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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:
key study
Study period:
2018/09/28 - 2018/10/04
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
yes
Remarks:
date of draft report delayed. No effect on study
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Bisguanidinium phosphate
EC Number:
226-552-4
EC Name:
Bisguanidinium phosphate
Cas Number:
5423-23-4
Molecular formula:
CH5N3.1/2H3O4P
IUPAC Name:
Diguanidinium hydrogen phosphate
Test material form:
solid: particulate/powder

In chemico test system

Details on the study design:
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.

Cinnamaldehyde dissolved in acetonitrile was used as positive control. 100 mM solutions of the positive control chemical in acetonitrile were prepared just before use. 100 mM solutions of the test chemical in ultrapure water were prepared just before use.

Results and discussion

Positive control results:
The acceptance criteria were met in case of the positive control with the cysteine peptide depletion value of 70.09 % and a mean lysine peptide depletion value of 57.24 %. The SD of the percent peptide depletions of the positive control was 0.65 % and 0.84 % for the cysteine and lysine depletion respectively.

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
other: 1
Parameter:
other: Cysteine peptide depletion [%]
Value:
6.55
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: Cysteine peptide depletion [%]
Value:
7.76
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Run / experiment:
other: 3
Parameter:
other: Cysteine peptide depletion [%]
Value:
12.74
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Run / experiment:
other: 1
Parameter:
other: Lysine peptide depletion [%]
Value:
0.23
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: Lysine peptide depletion [%]
Value:
0.53
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Run / experiment:
other: 3
Parameter:
other: Lysine peptide depletion [%]
Value:
-0.29
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Negative depletion values are substituted with zero when calculating mean peptide depletion
Run / experiment:
mean
Parameter:
other: Cysteine peptide depletion [%]
Value:
9.02
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Run / experiment:
mean
Parameter:
other: Lysine peptide depletion [%]
Value:
0.26
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Run / experiment:
mean
Parameter:
other: Peptide depletion [%]
Value:
4.64
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL 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.

ACCEPTANCE OF RESULTS:
Two individual tests were conducted with the cysteine peptide, since the first one did not meet the acceptance criteria and had to be rejected. The second run was valid and accepted. Only one individual run was needed with the lysine peptide, which has fulfilled all the acceptance criteria. All in all, the results of the two valid runs were used for the classification of the test item.

Applicant's summary and conclusion

Interpretation of results:
other: EU GHS criteria not met
Conclusions:
The mean percent peptide depletion value of the test itemwas 4.64 %, which corresponded to a negative outcome. Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item bisguanidinium phosphate indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitiser.
Executive summary:

This study was undertaken to evaluate the skin sensitisation potential of the test item bisguanidinium phosphate 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. At the beginning of the assay the solubility of the test chemical was assessed and ultrapure water was chosen as the appropriate solvent. Two individual tests were conducted with the cysteine peptide, since the first one did not meet the acceptance criteria and had to be rejected. The second run was valid and accepted. Only one individual run was needed with the lysine peptide, which has fulfilled all the acceptance criteria. All in all, the results of the two valid runs were used for the classification of the test item. The positive control replicates showed the expected percent peptide depletion values within acceptable limits with 70.09 % cysteine peptide depletion and 57.24 % lysine peptide depletion. The back-calculated values of the reference control replicates were within the expected molarity concentration range for the cysteine and lysine peptides, as well. The percent cysteine peptide depletion value of the test item was 9.02 % while the percent lysine peptide depletion was 0.26 %.The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides, therefore the Cysteine 1:10 / Lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers. The mean peptide depletion of the test item was 4.64 %, which did not exceed the 6.38 % threshold of the applicable prediction model. The mean percent peptide depletion value of the test item was 4.64 %, which corresponded to a negative outcome. Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item bisguanidinium phosphate indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitiser.