8,9,10-trinorborna-2,5-diene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October - December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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

Test material

Test material information

Constituent 1

Reference substance name:

8,9,10-trinorborna-2,5-diene

EC Number:

204-472-0

EC Name:

8,9,10-trinorborna-2,5-diene

Cas Number:

121-46-0

Molecular formula:

C7H8

IUPAC Name:

bicyclo[2.2.1]hepta-2,5-diene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch number of test material: N° 10006042
- Expiry date: 16/08/2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material under test conditions and during storage: stable

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

PREPARATION OF TEST ITEM DILUTIONS
- Preparation of the test chemical stock solution:
The test item was diluted in DMSO at 3.409 mM

- Preparation of the positive control:
The positive control was prepared at 200 mM in DMSO.

- Preparation of the 100 X plate (positive and negative control) :
A 100-fold concentrated dilutions series was prepared in 96-well plate.
Positive control :
100 µl of DMSO were distributed in row F (repetition 1 ) or G (repetition 2) from columns 7 to 10. 200 µl of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 µl of the column 1 1 in the column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
Negative control:
100 µl of DMSO were distributed in row F (repetition 1) or G (repetition 2) columns 1 to 6 and 12 and in the well H12.

- Preparation of the 4X dilution plate (positive and negative control):
The 100 X plate was diluted 25 fold in a new plate (4 X).

- Preparation of the 1X dilution (test item):
The test item was placed in one of the rows B to E.
1100 µl of treatment medium, 1% DMSO were distributed columns 1 to 11 in a masterblock. 2200 µl of the stock solution were placed in column 12 then the series dilutions were prepared by transferring 1100µl of the column 12 in the column 11 and so until the column 1.
Dilutions were mixed by repeated pipetting at least 3 times, between each concentration.


CONTACT BETWEEN THE CELLS AND THE TEST AND REFERENCE ITEM
-Positive and negative control:
In the 5 seeded plates, the medium was aspirated and replaced with 150 µl of treatment medium. Then the 4 X plate was replicated 5 times: 50 µl from the 4 X plate were placed in each of the three white plates and in the two transparent plates.

- Test item:
200µl of the test item 1X diluted were placed in each of the three white plates and in the two transparent plates.
The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37°C, 5% CO2).

-Luciferase activity:
After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate (luciferine + ATP + lysing agent) were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis.
The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

-Cell viability assessment with MTT method:
After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then, 225 µl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5 mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37°C, 5% CO2).
After this contact time, the staining solution was eliminated and the cells were treated with 200 µl of 10% SDS one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbances were measured at 540 nm.

EXPRESSION OF RESULTS
Two parameters are measured, the luciferase induction and the cytotoxicity.
The luciferase induction was measured using luminescence. Cytotoxicity was measured using the absorbance. These data were compared to negative control and blank.

Vehicle / solvent control:

DMSO

Negative control:

other: DMSO

Positive control:

cinnamic aldehyde [442D]

Results and discussion

In vitro / in chemico

Results

Key result

Run / experiment:

mean

Parameter:

other: Induction Imax

Value:

1.47

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

I max are lower than 1.5. No EC1.5 is determined.

Any other information on results incl. tables

Test item	VIABLITY	INDUCTION
	IC70 µM	Imax	Linear EC1.5 µM	EC1.5 Lin/Log µM
Rep 1	> 3409	1,46	-	-
Rep 2	>3409	1,47	-	-
Mean	-	1,47	-	-
Geometric mean	>3409	-	-	-

 

MEAN VIABILITY PERCENTAGE
Concentration µM	1.66	3.33	6.66	13.3	26.6	53.3	107	213	426	852	1704	3409
Rep 1	77.79	90.06	97.53	101.13	88.86	86.59	96.60	93.40	106.20	102.33	106.87	98.73
Rep 2	95.49	97.86	90.47	91.13	89.68	89.02	87.83	86.77	92.05	96.15	105.92	101.96
Viabili	86.6	94.0	94.0	96.1	89.3	87.8	92.2	90.1	99.1	99.2	106.4	100.3

 

MEAN INDUCTION
Concentration µM	1.66	3.33	6.66	13.3	26.6	53.3	107	213	426	852	1704	3409
Rep 1	0.82	1.23	1.35	1.28	1.46	1.28	1.39	1.37	1.24	1.32	1.17	0.85
Rep 2	1.00	1.01	0.96	1.06	0.99	0.99	1.06	1.26	1.12	1.05	1.47	1.05
Induction	0.91	1.12	1.16	1.17	1.23	1.13	1.22	1.31	1.18	1.19	1.32	0.95
SD	0.13	0.16	0.28	0.15	0.33	0.21	0.24	0.08	0.08	0.19	0.21	0.14

 

Student t-test
Rep 1	0.200	0.346	0.078	0.221	0.142	0.285	0.082	0.081	0.336	0.207	0.300	0.553
	0.958	0.932	0.540	0.513	0.896	0.749	0.654	0.116	0.097	0.545	0.194	0.765

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the retained experimental conditions BCHD BICYCLO 2.2.1 - HEPTA 2.5 DIENE may be classified as not skin sensitizer.
The test method KeratinoSensTM is considered scientifically valid to be used as part of an integrated approaches to testing and assessment. to support the identification of the sensitization potential of test item for hazard classification and labeling purposes