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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April-May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In chemico test system

Details on study design:
The objective of the study was to determine the reactivity of N-acetylglycil beta-alanine towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of GenoWhite with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Milli-Q water (MQ) was found to be an appropriate solvent to dissolve GenoWhite was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

Results and discussion

Positive control results:
The results of the positive control cinnamic aldehyde are presented in the following table:

SPCC Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

Sample code Peak area at 220 nm (μAU) Concentration (mM) SPCC Depletion
PCcys-1 1083065 0.126 74.5%
PCcys-2 1103315 0.129 73.9%
PCcys-3 1037781 0.120 75.7%
Mean 74.7%
SD 0.9%
SD = Standard Deviation.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 74.7% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Mean SPCC depletion
Run / experiment:
Mean
Value:
4.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Mean SPCL depletion
Run / experiment:
Mean
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Mean of SPCC and SPCL depletion
Run / experiment:
mean
Value:
2.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptability of the Cysteine Reactivity Assay

The correlation coefficient (r2) of the SPCC standard calibration curve was 0.994. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.511±0.005 mM, the mean peptide concentration of Reference Controls C was
0.494±0.012 mM and the mean peptide concentration of Reference Controls Cwater was 0.454±0.007 mM. The mean Reference Control samples A, and C and Cwater were all within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve N-acetylglycyl beta-alanine did not impact the Percent SPCC Depletion.

The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 2.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 18.40. The mean A220/A258 ratio ± 10% range was 16.56-20.24. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 74.7% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).


Results Cysteine Reactivity Assay for N-acetylglycyl beta-alanine

Preparation of a 100 mM the test item stock solution in MQ showed that the test item was dissolved completely. Upon preparation and after approximately 24 hours of incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No
precipitate was observed in any of the samples.
In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test
item with SPCC. For the 207182/B-cys samples, the mean SPCC A220/A258 area ratio was 18.61. Since this was within the 16.56-20.24 range, this again indicated that there was no coelution of the test item with SPCC.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls Cwater. The mean Percent SPCC Depletion for N-acetylglycyl beta-alanine was 4.5% ± 4.5%.


Acceptability of the Lysine Reactivity Assay

The correlation coefficient (r2) of the SPCL standard calibration curve was 0.993. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.488±0.015 mM, the mean peptide concentration of Reference Controls C was
0.500±0.006 mM and the mean peptide concentration of Reference Controls Cwater was 0.475±0.011 mM. The mean Reference Control samples A, and C and Cwater were all within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve N-acetylglycyl beta-alanine did not impact the Percent SPCL Depletion.

The CV of the peptide areas for the nine Reference Controls B and C was 3.1%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 13.91. The mean A220/A258 ratio ± 10% range was 12.52-15.30. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 60.9% ± 3.1%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).


Results Lysine Reactivity Assay for N-acetylglycyl beta-alanine
Preparation of a 100 mM N-acetylglycyl beta-alanine
stock solution in MQ showed that the test item was dissolved completely. Upon preparation and after approximately 24 hours of incubation, both the CC as well as the test item samples were visually inspected. No precipitate was observed
in any of the samples.

In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the 207182/B-lys samples, the mean SPCL A220/A258 area ratio was 13.56. Since this was within the 12.52-15.30 range, this again indicated that there was no coelution of the test item with SPCL.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls Cwater. The mean Percent SPCL Depletion for
N-acetylglycyl beta-alanine was 0.0% ± 0.0% .

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
All acceptability criteria were met, therefore this DPRA is considered to be valid.
N-acetylglycyl beta-alanine was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

The objective of this study was to determine the reactivity of N-acetylglycyl beta-alanine towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of N-acetylglycyl beta-alanine with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The study procedures described in this report were based on the most recent OECD guidelines.

Milli-Q water (MQ) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

Cysteine reactivity assay                                   Lysine reactivity assay

Acceptability criteria       Results forSPCC       Acceptability criteria       Results for SPCL

Correlation coefficient (r2)

standard calibration curve              >0.99                              0.994                     >0.99                            0.993

Mean peptide concentration

RC-A samples (mM)                     0.50 ± 0.05              0.511 ± 0.005              0.50 ± 0.05              0.488 ± 0.015

Mean peptide concentration

RC-C samples (mM)                     0.50 ± 0.05              0.494 ± 0.012              0.50 ± 0.05              0.500 ± 0.006

Mean peptide concentration

RC-Cwater samples (mM)             0.50 ± 0.05              0.454 ± 0.007              0.50 ± 0.05              0.475 ± 0.011

CV (%) for RC samples

B and C                                                 <15.0                        2.2                            <15.0                            3.1

Mean peptide depletion

cinnamic aldehyde (%)                      60.8-100                     74.7                            40.2-69.0                     60.9

SD of peptide depletion

cinnamic aldehyde (%)                     <14.9                            0.9                               <11.6                            3.1

SD of peptide depletion for

N-actylglycyl beta-alanine (%)         <14.9                            4.5                                <11.6                             0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation; NA = Not Applicable.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and Cwater, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for N-acetylgycyl beta-alanine were all within the acceptability criteria for the DPRA.

No co-elution of the test item with SPCC or SPCL was observed.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the tables below.

SPCC and SPCL Depletion and Reactivity Classification for N-acetylglycyl beta-alanine

SPCC depletion              SPCL depletion              Mean of SPCC and SPCL depletion                     Reactivity class

Mean       ± SD           Mean              ± SD                                                                             Cysteine 1:10 / Lysine 1:50 prediction model

4.5%       ±4.5%          0.0%              ±0.0%                             2.3%                                    No or minimal reactivity

SD = Standard Deviation.

In the cysteine reactivity assay N-acetylglycyl beta-alanine showed 4.5% SPCC depletion while in the lysine reactivity assay N-acetylglycyl beta-alanine showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.3% and as a result N-acetylglycyl beta-alanine was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. N-acetylglycyl beta-alanine was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.