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Diss Factsheets

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
• In the experiments 1 and 2 demineralized water was used instead of HPLC grade or Millipore Milli-Q grade water. This was seen as uncritical, because the reference con-trols showed that the used water has no negative impact on the test system. The deviation was assessed and signed by the study director on 27. May 2019.
Deviations:
yes
Remarks:
see Version/ remarks
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
This study was performed in order to estimate the skin sensitisation potential of N-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT) using a peptide model. The direct peptide reactivity assay (DPRA) is an in chemico assay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential. To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item was measured using HPLC. The assay was used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible.

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(N-methyl-p-toluidino)ethanol
EC Number:
220-638-5
EC Name:
2-(N-methyl-p-toluidino)ethanol
Cas Number:
2842-44-6
Molecular formula:
C10H15NO
IUPAC Name:
2-[methyl(4-methylphenyl)amino]ethan-1-ol
Test material form:
liquid: viscous
Specific details on test material used for the study:
Based on this non-GLP pre-test, the 100 mM test item solution was prepared by dissolving 49.8 mg (experiment 1) and 50.0 mg (experiment 3) test item in 3 mL acetonitrile for the Cys-peptide assay. For the Lys-peptide assay 49.8 mg (experiment 1) and 50.0 mg (experiment 2) were solved in acetonitrile. The solutions were vortexed until the test item was dissolved completely. 

Results and discussion

Positive control results:
Table 8.3 a Calculated peptide depletion values for the Cys-Peptide, experiment 1
Sample name Depletion [%]
Single Mean SD
Positive control Rep. 1 76.35 76.67 0.42
Positive control Rep. 2 76.51
Positive control Rep. 3 77.14

Table 8.3 b Calculated peptide depletion values for the Cys-Peptide, experiment 3
Sample name Depletion [%]
Single Mean SD
Positive control Rep. 1 77.25 77.55 0.44
Positive control Rep. 2 78.06
Positive control Rep. 3 77.33

Table 8.3 c Calculated peptide depletion values for the Lys-Peptide, experiment 1
Sample name Depletion [%]
Single Mean SD
Positive control Rep. 1 20.76 20.56 0.24
Positive control Rep. 2 20.29
Positive control Rep. 3 20.63

Table 8.3 d Calculated peptide depletion values for the Lys-Peptide, experiment 2
Sample name Depletion [%]
Single Mean SD
Positive control Rep. 1 20.19 20.66 0.69
Positive control Rep. 2 20.35
Positive control Rep. 3 21.45

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: experiment 1
Parameter:
other: Calculated peptide depletion values for the Cys-Peptide
Value:
9.34
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Key result
Run / experiment:
other: experiment 3
Parameter:
other: Calculated peptide depletion values for the Cys-Peptide
Value:
5.61
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Key result
Run / experiment:
other: experiment 1
Parameter:
other: Calculated peptide depletion values for the Lys-Peptide
Value:
1.06
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Key result
Run / experiment:
other: experiment 2
Parameter:
other: Calculated peptide depletion values for the Lys-Peptide
Value:
2.16
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Other effects / acceptance of results:
8.3.3 Acceptance criteria
a) The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100.0 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
b) The mean peptide depletion value for the positive control 2,3-Butanedione should be 10.0 % - 45.0 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
c) The standard deviation for the test item replicates should be < 14.9 % for the per-cent cysteine depletion and < 11.6 % for the percent lysine depletion.
8.3.4 Assessment
a) The mean peptide depletion with 76.67 % and 77.55 % a respective standard de-viation of 0.42 % and 0.44 % of the three replicates of the positive control cin-namaldehyde were in the acceptable range of 60.8 – 100.0 % and < 14.9 %, re-spectively, for the Cys-peptide assays.
b) The mean peptide depletion with 20.56 % and 20.66 % and a respective standard deviation of 0.24 % and 0.69 % of three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and < 11.6 %, respec-tively, for the Lys-peptide assays.
c) The standard deviation for the test item replicates with 0.54 and 2.58 % was < 14.9 % for the percent cysteine depletion for the test item in both experiments.
The standard deviation for the test item replicates with 0.97 % and 0.73 % was < 11.6 % for the percent lysine depletion for the test item.

Any other information on results incl. tables

According to the test guideline, the reactivity is classified as “high”, “moderate”, “low” or “minimal” using the Cysteine 1:10/Lysine 1:50 prediction model shown in Table8.4–a.

 

Table8‑eEvaluation of results according to the Cysteine 1:10/Lysine 1:50 prediction model

Mean peptide depletion
[%]

Reactivity Class

DPRA Prediction

0 – ≤ 6.38

No or Minimal

Negative

> 6.38 – ≤ 22.62

Low

Positive

> 22.62 – ≤ 42.47

Moderate

> 42.47 - ≤ 100

High

 

The mean peptide depletion in the Lys-peptide and Cys-peptide assay was 5.20 %, therefore the test had to be repeated for verification of this result. The mean peptide depletion in the Lys-peptide and Cys-peptide in the repeated experiments was 3.88 %. The result of the first experiment was considered verified and therefore the test item was classified with:

 

DPRA Prediction: Negative

Reactivity class: No or Minimal

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
All acceptance criteria were fulfilled; therefore, the test was considered valid.

Experiment 1 was valid for both peptide assay, but the mean peptide depletion of both peptides falls in the range of 3 % to 10 %. This means it is a borderline result, close to the threshold used to discriminate between positive and negative results. Therefore, it was necessary to perform a second run to verify the DPRA prediction.
In Experiment 2 the mean peptide concentration of the reference control A of the Cys-peptide assay was not in the given range. Therefore, this experiment was invalid for the Cys-peptide assay. For the Lys-peptide assay experiment 2 was valid and the peptide-depletion value could be used for calculation.
After experiment 2 an error was detected in the preparation of the peptide buffers. Demin-eralized water was used instead of HPLC grade or Millipore Milli-Q grade water. This was seen as uncritical, because the reference controls showed that the water has no impact on the test system.
The third experiment was performed with the correct buffer preparation and was valid for the Cys-peptide.
The mean peptide depletion value calculated with the depletion values of the Lys-peptide of experiment 2 and the Cys-peptide of experiment 3 confirmed the DPRA prediction of the first experiment and lead to an overall negative DPRA prediction.
In conclusion, the DPRA prediction is “negative” according to the Cysteine 1:10/Lysine 1:50 prediction model. Thus, under the experimental conditions reported, the test item N-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT) shows no reactivity towards the two model synthetic peptides.
This assignment supports the discrimination between skin sensitisers and non-sensitisers in the framework of an integrated approach (IATA).
For sensitising potency assessment within an IATA, the test item N-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT) could be assigned to the reactivity class that covers no or min-imal reactivity.
No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.

Executive summary:

The study was performed in order to evaluate the reactivity of the test itemN-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT)towards cysteine (Cys-) and lysine (Lys-) containing peptides. The results are used for supporting the discrimination between skin sensitisers and non-sensitisers.The DPRA is part of a tiered strategy for the evaluation of skin sensitisation potential in the context ofan integrated approach to testing and assessment (IATA).

The test item was incubated for 22 h at 25 °C together with Cys- and Lys-peptides, respectively. The peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

 

Three experiments were performed.

Experiment 1 was valid, but the mean peptide depletion of both peptides falls in the range of 3 % to 10 %. This means it is a borderline result, close to the threshold used to discriminate between positive and negative results. Therefore, a second run was performed to verify the DPRA prediction.

In Experiment 2 the mean peptide concentration of the reference control A of the Cys-peptide assay was not in the given range. Therefore, this experiment was invalid for the Cys-peptide assay. For the Lys-peptide assay experiment 2 was valid and the results are reported here.

The third experiment was valid for the Cys-peptide assay and the results are reported here.

The mean peptide depletion value calculated with the depletion values of the Lys-peptide of experiment 2 and the Cys-peptide of experiment 3 confirmed the DPRA prediction of the first experiment and lead to an overall negative DPRA prediction.

The invalid experiment is not reported in this report, but the raw data are kept in the test facility in the GLP- archive.


The peptide depletion values after incubation are shown in Table3‑a:

Table3‑a        Results

 

Cys-peptide
depletion [%]

Lys-peptide
depletion [%]

Mean peptide
depletion [%]

Experiment 1

9.34

1.06

5.20

Experiment 2

Invalid

2.16

-*

Experiment 3

5.61

2.16**

3.88**

*Note: Due to the invalid Cys-peptide assay no calculation of the mean peptide depletion was possible.

**Note: In experiment 3, the Lys-peptide assay was not performed, because a valid result could be obtained in experiment 2. The Lys-peptide result of experiment 2 was used for calculation of the mean peptide depletion together with the result of the Cys-peptide assay of experiment 3 .

 

In conclusion, the DPRA prediction is “negative” according to the Cysteine 1:10/Lysine 1:50 prediction model. Thus, under the experimental conditions reported, the test itemN-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT)shows no reactivity towards the two model synthetic peptides.

This assignment supports the discrimination between skin sensitisers and non-sensitisers in the framework of an integrated approach (IATA).

For sensitising potency assessment within an IATA, the test itemN-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT) could be assigned to the reactivity class that covers “no or minimal” reactivity.