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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 Nov - 21 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Departement Of Health Of The Government Of The United Kingdom
Type of study:
activation of keratinocytes

Test material

Constituent 1
Chemical structure
Reference substance name:
ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol
EC Number:
500-276-7
EC Name:
ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol
Cas Number:
89800-10-2
Molecular formula:
{(C10H16O7).(C3H3O)b.[(C6H10O2)m.(C3H3O)a]} (i.e., UVCB substance)
IUPAC Name:
ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol

In vitro test system

Details on the study design:
TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance using transgenic keratinocytes which were stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up­regulated by contact sensitisers. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Cell number at seeding: 10000 cells per well (96 well plates)
- Passage number: 14

CELL CULTURE CONDITIONS
- Type and identity of media: DMEM (Dulbecco's Modified Eagle's Medium) with GlutaMAX™ I, 1000 mg/L D-glucose, sodium pyruvate, human serum, (no geneticin used)
- Temperature (°C): 37
- CO2 (%): 5.0
- Relativ humidity: 95%

TEST CONCENTRATIONS
0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25.0, 50.0, 100.0, 200.0 and 400.0 μg/mL

CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1%

Positive control
- Substance: cinnamic aldehyde
- Final concentration: 8, 16, 32, 64 and 128 μM

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 2 h

NUMBER OF REPLICATIONS: 3 runs (with 3 replicates (for luminescence and 2 for MTT))

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- Details on MTT assay design: After removal of the medium, cells were washed with HBSS. The working stock of MTT solution (1:5 dilution in DMEM from stock) was then applied to each well of the appropriate 96 well plates for 3 h prior to solubilisation using MTT solubilisation solution (50 µL). This was then mixed by shaking and read at 570 nm.

DETERMINATION OF LUMINESCENCE
- Device: luminometer
- Details on luminescence assay design: Cells were washed with PBS and then passive lysis buffer (1x; diluted from 5x using sterile water) was added, shaken for 1 min and incubated for 20 min. Following this, luciferase substrate (50 µL) was added by the luminometer and then read.

Results and discussion

Positive control results:
The mean of the positive control cinnamic (32 µM) acid was 2.11 (fold-induction).

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: experiment 1, test substance
Parameter:
other: maximum luciferase activity induction (Imax) (mean of 3 replicates)
Value:
28.765
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: at 3.125 μg/mL
Key result
Run / experiment:
other: experiment 1, test substance
Parameter:
other: EC1.5 [µg/mL]
Remarks:
(interpolated concentration for a 1.5 fold luciferase induction)
Value:
0.36
Vehicle controls validity:
not applicable
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Run / experiment:
other: experiment 2, test substance
Parameter:
other: maximum luciferase activity induction (Imax) (mean of 3 replicates)
Value:
45.011
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: at 6.25 μg/mL
Key result
Run / experiment:
other: experiment 2, test substance
Parameter:
other: EC1.5 [µg/mL]
Remarks:
(interpolated concentration for a 1.5 fold luciferase induction)
Value:
0.28
Vehicle controls validity:
not applicable
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Run / experiment:
other: experiment 3, test substance
Parameter:
other: maximum luciferase activity induction (Imax) (mean of 3 replicates)
Value:
29.843
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: at 6.25 µg/mL
Key result
Run / experiment:
other: experiment 3, test substance
Parameter:
other: EC1.5 [µg/mL]
Remarks:
(interpolated concentration for a 1.5 fold luciferase induction)
Value:
0.55
Vehicle controls validity:
not applicable
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and min 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours. An increase in metabolic activity (cell viability > 150%) was observed in cells showing a positive maximal luciferase intensity (starting at 500 μM in experiments 1 and 2).

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: The luciferase activity induced by the positive control at a concentration of 32 μM was between 1.6 and 3.0 (mean = 2.11) and the calculated EC1.5 value was between 6 and 39 μM (mean = 11.5 μM).
- Acceptance criteria met for variability between replicate measurements: The average CV of the luminescence reading for the blank values control was < 20% (mean = 13.06%).

Any other information on results incl. tables

Table 1: Results of the cytotoxicity measurement and overall induction of luciferase activity

Concentration
(µM)

Cell viability (Mean and SD)

Fold Induction (Mean and SD)

Experiment No.

Experiment No.

1

2

3

1

2

3

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Vehicle control

-

100.00

-

100.00

-

100.00

-

1.00

-

1.00

-

1.00

-

Positive control

8

99.80

7.82

114.92

6.63

110.52

1.82

1.45

0.07

2.50

0.21

1.06

0.06

16

113.04

2.66

128.44

9.26

121.17

11.60

1.88

0.03

6.38

0.89

1.09

0.06

32

107.62

1.34

206.35

15.66

114.38

3.07

2.84

0.22

44.71

1.94

1.39

0.05

64

120.75

2.36

-4.85

0.77

124.36

5.21

8.58

0.43

0.00

0.00

2.67

0.31

128

127.15

10.11

-1.04

0.36

148.96

13.64

47.44

2.45

0.00

0.00

8.73

0.69

Test substance

0.195

129.46

7.76

110.39

4.83

100.69

4.52

1.21

0.20

1.24

0.15

0.98

0.08

0.391

115.23

2.22

107.26

3.74

98.48

1.09

1.56

0.15

1.84

0.11

1.34

0.13

0.781

127.01

4.59

121.50

7.77

108.84

2.46

3.31

0.51

1.40

0.16

1.73

0.05

1.563

129.60

2.24

146.27

21.64

128.19

1.38

9.19

2.05

1.97

0.15

5.59

0.59

3.125

114.03

17.81

138.69

1.12

144.40

10.49

28.77

1.50

9.03

4.45

24.32

0.79

6.250

-9.66

0.24

-3.15

2.47

35.23

3.92

0.06

0.04

45.01

14.42

29.84

3.04

12.500

-4.14

0.10

-0.04

2.77

-4.71

2.10

0.00

0.00

5.29

5.27

0.02

0.00

25.000

-14.47

0.32

-5.57

2.16

-8.15

2.06

0.01

0.00

0.01

0.00

0.00

0.00

50.000

-14.47

0.32

-7.96

0.47

-12.01

3.31

0.00

0.00

0.01

0.00

0.01

0.00

100.000

-9.56

3.90

-4.50

0.42

-5.58

1.23

0.00

0.00

0.01

0.00

0.00

0.00

200.000

0.74

2.09

-0.01

0.69

-3.00

1.26

0.01

0.00

0.01

0.00

0.00

0.00

400.000

-0.71

0.71

-1.08

2.44

-0.42

1.29

0.01

0.00

0.01

0.00

0.01

0.00

Table 2: Results of different sensitisation parameters of the test substance

 

Experiment No.

1

2

3

Imax

28.765

at 3.125 µg/mL

45.011

at 6.25 µg/mL

29.843

at 6.25µg/mL

EC1.5

0.36 µg/mL

0.28 µg/mL

0.55 µg/mL

IC50

4.743 µg/mL

5.079 µg/mL

5.827 µg/mL

IC30

4.237 µg/mL

4.638 µg/mL

5.255 µg/mL

Imax = maximal induction factor of luciferase activity compared to the solvent (negative) control measured at any test chemical concentration

EC1.5 = interpolated concentration for a 1.5 fold luciferase induction

IC50/30 = concentration effecting a reduction of cellular viability by 50/30%

 

Table 3: Results of different sensitisation parameters of the vehicle and positive control

 

Experiment No.

1

2

3

Acceptance criterion

Range

Mean

SD

Mean

SD

Mean

SD

CV% of vehicle control

< 20%

8.85

4.47

6.49

4.26

9.54

2.81

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

4

-

3

-

2

-

EC1.5 positive control

6 < x < 39

8.98

-

8

-

34.87

-

Induction positive control at 32 µM

1.6 < x < 3

2.84

0.215

44.714

1.939

1.39

0.051

CV= Coefficient of variation(a measure of variability that is calculated for a group of replicate data by dividing the standard deviation by the mean, multiplied by 100 for expression as a percentage)

Applicant's summary and conclusion

Interpretation of results:
other: skin sensitising potential based on the key event “inflammatory response in keratinocytes”
Conclusions:
Under the conditions of the test, the test substance did have a keratinocyte activating potential. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.