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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 21-May 14, 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 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Test method is considered able to detect chemicals that cause skin sensitization when used with an integrated approach. This is an in vitro test method that does not require vertebrate testing.

Test material

Constituent 1
Chemical structure
Reference substance name:
(Z)-non-6-enal
EC Number:
218-900-9
EC Name:
(Z)-non-6-enal
Cas Number:
2277-19-2
Molecular formula:
C9H16O
IUPAC Name:
(Z)-non-6-enal
Test material form:
liquid

In vitro test system

Details on the study design:
Skin sensitisation (In vitro test system)
- Details on study design: The test item was dissolved in DMSO with mixing to create a stock solution of 200 mM. Dilutions were then performed from the stock solution. The final concentration of solvent in test solutions was 1% (v/v). A blank control was performed, along with a negative control of 1% (v/v) DMSO and a positive control of 4 to 64 uM of cinnamic aldehyde in DMSO (1% (v/v)). Human keratinocytes maintained at 37 degrees C and 5% CO2 were used. Dulbecco’s Modified Eagle Medium augmented with 10% fetal bovine calf serum was used for the assay. Dulbecco’s Modified Eagle Medium augmented with 1% fetal bovine calf serum was used for the test item medium.

Three replicates of the negative control and each test concentration was performed. Six replicates of the positive control were performed. 1x10^4 cells per well were added. A cell viability test was also performed with cells in transparent wells, for the other plates, luciferase was added. Cells were incubated for 24 hrs at 37 degrees C with assay medium. Assay medium was removed and 150 uL test substance medium was then added and incubation performed for 48 hrs at 37 degrees C with the plates sealed to prevent loss and cross-contamination. After incubation, wells were washed with DPBS. 50 uL of luciferase was added to wells and luciferase activity was recorded.

For the cell viability test, assay medium was replaced with 200 uL test medium and 27 uL MTT solution. The plates were then incubated for 4 hrs at 37 degrees C sealed. Test medium was then removed and replaced with 200 uL 10% SDS solution. This was incubated for the weekend sealed at 37 degrees C, and then shaken for 10 minutes. The optical density was then measured at 600 nm.

Test was considered positive if the following conditions were met in two independate replicates: 1) Imax showed a 1.5 times fold or greater and statistically significant increase over negative controls; 2) cell viability at least greater than 70% at the lowest concentration with an inducation of luciferase activity of greater than 1.5; 3) the EC1.5 value is < 1000 uM; and 4) an apparent overall dose dependent response for luciferase inducation.

The test was considered valid if the following conditions were met: 1) the luciferase inducation in the positive control is statistically significant increased 1.5 times that of at least one tested concentration; 2) the average induction of the 64 uM positive control is 2-8; 3) the EC1.5 is within two statistical deviations of the historical mean; and 4) the average coefficient of variation of the luminescence reading for the negative control is less than 20% in each repetition.

Results and discussion

Positive control results:
Sensitizing

In vitro / in chemico

Results
Key result
Run / experiment:
other: 1 and 2
Parameter:
other: luciferase inducation
Value:
1.58
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Concentration

Cell Viability (%)

Mean Induction of Luciferase Activity Experiment 1

Mean Induction of Luciferase Activity Experiment 2

Mean Induction of Luciferase Activity

Solvent Control

 

 

 

 

--

100

1.00

1.00

1.00

Positive Control

 

 

 

 

4.00

103.2

1.41

1.25

1.33

8.00

105.2

1.46

1.28

1.37

16.00

107.4

1.43

1.25

1.34

32.00

107.6

1.65

1.66

1.65

64.00

108.2

2.03

2.19

2.11

Test Substance

 

 

 

 

0.99

96.6

1.05

1.01

1.03

1.97

112.7

1.49

1.21

1.35

3.95

113.6

1.64

1.24

1.44

7.90

111.6

1.80

1.26

1.53

15.80

110.8

2.08

1.40

1.74

31.59

114.4

2.23

1.43

1.83

63.19

110.8

2.33

1.45

1.89

126.38

104.2

3.11

1.58

2.34

252.75

31.8

2.93

2.76

2.84

505.50

0.1

0.00

0.00

0.00

1011.0

0.2

0.00

0.00

0.00

2022.0

0.2

0.00

0.00

0.00

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
The test substance is potentially skin sensitizing.
Executive summary:

The skin sensitization potential of the test substance was determined in an OECD Guideline 442D human keranitocyte test using luciferase activity. Test concentrations of 2022, 1011, 505.50, 252.75, 126.38, 63.19, 31.95, 15.80, 7.90, 3.95, 1.97, and 0.99 µM were tested along with blank, positive, and negative controls. The results of the test show that the test substance is potentially a skin sensitizer.