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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:
key study
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:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
yes
Remarks:
Minor deviation which was considered uncritical and which is not an acceptance criterium for the study.
GLP compliance:
yes (incl. certificate)
Type of study:
other: Direct Peptide Reactivity Assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In chemico test system

Details on study design:
Positive controls were treated identically as the test item. The following positive controls were used:
• Cinnamaldehyde (CAS 104-55-2, food grade ≥95 %, batch no. MKBT8955V) was used as 100 mM (± 10 %) solution in acetonitrile for the Cys-peptide.
• 2,3-Butanedione (CAS 431-03-8, ≥99 %, batch no. BCBM5232V) was used as 100 mM (± 10 %) solution in acetonitrile for the Lys-peptide
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments per-formed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing mid-range depletion for the lysine peptide.

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12
samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis
sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was
used for calculation of the peptide depletion.

Co-elution control
Sample prepared from the respective peptide buffer and the test item, but without peptide

Incubation
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber
for 23 h for the Cys-peptide and 22.5 h for the Lys-peptide, respectively, at 25 °C.
In experiment 2 the incubation time was 22 h for the Cys-peptide and 22 h and 5 min. for the Lys-peptide. None of the samples were turbid after incubation.

Results and discussion

Positive control results:
Acceptance criteria
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.
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.
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.

Assessment
The mean peptide depletion with the values 80.01 % and 80.87 % and a standard deviation of 2.28 % and 1.57 % for the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and < 14.9 %, respectively, for the Cys-peptide.
The mean peptide depletion with a value of 13.58 % and 24.62 % and a standard deviation of 1.25 % and 4.53 % for the 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.
The standard deviation for the test item replicates with 1.99 % and 2.32 % was < 14.9 % for the percent cysteine depletion for the test item. The standard deviation for the test item replicates with 0.14 % and 0.05 % was < 11.6 % for the percent lysine depletion for the test item.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Cys-Peptide Depletion
Run / experiment:
Rep. 1-3 / Exp. 1, Mean Value
Value:
7.03
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Cys-Peptide Depletion
Run / experiment:
Rep. 1-3 / Exp. 2 (mean)
Value:
4.78
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Lys-Peptide Depletion
Run / experiment:
Rep. 1-3 / Exp. 1 (mean)
Value:
0.27
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Lys-Peptide Depletion
Run / experiment:
Rep. 1-3 / Exp. 2 (mean)
Value:
0.03
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The sensitizing potential of the test item was examinied according to OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)). In conclusion, the DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item 3,3'-Oxybis-1 -propanol possesses no or minimal skin sensitisation potential.
Executive summary:

A study was performed in order to evaluate the reactivity of the test item 3,3'-Oxybis-1-propanoltowards cysteine (Cys-) and lysine (Lys-) containing peptides.

A test item solution in acetonitrile and the respective peptide was incubated 23 h at 25 °C for the Cys-peptide and 22.5 h for the Lys-peptide, respectively. In experiment 2 the incubation time was 22 h for the Cys-peptide and 22 h and 5 min. for the Lys-peptide. 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.

Two valid experiment were performed.

In experiment 1 the mean percent area ratio 220 nm/258 nm of the positive control 2,3-butanedione is 120 %. This is marginal out of the given range 90-110 % and shows a peak impurity and co-elution. This was considered uncritical, because the value of the peptide depletion was reported as “co-elution – percent depletion estimated”, it is not an acceptance criterium for the study. A second experiment was performed to verify the results of experiment 1, because the mean percent peptide depletion was 3.65 % and thereforeit is a borderline resultbetween 3 % and 10 %. In this case it is considered to repeat the experiment. The peptide depletion values after incubation are shown in the Table:

Table3        Results

 

Cys-peptide
depletion [%]

Lys-peptide
depletion [%]

Mean peptide
depletion [%]

Experiment 1

7.03

0.27

3.65

Experiment 2*

4.78

0.03

2.40

*Note: Experiment 2 was performed to verify the result of experiment 1.

In conclusion, the DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item 3,3'-Oxybis-1 -propanol possesses no or minimal skin sensitisation potential.