Reaction mass of N-butylphthalimide and N-sec-butylphthalimide and N-propylphthalimide

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

4 February 2015

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

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. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

1

Reference substance name:

Reaction mass of N-butylphthalimide and N-sec-butylphthalimide and N-propylphthalimide

Molecular formula:

C11 H11 N O2 + C12 H13 N O2

IUPAC Name:

Reaction mass of N-butylphthalimide and N-sec-butylphthalimide and N-propylphthalimide

Test material form:

liquid

Specific details on test material used for the study:

Expiring date: 16 May 2018

In chemico test system

Details on the 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.

Results and discussion

Positive control results:

The positive control replicates showed the expected percent peptide depletion values within acceptable limits.

In vitro / in chemico

Results

Key result

Run / experiment:

other: 1-3

Parameter:

other: peptide depletion value

Remarks:

mean %

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.