Registration Dossier

Administrative data

Description of key information

In a weight of evidence approach, the substance 1,3-Diiodopropane was tested in two in chemico/in vitro test systems for binding activity to proteins (adverse outcome pathway key event 1, DPRA study) and for the potential to activate the Nrf2 transcription factor (adverse outcome pathway key event 2, LuSens study). Testing for a third key event was not required.

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.

In the LuSens study, a statistically significant and reproducible dose-dependent increase in luciferase induction >1.5 fold in more than two non-cytotoxic consecutive test item concentrations was observed in both repetitions. Therefore, under the experimental conditions of this study, the test item, 1,3-Diiodopropane, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor.

Resulting from moderate, positive outcome of the DPRA study and the positive outcome from the LuSens study on 1,3 -Diiodopropane, it can be concluded that the test substance is a moderate skin sensitizer and no further studies to assess skin sensitization are required.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
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)
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).
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

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.

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.

Endpoint:
skin sensitisation: in vitro
Remarks:
In Vitro Skin Sensitisation assays addressing the AOP Key Event on Keratinocyte activation
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
25.6.2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Run / experiment:
other: repetition II, conc. 2000 µM
Parameter:
other: fold increase in luciferase induction
Value:
29.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: repetition III, conc. 2000 µM
Parameter:
other: fold increase in luciferase induction
Value:
28.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
"In the case of a cytotoxic result, the concentrations for the experiment should be determined so that at least one of them is in the cytotoxic range." In the main experiment, concentrations were chosen which were expected to produce at least one cytotoxic concentration. However, in both repetitions all concentrations were in the non-cytotoxic range. This deviation was considered uncritical since a statistically significant increase ≥1.5 fold in luciferase induction was measured in all concentrations. Only in the case a result is to be considered negative, at least one concentration should be cytotoxic.

Results of Repetition II: All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values >100%). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 1.0 fold, negative control: 1.0 fold). However, the positive control induced a clear effect with an induction value of 6.5 fold in comparison to the solvent control. No cytotoxic effect was observed up to the highest test item concentration. The viability values were all >100% and therefore analysable for luciferase induction.

In the Luciferase assay, all of the tested concentrations induced a statistically significant and extremely high increase (13.8 fold to 29.8 fold) in luciferase induction above the threshold of 1.5 fold in comparison to the solvent control.

Results of repetition II

Parameter

Concentration [µM]

Induction of Luciferase

Viability of the Cells

Mean Induction [fold]

Standard Deviation

Standard Deviation [%]

Mean Relative Viability [%]

Standard Deviation

Standard Deviation [%]

Solvent Control

-

1.0

0.08

8.08

100.0

4.81

4.81

Growth Control

-

1.0

0.08

7.57

125.3

5.92

4.73

Negative Control

5000

1.0

0.11

10.21

108.4

6.98

6.44

Positive Control

120

6.5

0.94

14.46

102.7

2.85

2.78

Test item

269

15.5

1.83

11.78

132.1

1.95

1.47

Test item

323

13.8

2.50

18.17

118.3

4.03

3.40

Test item

388

16.7

2.24

13.40

124.8

7.12

5.70

Test item

465

16.4

1.56

9.54

123.0

4.76

3.87

Test item

558

16.2

1.15

7.09

126.6

3.78

2.99

Test item

670

18.5

2.04

11.03

125.5

3.28

2.62

Test item

804

18.4

0.62

3.35

128.4

10.02

7.81

Test item

965

19.6

1.96

9.99

126.2

1.62

1.29

Test item

1157

22.2

3.57

16.06

125.4

4.78

3.81

Test item

1389

24.4

3.24

13.25

126.5

4.83

3.82

Test item

1667

22.8

2.02

8.84

121.7

5.29

4.34

Test item

2000

29.8

5.10

17.12

129.1

3.86

2.99

 

Results of Repetition III: All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥99%). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 1.1 fold, negative control: 1.0 fold). However, the positive control induced a clear effect with an induction value of 6.1 fold in comparison to the solvent control. No cytotoxic effect was observed up to the highest test item concentration. The viability values were all >100% and therefore analysable for luciferase induction.

In the Luciferase assay, all of the tested concentrations induced a statistically significant and extremely high increase (22.4 fold to 28.8 fold) in luciferase induction above the threshold of 1.5 fold in comparison to the solvent control.

Results of repetition III

Parameter

Concentration [µM]

Induction of Luciferase

Viability of the Cells

Mean Induction [fold]

Standard Deviation

Standard Deviation [%]

Mean Relative Viability [%]

Standard Deviation

Standard Deviation [%]

Solvent Control

-

1.0

0.08

7.72

100.0

3.64

3.64

Growth Control

-

1.1

0.09

8.16

142.8

5.52

3.87

Negative Control

5000

1.0

0.05

4.72

106.5

3.09

2.90

Positive Control

120

6.1

0.22

3.60

98.7

1.49

1.51

Test item

269

22.4

1.51

6.76

132.6

3.79

2.86

Test item

323

24.5

2.93

11.94

129.0

3.44

2.67

Test item

388

23.4

1.21

5.16

137.7

7.67

5.57

Test item

465

22.7

3.20

14.09

136.7

5.56

4.06

Test item

558

23.6

0.64

2.72

134.2

6.43

4.79

Test item

670

24.3

1.49

6.12

133.8

4.64

3.47

Test item

804

26.2

2.25

8.59

138.1

6.50

4.70

Test item

965

25.9

2.41

9.30

136.5

2.68

1.97

Test item

1157

26.3

1.34

5.10

136.7

4.03

2.95

Test item

1389

25.6

2.68

10.45

134.1

3.07

2.29

Test item

1667

26.6

1.59

5.98

133.7

4.42

3.30

Test item

2000

28.8

6.09

21.14

132.0

6.96

5.27

 

EVALUATION

Acceptability: In the following table the criteria for acceptability as well as the corresponding results in repetition II and III are given.

 

Acceptability of repetition II and III

Criteria

Found in repetition II

Found in repetition III

The average induction for the positive control should be ≥2.5 fold and it should have a relative viability of at least 70%.

Positive control Fold induction: 6.5, Relative viability: 102.7%

Positive control Fold induction: 6.1, Relative viability: 98.7 %

The induction triggered by the negative control and growth control should be <1.5 fold as compared to the induction of the solvent control and the viability should be above 70%.

Negative control: 1.0 Fold induction, 108.4% Relative viability; Growth control: 1.0 Fold induction, 125.3% Relative viability

Negative control: 1.0 Fold induction, 106.5% Relative viability; Growth control: 1.1 Fold induction, 142.8% Relative viability

The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20%.

8.08%

7.72%

At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70%.

12 concentrations are analysable

12 concentrations are analysable

In case a result is to be considered negative, at least one concentration should be cytotoxic, i.e. have a cell viability <70%, or the maximum concentration of 2000 µM (2000 µg/mL) should have been tested

The result is positive and the maximum concentration of 2000 µM (2000 µg/mL) was tested

The result is positive and the maximum concentration of 2000 µM (2000 µg/mL) was tested

All validity criteria were met. Therefore, the study is valid.

Prediction Model: Each valid experiment (i.e. meeting all acceptance criteria, according to the procedure described above) is interpreted as follows: A test compound is considered to have the potential to activate the Nrf2 transcription factor if the luciferase induction is ≥1.5 fold and statistically significant compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic (relative viability ≥70%) tested concentrations whereby at least three tested concentrations must be non-cytotoxic in two independent valid repetitions. A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the effects mentioned above are not observed. A negative result obtained with test chemicals that do not form a stable dispersion and/or were not tested up to 2000 µM (or 2000 µg/mL for test chemicals with no defined molecular weight) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive.

In order to come to a conclusion, a minimum of two valid and independent repetitions need to indicate a positive or negative result according to the criteria described above. If the first two repetitions come to the same result (i.e. either being negative or being positive) no further testing is required. In case that the first two repetitions give discordant results (i.e. one is negative and the other is positive), a third independent repetition needs to be conducted to complete the study. The potential to activate the Nrf2 transcription factor of a test substance is determined by the result of the majority of the repetitions of an experiment. If two of two or two of three repetitions are negative/positive, the substance is considered as negative/positive.

The luciferase induction was ≥ 1.5 fold and statistically significant compared to the solvent control in 2 (or more) consecutive non-cytotoxic tested concentrations in repetitions II and III. Therefore, the test item 1,3-Diiodopropane is considered to have the potential to activate the Nrf2 transcription factor under the conditions of the LuSens test.

Interpretation of results:
other: positive for key event 2 of adverse outcome pathway for skin sensitisation
Conclusions:
1,3-Diiodopropane was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor.
Executive summary:

This in vitro study evaluates the potential of the test item 1,3-Diiodopropane to activate the Nrf2 transcription factor by using the LuSens cell line which is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. The LuSens test is an ARE Reporter Gene Assay performed according to the OECD 442D Guideline with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on Keratinocyte activation”.

The assay performed included a cytotoxicity range finder test (CRFT) and one experiment, consisting of two independent repetitions (repetition II and III) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on these results, the concentrations to be tested in the repetitions were determined.

In the experiment (repetition I, II and III) the highest nominal applied concentration (2000 µM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the repetitions. The first repetition was invalid (one acceptance criteria was not fulfilled as the growth control induced an induction of 1.5 fold). Those data are not reported, all documentation is kept with the raw data and will be archived at the GLP test facility.

DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.

A statistically significant and reproducible dose-dependent increase in luciferase induction >1.5 fold in more than two non-cytotoxic consecutive test item concentrations was observed in both repetitions.

Therefore, under the experimental conditions of this study, the test item, 1,3-Diiodopropane, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the outcome of two key event studies in the adverse outcome pathway approach, the substance is to be classified as a moderate skin sensitiser, category 1B according to CLP (Regulation EC No 1272/2008).