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Description of key information

One Guideline study on the endpoint "in vitro testing on skin sensitisation" available,

a second Guideline study is in progress.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June - November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Principles of method if other than guideline:
The LuSens cell line was specially designed for this test system by the BASF SE (Lud-wigshafen, Germany). It employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
16JSVA015
- Expiration date of the lot/batch:
14. September 2018
- Purity test date:
not state

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room Temperature: (20 ± 5°C), keep container tightly closed, store under inert gas
- Stability under test conditions:
assumed stable


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none
Details on study design:
Cell Cultures
For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitro-gen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 7 were used. For the main ex-periments cells of passage 9 were used. After thawing the cells were cultivated in DMEM (9 % FCS) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Cytotoxicity Range Finder Test
A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concen-tration range applicable for experiment I and II. In the CRFT cytotoxicity was determined by measuring the cell viability with MTT. This yellow tetrazole is reduced to purple forma-zan in viable cells and can therefore be used for assessing of the cell metabolic activity. A reduction of the viability below 70 % is defined as a cytotoxic effect.
In the CRFT the following 12 nominal concentrations of the test item were tested:
0.98 μM, 1.95 μM, 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM, 2000 μM

Dose Selection for Experiment I and II
Since a cytotoxic reaction was observed in the CRFT the following 12 nominal concentra-tions were chosen for experiment I and II: 1.05 μM, 1.26 μM, 1.51 μM, 1.82 μM, 2.18 μM, 2.62 μM, 3.14 μM, 3.77 μM, 4.52 μM, 5.42 μM, 6.51 μM, 7.81 μM
The real test item concentrations are given in chapter 19, page 42.
In the main experiments, a reduction of the viability below 70 % is considered as cytotoxic and is not allowed to be evaluated for luciferase induction.

Experimental Parameters of Experiment I and II
Experimental Performance
Experiment I and II were performed in the same way. Experiment II serves only to confirm the results of experiment I. The exposure dates were 18. Jul. 2017 and 19. Jul. 2017.
At the time of seeding the cells were 90 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium no. 2 was added. After centrifuga-tion (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2. After quantification, the cell suspension was adjusted to 83 000 (±10 %) cells per mL. 120 μL of the cell suspension (≙ 10 000 cells) were seeded in two clear flat bottom 96 well plates (one for viability and one for luciferase induction measurement). Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 23 h and 15 min in Experiment I and 24 h in Experiment II.

For the evaluation of the viability, one of the plates will be used:
The MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 μL MTT working solution were added to each well. The plates were incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was re-moved and 100 μL of lysis buffer were added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5-Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow color) to its insoluble formazan (purple color) in living cells and therefore indicates the amount of living cells. After the measurement of the color change, the values were transferred in a validated spreadsheet for the calculation of the viability (see chapter 7.3.2).
For the evaluation of the Luciferase induction, the second plate will be used:
For the evaluation of the Luciferase expression all solutions were removed from the wells and the cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Afterwards 100 μL per well of a Lysis buffer were added to the cells and incubated for 5 min at room temperature. During this process, the plate was slightly moved. Afterwards 100 μL Steady-Glo® Reagent were added to each well and the plate was shaken again slowly for 5 min at room temper-ature. Then, 160 μL per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometer.
For calculation of the luciferase induction as well as the relative viability (see chapter 7.3.2) a validated Microsoft Excel® file was used.
Positive control results:
the positive control induced a clear effect with an induction value of 5.0 fold in comparison to the sol-vent control.
Key result
Parameter:
other: Induction of Luciferase, expressed as fold in comparision to solvent control
Run / experiment:
Experiment II
Value:
>= 1.1 - <= 1.6
Vehicle controls validity:
other: the growth con-trol and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control
Negative controls validity:
other: the growth con-trol and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control
Positive controls validity:
valid
Key result
Parameter:
other: Induction of Luciferase, expressed as fold in comparision to solvent control
Run / experiment:
Experiment II
Value:
>= 0.9 - <= 1.5
Vehicle controls validity:
other: the growth con-trol and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control
Negative controls validity:
other: the growth con-trol and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptability of experiment I and II
Criteria
The average induction for the positive control should be ≥ 2.5 fold and it should have a rela-tive viability of at least 70 %.
Found in experiment I
Positive control Fold induction: 5.0 Relative viability: 83.8 %
Found in experiment II
Positive control Fold induction: 6.0 Relative viability: 75.5 %
Criteria
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%.
Found in experiment I
Negative control: Fold induction: 0.9 Relative viability: 107.0 %
Growth control: Fold induction: 1.2 Relative viability: 136.4 %
Found in experiment II
Negative control: Fold induction: 0.9 Relative viability: 105.2 %
Growth control: Fold induction: 1.2 Relative viability: 142.4 %
Criteria
The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.
Found in experiment I
8.44
Found in experiment II
9.91
Criteria:
At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.
Found in experiment I
10 concentrations are analysable
Found in experiment II
9 concentrations are analysable

All validity criteria were met. Therefore, the study is valid.
Interpretation of results:
study cannot be used for classification
Conclusions:
Under the experimental conditions of this study, the test item, Bis(neodecanoyloxy) dioctylstannane, was negative in the LuSens assay in accordance to the protocol of the BASF SE and inconclusive in accordance to the OECD 442 D.
Executive summary:

This in vitro study evaluates the sensitizing potential of the test item Bis(neodecanoyloxy)dioctylstannane by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitisation 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 as-sessment.

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens As-say). The assay differs in some points from the OECD guideline however the proficiency of the LuSens test was demonstrated (see chapter 6.5, page 13).

The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the con-centrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (7.81 μM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilu-tions thereof was prepared. Precipitation of the test item was not visible in any of the ex-periments.

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 pos-itive control.

The evaluated experimental points and the results are summarised in chapter 8 page 20ff.

No substantial dose dependent increase in luciferase induction ≥ 1.5 fold was observed in a minimum of 2 consecutive non-cytotoxic concentrations in both experiments up to the maximal concentration of the test item. Therefore, according to the protocol of the BASF SE the LuSens test is negativ.

According to the OECD 442D the result is inconclusive.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
16 May 2012 to 29 May 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions. Since the study was conducted using the read across substance dioctyltin oxide which was considered suitable based on its structural similarities. The study was assigned a reliability score of 2, on this basis.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: Individually.
- Diet: ad libitum.
- Water: Mains tap water ad libitum.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): Approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours darkness.
Vehicle:
propylene glycol
Concentration:
5, 10 or 25 % w/w
No. of animals per dose:
Animals were tested in groups of four.
Details on study design:
RANGE FINDING TESTS: a preliminary screening test was performed on one mouse. The mouse was treated by daily application of 25 µL of the test material at a maximum attainable concentration of 25 % w/w in propylene glycol, to the dorsal surface of each ear for three consecutive days. The mouse was observed twice daily on days 1, 2 and 3 and once daily on days 4, 5 and 6. Local skin irritation was scored daily according to the scale outlined in the field “Any other information on materials and methods incl. tables”. Any signs of toxicity, if present, were also recorded.
The thickness of each ear was measured using an Oditest micrometer pre-dose on day 1, post dose on day 3 and again on day 6. Any changes in ear thickness were noted.
- Compound solubility: 25 % w/w was selected as the highest dose investigated in the main test, reflecting the maximum attainable concentration of test material in vehicle. The vehicle was chosen as it produced the highest concentration of test material that was suitable for dosing.


TREATMENT PREPARATION AND ADMINISTRATION: Main test
Approximately 25 µL of 5 %, 10 % or 25 % w/w preparation of the test material in propylene glycol was applied using an automatic micropipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using propylene glycol alone. The procedure was repeated daily for 3 consecutive days.

Five days after the first application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing approximately 80 µCi/mL of 3H-methyl thymidine with a specific activity of 2.0 Ci/mmol (total dose 20 µCi per mouse). Approximately 5 hours later, the animals were humanely killed by carbon dioxide asphyxiation. The draining aurcular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed twice by centrifugation with approximately 10 mL of PBS. The cells suspensions were pelleted again and resuspended in approximately 3 mL of 5 % w/v trichloroacetic acid (TCA), and precipitated overnight (approximately 18 hours) at 4 °C. The samples were again pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA. The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to β-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation counter.

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE:
The results are expressed as disintegrations per minute (dpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test/control ratio for each concentration. The criterion for a positive response is that one or more concentrations of the test material should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. A test material which does not fulfil the above criterion is designated as unlikely to be a sensitiser.
Positive control substance(s):
other: phenylacetaldehyde (90 %) as a solution in propylene glycol at a concentration of 2.5 % v/v
Positive control results:
The application of phenylacetaldehyde at concentrations of 2.5 % v/v in propylene glycol resulted in a greater than 3-fold increase in isotope incorporation. Therefore, phenylacetaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The stimulation index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, is presented in table 2 in 'Any other information on results incl. tables'. The application of the test material at concentrations of 5 %, 10 % and 25 % w/w in propylene glycol resulted in an isotope incorporation which was less than 3-fold at all three concentrations. Consequently, the test material was designated as unlikely to be a sensitiser under the conditions of the test. However, there was an statistical significant increase of the dpm in the 25 % w/w dose group; due to this increase it is plausible to presume that higher doses of test material could cause a stimulation index >3. So it can be only concluded, that doses of <25 w/w % are not sensitizing.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
The pooled disintegrations per minute, disintegrations per minute per lymph node and stimulation index are presented in table 2 in 'Any other information on results incl. tables'. The test material is designated as unlikely to be a sensitiser under the conditions of the test.

Preliminary Screening Test Results

No signs of systemic toxicity, visual local skin irritation or excessive irritation indicated by an equal to or greater than 25 % increase in mean ear thickness were noted. Based on this information the dose levels selected for the main test were 5, 10 and 25 % w/w test material in propylene glycol.

Main Test Results

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the study. Furthermore, bodyweight changes of the test animals between day 1 and day 6 were comparable to those observed in the corresponding control group animals over the same period.

Positive Control Test Results

The stimulation index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group was 3.49. Therefore, phenylacetaldehyde (90 %) was considered to be a sensitiser under the conditions of the study.

Table 2: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in vehicle

dpm

dpm/node*

stimulation index #

Result

Vehicle

7746.32

968.29

na

na

5

7152.44

894.06

0.92

negative

10

6866.45

858.31

0.89

negative

25

13714.73

1714.34

1.77

negative

dpm = Disintegrations per minute

* Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

# Stimulation index of 3.0 or greater indicates a positive result

na = not applicable

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the isotope concentration was less than three-fold at all test concentrations and therefore, the test material is considered to be unlikely to be a skin sensitiser. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The skin sensitisation potential of the test material was determined in accordance with the standardised guidelines OECD 429 and EU Method B.42 using the mouse Local Lymph Node Assay. The test material was applied as a 5 %, 10 % or 25 % w/w preparation in propylene glycol. The positive control was shown to have the capacity to cause skin sensitisation confirming the validity of the protocol used for this study. Under the conditions of the test the isotope concentration induced by the test material was less than three-fold at all test concentrations. Therefore the test material is considered to be unlikely to be a skin sensitiser when administered as a preparation at ≤ 25 % w/w.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)
Additional information:

An Endpoint conclusion can be made based on the results of two out of three or three studies targeting the three identified steps in the adverse outcome pathway which is the basic principle for the OECD Guidelines 442C, 442D and 442E

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification