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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:
supporting study
Study period:
22 September 2017 - 08 December 2017
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: outside the applicability domain of the OECD442D test

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)
Deviations:
yes
Remarks:
Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The existing knowledge of the chemical and biological mechanisms associated with skin sensitization has been summarized in the form of an Adverse Outcome Pathway (AOP). The second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways.

In vitro test system

Details on the study design:
Two independent experiments were performed. A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element were used) were used in these experiments incubated with Dilithium tetraborate in a concentration range of 0.49 – 1000 µM (2-fold dilution steps) for 48 hours. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.
The positive control used was Ethylene dimethacrylate glycol and the negative control is the vehicle of the test item (i.e. the exposure medium).The solvent control was 1% DMSO in exposure medium and DMEM. Eighteen wells were tested per plate.

For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.


In the main experiments the test item was suspended in DMEM at 4 mM. The stock was vortexed and sonicated (49 min with a maximum temperature of 22.0 °C in experiment 1 and for 23 minutes with a maximum temperature of 36.0 °C). From this stock 11 spike solutions in DMEM were prepared (2-fold dilution series). At 2 mM the test item formed a suspension in experiment 1 whereas at 2 mM in experiment 2 it was fully soluble. At 1 mM and lower the test item was fully soluble. The stock and spike solution were diluted 4-fold in the assay resulting in final test concentrations of 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98 and 0.49 µM. All concentrations of the test item were tested in triplicate.
No precipitation was observed at the start and end of the incubation period in the 96-well plates.
Test item concentrations were used within 3 hours after preparation.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: I (max)
Value:
5.73
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: EC1.5 = 140 µM
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: I (max)
Value:
1.93
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: EC1.5 = 100 µM
Other effects / acceptance of results:
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) The average coefficient of variation of the luminescence reading for the negative (solvent) control exposure medium should be below 20% in each repetition which consists of 18 wells. If the variability is higher, results should be discarded.
d) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Any other information on results incl. tables

Table 1          Overview Luminescence Induction and Cell Viability of Dilithium tetraborate in Experiment 1 and 2

Concentration (µM)

0.49

0.98

2.0

3.9

7.8

16

31

63

125

250

500

1000

Exp 1 luminescence

0.91

1.02

1.10

1.12

1.02

1.02

1.06

1.13

1.44

1.98***

3.25***

5.73***

Exp 1 viability (%)

104.4

92.6

96.2

96.0

94.1

92.6

90.4

92.7

91.6

119.3

124.7

110.7

Exp 2 luminescence

1.05

1.04

1.05

1.13

1.07

1.06

1.12

1.11

1.27

1.55***

2.22***

4.27***

Exp 2 viability (%)

106.4

103.8

97.5

99.1

83.9

88.4

97.6

92.1

93.2

93.1

97.4

117.1

***p<0.001 Student’s t test

 

Table 2          Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)

7.8

16

31

63

125

250

Exp 1 luminescence

0.98

0.93

1.02

1.22

1.69***

1.93***

Exp 1 viability (%)

96.4

103.6

105.4

107.3

103.9

105.4

Exp 2 luminescence

0.92

1.15

1.38

1.58***

1.84***

2.68***

Exp 2 viability (%)

96.6

96.7

102.8

102.3

109.8

112.0

***p<0.001 Student’s t test

 

Table 3          Overview EC1.5, Imax, IC30and IC50Values

 

EC1.5(µM)

Imax

IC30(µM)

IC50(µM)

Test item Experiment 1

140

5.73

NA

NA

Test item Experiment 2

227

4.27

NA

NA

Pos Control Experiment 1

100

1.93

NA

NA

Pos Control Experiment 2

50

2.68

NA

NA

NA = Not applicable

Applicant's summary and conclusion

Interpretation of results:
other: Dilithium tetraborate is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Conclusions:
Dilithium tetraborate is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions which were used and according to OECD TG442D.
Executive summary:

In an OECD TG442D Dilithium tetraborate was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. 

Two independent experiments were performed. The cells were in these experiments incubated with Dilithium tetraborate in a concentration range of 0.49 – 1000 µM (2-fold dilution steps) for 48 hours. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. 

Dilithium tetraborate is classified as positive in the KeratinoSensTM assay (OECD TG 442D) since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.