1,3-diiodopropane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Remarks:

direct peptide reactivity assay (DPRA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18.6.2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-diiodopropane

EC Number:

210-993-4

EC Name:

1,3-diiodopropane

Cas Number:

627-31-6

Molecular formula:

C3H6I2

IUPAC Name:

1,3-diiodopropane

impurity 1

Reference substance name:

1-chloro-3-iodopropane

EC Number:

230-088-8

EC Name:

1-chloro-3-iodopropane

Cas Number:

6940-76-7

Molecular formula:

C3H6ClI

IUPAC Name:

1-chloro-3-iodopropane

impurity 2

Reference substance name:

Unknown Impurity

IUPAC Name:

Unknown Impurity

Test material form:

liquid

Details on test material:

Batch 90423 / CME0032526

In chemico test system

Details on the study design:

The OECD AOP (The adverse outcome pathway for skin sensitisation) is initiated by key event 1 (Covalent interaction with skin proteins), which is followed sequentially by three key events (KE): (KE2) keratinocyte activation, (KE3) dendritic cell activation, and (KE4) proliferation of antigen-specific T cells. However, none of the assays addressing the different KEs is currently accepted as stand-alone test method and may not be sufficient to conclude on the presence or absence of skin sensitisation potential of chemicals, but may support the discrimination between skin sensitisers and non sensitisers in combination with other complementary data.
The direct peptide reactivity assay (DPRA) addresses the molecular initiating event (KE1) of the AOP. It is an in chemico assay to quantify the depletion of synthetic model peptides caused by known amounts of the test item measured by HPLC.
This study is performed in order to evaluate the reactivity of 1,3-Diiodopropane towards cysteine and lysine containing peptides. The peptide depletion compared to the solvent controls is calculated and leads to a DPRA prediction (reactive/positive or non-reactive/negative) that could be used to support the discrimination between skin sensitisers and non-sensitisers. Additionally, an assignment to one of four reactivity classes could be made in order to possibly support a potency assessment.
The DPRA is part of a tiered strategy for the evaluation of the skin sensitisation potential. Thus, all data generated with the present Test Guideline OECD 442C and EU-Method B.59 should be used in the context of an integrated approach to testing and assessment (IATA).

Results and discussion

In vitro / in chemico

Results

Run / experiment:

other: Mean of triplicate measurements

Parameter:

other: Mean peptide depletion [%]

Value:

32.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: DPRA prediction is positive and reactivity class moderate

Any other information on results incl. tables

Measurements

Measurements were performed using the HPLC method described before and results are shown in the following two tables.

Measurements Cys-Peptide, experiment 1

Sample name	Peak area 220 nm [mAU*min]	Peak area 258 nm [mAU*min]	Area ratio 220 nm/258nm [dimensionless]
Blank	n.a.*	n.a.*	n.c.**
Standard 0.0167 mM	0.6955	n.a.*	n.c.**
Standard 0.0334 mM	1.3448	0.0425	31.64
Standard 0.0667 mM	2.8552	0.0915	31.20
Standard 0.1335 mM	5.9296	0.1875	31.63
Standard 0.267 mM	11.8836	0.3730	31.86
Standard 0.534 mM	23.3031	0.7417	31.42
Reference A Rep. 1	21.9809	0.6971	31.53
Reference A Rep. 2	22.0877	0.7011	31.51
Reference A Rep. 3	22.1613	0.7023	31.55
Co-elution control positive control	n.a.*	n.a.*	n.c.**
Co-elution control test item	n.a.*	n.a.*	n.c.**
Reference B Rep. 1	22.1472	0.7018	31.56
Reference B Rep. 2	21.8587	0.6943	31.48
Reference B Rep. 3	22.1524	0.6956	31.85
Reference C ACN Rep. 1	21.6619	0.6866	31.55
Positive control Rep. 1	6.5846	0.1973	33.37
Test item Rep. 1	8.7632	0.2687	32.61
Reference C ACN Rep. 2	21.6949	0.6938	31.27
Positive control Rep. 2	6.6146	0.2018	32.78
Test item Rep. 2	7.2736	0.2229	32.63
Reference C ACN Rep. 3	21.7751	0.6916	31.49
Positive control Rep. 3	6.8072	0.2054	33.14
Test item Rep. 3	6.7872	0.2062	32.91
Reference B Rep. 4	22.3911	0.7094	31.56
Reference B Rep. 5	22.3782	0.7070	31.65
Reference B Rep. 6	22.2062	0.7043	31.53
Mean peak area ratio of reference controls A, B and C (ACN)***			31.54

* n.a. = no peak detected

** n.c. = not calculable

*** Used as reference for calculation of peak purity

Measurements Lys-Peptide, experiment 2

Sample name	Peak area 220 nm [mAU*min]	Peak area 258 nm [mAU*min]	Area ratio 220 nm/258nm [dimensionless]
Blank	n.a.*	n.a.*	n.c.**
Standard 0.0167 mM	0.7883	n.a.*	n.c.**
Standard 0.0334 mM	1.4845	0.0464	32.01
Standard 0.0667 mM	2.9523	0.0951	31.04
Standard 0.1335 mM	5.8159	0.1905	30.54
Standard 0.267 mM	11.6027	0.3934	29.49
Standard 0.534 mM	21.5994	0.7420	29.11
Reference A Rep. 1	21.2396	0.7285	29.15
Reference A Rep. 2	21.3696	0.7271	29.39
Reference A Rep. 3	21.4059	0.7203	29.72
Co-elution control positive control	n.a.*	0.0660****	n.c.**
Co-elution control test item	n.a.*	n.a.*	n.c.**
Reference B Rep. 1	21.3720	0.7255	29.46
Reference B Rep. 2	21.2810	0.7154	29.75
Reference B Rep. 3	21.2179	0.7117	29.81
Reference C ACN Rep. 1	21.1301	0.7154	29.54
Positive control Rep. 1	16.2577	0.6106	26.63
Test item Rep. 1	21.3513	0.7232	29.53
Reference C ACN Rep. 2	21.2763	0.7138	29.81
Positive control Rep. 2	16.0271	0.6051	26.49
Test item Rep. 2	20.9877	0.7221	29.07
Reference C ACN Rep. 3	21.2097	0.7165	29.60
Positive control Rep. 3	16.0423	0.5982	26.82
Test item Rep. 3	21.1763	0.7273	29.12
Reference B Rep. 4	21.0273	0.7106	29.59
Reference B Rep. 5	21.0179	0.7058	29.78
Reference B Rep. 6	20.9609	0.7036	29.79
Mean peak area ratio of reference controls A, B and C (ACN)***			29.61

* n.a. = no peak detected

** n.c. = not calculable

*** Used as reference for calculation of peak purity

**** Probable sign for co-elution. See discussion

Results

Calibration curve determination: From the peak areas of the peptide calibration standards detected at 220 nm, linear calibration curves in the form y = b * x + a were calculated for both peptides using validated Microsoft Excel® spreadsheets, with

y = Measured peak area [mAU*min]

b = Slope [mAU*min/mM]

a = Intercept [mAU*min]

x = Standard concentration [mM]

Linear calibration curves

Cys-Peptide: Slope b = 43.86465; intercept a = -0.01718; r² = 0.99982

Lys-Peptide: Slope b = 40.40715; intercept a = 0.29384; r² = 0.99869

Acceptance criteria

The r² of linear calibration should be > 0.99. The calibration curve was linear with acceptable coefficient of determination 0.99982 for the Cys-peptide and 0.99869 for the Lys-peptide, respectively.

Solvent controls

The peptide concentrations in the solvent controls were calculated using the linear regression (a = intercept, b = slope): Peptide concentration [mM] = (Peak area [mAU*min]-a)/b

Calculated peptide concentration for solvent controls

Sample name	Cys-Peptide concentration [mM]	Lys-Peptide concentration [mM]
Reference A Rep. 1	0.501	0.518
Reference A Rep. 2	0.504	0.522
Reference A Rep. 3	0.506	0.522
Reference B Rep. 1	0.505	0.522
Reference B Rep. 2	0.499	0.519
Reference B Rep. 3	0.505	0.518
Reference B Rep. 4	0.511	0.513
Reference B Rep. 5	0.511	0.513
Reference B Rep. 6	0.507	0.511
Reference C (ACN) Rep. 1	0.494	0.516
Reference C (ACN) Rep. 2	0.495	0.519
Reference C (ACN) Rep. 3	0.497	0.518
Mean concentration of Reference controls A and C [mM]	A: 0.50 C: 0.50	A: 0.52 C: 0.52
Variation coefficient (RSD) of Reference controls B and C (ACN) [%]	1.3	0.7

 

Acceptance criteria: The mean peptide concentration of solvent control samples of sets A and C (ACN) should be 0.50 ± 0.05 mM and the variation coefficient (relative standard deviation, RSD) of measured values of the nine samples from sets B1, B2 and C should be <15%

Assessment: The mean peptide concentration of all solvent controls (Reference A and Reference C) were with 0.50 mM and 0.50 mM for the Cys-peptide and 0.52 mM and 0.52 mM for the Lys-peptide in the acceptable range of 0.50 ±0.05 mM and the variation coefficients (RSD) of the measured values of Reference controls B and C in acetonitrile were in the acceptable range with 1.3% for the Cys-peptide and 0.7% for the Lys-peptide, respectively.

Calculations of Peptide depletion: The peptide depletion was calculated for each individual sample using the following equations (shown for Cys-Peptide, Lys-peptide is calculated analogously):

 

Peptide depletion(Cys,i) = (1 - measured peptide peak area in sample/mean peptide peak area in Solvent controls C ) * 100%

The mean peptide depletion of the Cys-peptide was calculated as follows:

Peptide depletion(Cys) = (∑_Cys,I =1,2,3 Peptide depletion_i)/3

The mean peptide depletion of the test item was calculated using the following equation:

Mean peptide depletion [%] = (Peptide depletion(Cys) [%] + Peptide depletion(Lys [%] )/2

 

Results

Calculated peptide depletion values for the Cys-Peptide

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	69.67	69.28	0.56
Positive control Rep. 2	69.53
Positive control Rep. 3	68.65
Test item Rep. 1	59.64	64.96	4.74
Test item Rep. 2	66.50
Test item Rep. 3	68.74

 

Calculated peptide depletion values for the Lys-Peptide

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	23.33	24.03	0.61
Positive control Rep. 2	24.42
Positive control Rep. 3	24.35
Test item Rep. 1	0 (-0.69)*	0.39	0.56
Test item Rep. 2	1.03
Test item Rep. 3	0.14

* Note: Negative depletion values were considered as “zero” when calculating the mean.

Mean depletion of both peptides after incubation with the test item: 32.67 %

 

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 with 69.28 % and a standard deviation of 0.56 % of 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 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 mean peptide depletion with 24.03% and a standard deviation of 0.61% of 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.

- The standard deviation for the test item replicates should be <14.9% for the percent cysteine depletion and <11.6% for the percent lysine depletion. The standard deviation for the test item replicates with 4.74% was <14.9% for the percent cysteine depletion for the test item. The standard deviation for the test item replicates with 0.56% was <11.6% for the percent lysine depletion for the test item.

Evaluation of results

According to the test guideline OECD 442C and EU-Method B.59, the reactivity is classified as “high”, “moderate”, “low” or “minimal” using the Cysteine 1:10/Lysine 1:50 prediction model as follows:

Evaluation 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 Cys-peptide and Lys-peptide assay was 32.67 %, therefore the test item was classified with DPRA Prediction positive and reactivity class moderate.

Applicant's summary and conclusion

Interpretation of results:

other: positive for key event 1 of adverse outcome pathway for skin sensitisation

Conclusions:

Under the experimental conditions reported, the test item 1,3-Diiodopropane shows a positive, moderate reactivity towards the two model synthetic peptides.

Executive summary:

This in chemico study was performed in order to evaluate the reactivity of the test item 1,3-Diiodopropane towards cysteine (Cys-) and lysine (Lys-) containing peptides. The calculated peptide depletion could be used to support 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 of an 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.

Two experiments were performed.

Experiment 1 was not valid for the Lys-peptide assay, because the peptide calibration values and the reference controls A and B values couldn’t be analyzed as the retention time of the Lys-peptide was not in the normal range. For the Cys-peptide experiment 1 was valid.

In Experiment 2, only the Lys-peptide assay was performed and was found valid. The result of the Cys-peptide assay of experiment 1 and of the Lys-peptide assay of experiment 2 were used to calculate the mean peptide depletion. 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 were 64.96% for Cys-peptide and 0.39% for Lys-peptide, resulting in amean depletion of 32.67%

Thus, the DPRA prediction is ”positive” according to the Cysteine 1:10/Lysine 1:50 prediction model and under the experimental conditions reported, the test item 1,3-Diiodopropane shows a 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 1,3-Diiodopropane could be assigned to the reactivity class that covers a moderate reactivity under the conditions of this study.