Enoxolone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

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:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Enoxolone

EC Number:

207-444-6

EC Name:

Enoxolone

Cas Number:

471-53-4

Molecular formula:

C30H46O4

IUPAC Name:

3-hydroxy-11-oxoolean-12-en-30-oic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name Enoxolone
Appearance white powder
Composition Enoxolone >98%
CAS No. 471-53-4
EINECS-No. 207-444-6
Molecular formula C30H46O4
Molecular weight 470.68 g/mol
Purity > 98%
Homogeneity homogeneous

In vitro test system

Details on the study design:

Test System
Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, were used.
1 Cys-Peptide (Cysteine)
Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Batch no.:P170415-2-LR569638
Purity: 98.10%
2 Lys-Peptide (Lysine)
Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol)
Batch no.: P170415-2-LR569640
Purity: 98.85%


Positive control
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 solution in acetonitrile for the Cys-peptide.
• 2,3-Butanedione (CAS 431-03-8, ≥99 %, batch no. BCBS3560V) was used as 100 mM 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 of positive controls. Additionally, a solvent control containing isopropanol instead of test item solution was prepared in triplicate (set C(isopropanol)) and also incubated and analysed together with the samples and was used for calculation of the depletion of the test item.

Co-elution controls
Samples prepared from the respective peptide buffer and the test item, but without peptide.

Results and discussion

Positive control results:

a) The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde was not assessed because the test was invalid anyway.
b) The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide for batches 20180214 and 20180426. For batch 20180607 the peptide depletion was out of the acceptable range with 64.72 %.

In vitro / in chemico

Results

Remarks on result:

other: see “onther infromation on results”

Any other information on results incl. tables

1.1.1    Results

As reference control C was out of the acceptable range for all experiments with the Cys-peptide, the peptide depletion could not be calculated and these tests were invalid.

Only peptide depletion values for the Lys-peptide are reported therefore.

Table8.3–a   Calculated peptide depletion values for the Lys-Peptide batch 20180214

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	30.20	31.57	1.48
Positive control Rep. 2	31.39
Positive control Rep. 3	33.14
Test item Rep. 1	4.17	1.83	2.13
Test item Rep. 2	0.00
Test item Rep. 3	1.33

 

Table8.3–b  Calculated peptide depletion values for the Lys-Peptide batch 20180426

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	33.60	36.24	2.91
Positive control Rep. 2	39.36
Positive control Rep. 3	35.75
Test item Rep. 1	0.00	0.04	0.08
Test item Rep. 2	0.13
Test item Rep. 3	0.00

 

Table8.3–c   Calculated peptide depletion values for the Lys-Peptide batch 20180607

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	62.70	64.72	2.12
Positive control Rep. 2	64.53
Positive control Rep. 3	66.93
Test item Rep. 1	0.00	0.04	0.07
Test item Rep. 2	0.00
Test item Rep. 3	0.12

 

1.1.2    Acceptance criteria

a)   The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100 % 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 % - 45 % with a maximum standard deviation < 11.6 % for the Lys-peptide.

c)   The maximum standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion

1.1.3    Assessment

a)   The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde was not assessed because the test was invalid anyway.

b)   The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide for batches 20180214 and 20180426. For batch 20180607 the peptide depletion was out of the acceptable range with 64.72 %.

c)   The maximum standard deviation for the test item replicates was not assessed for the percent cysteine depletion for the test item because the test was invalid anyway.

The maximum standard deviation for the test item replicates was < 11.6 % for the percent lysine depletion for the test item in all experiments.

1.2     Evaluation of results

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

 

Table8.4–a   Evaluation of results according to the Cysteine 1:10/Lysine 1:50 prediction model. Values in % are peptide depletions

Cysteine 1:10 Prediction model		Cysteine 1:10/lysine 1:50 Prediction model
lower limit (>)		upper limit (≤)		lower limit (>)		upper limit (≤)		Reactivity class		DPRA Prediction
0.00 %	13.89 %	0.00 %	6.38 %	Minimal	negative
13.89 %	23.09 %	6.38 %	22.62 %	Low	positive
23.09 %	98.24 %	22.62 %	42.47 %	moderate	positive
98.24 %	100.00 %	42.47 %	100.00 %	high	positive

 

As the Cys-peptide depletion was not evaluable for all three assays and no prediction model exists for Lys-peptide only results, the test is not evaluable for the test item Enoxolone.Table8.4–bshows a summary of all individual test results.

 

Table8.4–b  Summary of results

	Cys-peptide depletion [%]	Lys-Peptide depletion [%]	Mean peptide depletion [%]
First experiment	(invalid, batch 20180215)	1.83 (batch 20180214)	Not evaluable
Verification 1	(invalid, batch 20180308)	0.48 (batch 20180426)	Not evaluable
Verification 2	(invalid, batch 20180426)	(invalid, batch 20180607)	Not evaluable
Verification 3	(invalid, batch 20180606)	(not performed)	Not evaluable

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study was performed in order to evaluate the reactivity of the test item Enoxolone to-wards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in isopro-panol was incubated 24 ± 2 h at 25 °C together with cysteine and lysine peptides, respec-tively, and the peptide concentration after the incubation was measured using HPLC-UV.
As repeated Cys-peptide experiments using isopropanol were invalid, the additional sol-vents acetone and acetonitrile/acetone (50/50 % v/v) were tried. As the test item was in-soluble in both solvents, these samples were not used for incubation.
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 in parallel.
Three experiments were performed which were invalid for the Cys-peptide each time.

As no prediction model exists for the DPRA test based on Lys-peptide depletion alone, no classification can be given for the test item Enoxolone.