDUCTION

CI 70 µM                                          Imax              EC1.5lin       EC1.5lin/lig

Rep1                >400                        1.07              -                     -

Rep2              >400                     0.96              -                     -

Mean                      -                        1.01              -                     -

Geometric mean >400                             -              -                     -

Imax are lower than 1.5 no EC 1.5 is determined.

Under the retained conditions Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide code ID-17/08402 may be classified as not skinsensitizer.

 

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16, October, 2017- 16, January, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 442E
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
other: Human cell activation test (h-CLAT)
Details on 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 Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide and was observed from the 1st activation test. In the followings the dose ranges were between 32.4-200ug/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: 84%)
NiSo4(CV: crit:>50%; result: 82%)
According to the results the testsystem was validated.
Key result
Parameter:
other: MIT ug/ml
Run / experiment:
1-3
Value:
44.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: Activation test (ug/ml)
Run / experiment:
1-3
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

Based on the solubility and the cell toxicity assays the max dose levels selected for the main study was 200ug/ml.

In the assay conditiions reproducible increase of CD54 expression compared to the negative controll at least for five levels of Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide.

In the 1st exp: dose response realtionship was observed for CD54 at any of the tested test item concentration, but without of increase of the CD86 expression.

In the 2nd exp:dose response realtionship was observed for CD54

expression compared with the negative control: increasing 2.24 -4.25 fold,

but without of increase of the CD86 expression.

In the 3rd exp:dose response realtionship was observed for CD54

expression compared with the negative control: increasing 2.28 -4.09 fold,

but with slight increase of the CD86 expression.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on the results of the test item Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide demonstrated an in vitro sensitizing potential with miT of 44 ug/ml (in active substance) in conditioin 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 Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide and was observed from the 1st activation test. In the followings the dose ranges were between 32.4-200ug/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.

Based on the solubility and the cell toxicity assays the max dose levels selected for the main study was 200ug/ml.

In the assay conditiions reproducible increase of CD54 expression compared to the negative controll at least for five levels of Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide.

In the 1st exp: dose response realtionship was observed for CD54 at any of the tested test item concentration, but without of increase of the CD86 expression.

In the 2nd exp:dose response realtionship was observed for CD54 expression compared with the negative control: increasing 2.24 -4.25 fold,

but without of increase of the CD86 expression.

In the 3rd exp:dose response realtionship was observed for CD54 expression compared with the negative control: increasing 2.28 -4.09 fold,

but with slight increase of the CD86 expression.

Based on the results of the test item Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide demonstrated an in vitro sensitizing potential with miT of 44 ug/ml (in active substance) in conditioin of the experimental human Cell Line Activatioin Test during the study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Results obtained from In Chemico Determination of Skin Sensitisation (Direct Peptide Reactivity Assay – DPRA, OECD 442C) and human Cell Line Activation Test (h-CLAT, OECD 442E) with the test item Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide indicated that the test item is a potential skin sensitiser. Under the experimental conditions (KeratinoSens, OECD 442D) Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide showed no indication of a skin sensitisation.

Overall, based on the test results the substance is considered a skin sensitizing substance.