Hexane, 1,6-diisocyanato-, homopolymer, 2-hydroxyethyl acrylate- and propylene glycol monoacrylate-blocked

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 17, 2017 to January 27, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

yes

Remarks:

deviations do not affect the results of this study, see ''principles of method if other than guideline"

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

- Cytotoxicity range finding test:
Preparation of the keratinocyte cultures: cell suspension was adjusted to 83000 (± 10%) cells per mL. 120 µL of the cell suspension (= 10000 cells) were seeded in a clear flat bottom 96 well plate. The plate was incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 in a humidified atmosphere for 24 h. After the incubation time the medium was replaced. 50 µL of the single test substance concentrations (in three replicates) as well as controls were added to the cells. Afterwards the plate was incubated for 48 h at 37 ± 1°C in a humidified atmosphere containing 5.0 ± 0.5% CO2.
Viability assay (MTT): The cell culture medium was removed from all wells of the 96 well plate and 200 µL MTT working solution was added to each well. The plates were incubated for 2 h and 15 min h at 37 ± 1°C and 5.0 ± 0.5% CO2 in a humidified atmosphere. Finally the solution was removed and 100 µL MTT-lysis buffer was added to each well. The plate was agitated for 3 h and 45 min, before it was measured at 570 nm and at 690 nm at the photometer. For calculation of the relative viability a validated Microsoft Excel file was used.

- Main test:
Keratinocyte cultures and MTT test were prepared as above. The assay was performed in two independent experiments. 12 concentrations of the test substance were evaluated. The exposure time was 48 h. The following real concentrations of the test substance were investigated in experiment I and II: 0, 0.8, 1.0, 1.2, 1.5, 1.7, 2.1, 2.5, 3.0, 3.6, 4.3, 2.5 and 6.3 µg/mL. None of the treatment concentrations in both experiments deviated more than 10% from the nominal concentration. Precipitation of the test substance was not visible up to the highest concentration. 12 wells were used as growth control, 24 wells were used as solvent control, 6 wells were used as negative control and 5 wells were used as positive control.
For the evaluation of the luciferase expression the medium was 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 given to the cells and incubated for 5 min at room temperature. During this process the plate was slightly moved. After lysis 100 µL Steady-Glo Reagent were added to each well and the plate was shaken slowly for 5 min at room temperature. Then, 170 µL per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 sec using a luminometer.
- Luciferase induction as well as the relative viability measurements.

Positive control results:

EGDMA (120 μM) was used as positive control. The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70%.
Experiment I: Fold induction: 5.3; Relative viability: 93.2%
Experiment II: Fold induction: 4.1; Relative viability: 94.7%

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

1 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

- Cytotoxicity range-finding test:
No cytotoxic effect was observed with the controls. A dose-dependant effect was observed with the test substance. At the concentrations of 0.2 µg/mL to 1.6 µg/mL no negative effect was detected in comparison to the solvent control. At the concentration 3.1 µg/mL a slight reduction to 85.1% was observed. At the next lower concentration (6.3 µg/mL) a clear cytotoxic effect was measured with a relative viability value of 35.6%. All higher test substance concentrations (12.5 µg/mL - 400 µg/mL) induced complete cytotoxicity with relative viability values below 2%.

- Main test:
In experiment I a cytotoxic effect was observed at the concentration 6.3 µg/mL. In experiment II, a cytotoxic effect was observed at 6.3 µg/mL and 5.2 µg/mL. Those concentrations were then excluded for the evaluation of the luciferase induction. Finally the following test substance concentration showed a viability ≥ 70% and could therefore be evaluated for luciferase induction:
Experiment I: 5.2 µg/mL, 4.3 µg/mL, 3.6 µg/mL, 3.0 µg/mL, 2.5 µg/mL, 2.1 µg/mL, 1.7 µg/mL, 1.5 µg/mL, 1.2 µg/mL, 1.0 µg/mL, 0.8 µg/mL;
Experiment II: 4.3 µg/mL, 3.6 µg/mL, 3.0 µg/mL, 2.5 µg/mL, 2.1 µg/mL, 1.7 µg/mL, 1.5 µg/mL, 1.2 µg/mL, 1.0 µg/mL, 0.8 µg/mL;
A substantial and reproducible dose dependent increase in luciferase induction was measured in nearly all test substance concentrations in both experiments. Even at the lowest test substance concentration the induction value exceeded the threshold of 1.5 fold in comparison to the solvent control.

Interpretation of results:

other: The substance is a sensitizer and in vivo follow up is needed to assess the subcategorization.

Conclusions:

Under the study conditions, the substance produced positive responses in the LuSens assay, which is indicative of the potential to activate the Nrf2 transcription factor.

Executive summary:

A study was conducted to determine the potential of the substance to activate the Nrf2 transcription factor in the “LuSens Assay” similar to OECD Guideline 442D, in compliance with GLP. The measured endpoint in this assay was the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. The relative viability was recorded as well using a MTT test. Keratinocyte cultures were exposed in experiment I and II to concentrations of 0.8, 1.0, 1.2, 1.5, 1.7, 2.1, 2.5, 3.0, 3.6, 4.3, 5.2 and 6.3 µg/mL (based on a dose-range finding experiment). A substantial and reproducible dose-dependent increase in luciferase induction was measured in nearly all test substance concentrations in both experiments. Even at the lowest test substance concentration the induction value exceeded the threshold of 1.5 fold in comparison to the solvent control. Under the study conditions, the substance showed positive responses in the LuSens assay, which is indicative of the potential to activate the Nrf2 transcription factor (Fruhmesser, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation - in vitro:

A study was conducted to determine the potential of the substance to activate the Nrf2 transcription factor in the “LuSens Assay” similar to OECD Guideline 442D, in compliance with GLP. The measured endpoint in this assay was the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. The relative viability was recorded as well using a MTT test. Keratinocyte cultures were exposed in experiment I and II to concentrations of 0.8, 1.0, 1.2, 1.5, 1.7, 2.1, 2.5, 3.0, 3.6, 4.3, 5.2 and 6.3 µg/mL (based on a dose-range finding experiment). A substantial and reproducible dose-dependent increase in luciferase induction was measured at nearly all test substance concentrations in both experiments. Even at the lowest test substance concentration the induction value exceeded the threshold of 1.5 fold in comparison to the solvent control. Under the study conditions, the substance showed positive responses in the LuSens assay, which is indicative of the potential to activate the Nrf2 transcription factor (Fruhmesser, 2017).

Skin sensitisation - in vivo:

A study was conducted to determine the skin sensitisation potential of the substance according to OECD Guideline 406, in compliance with GLP. Thirty-nine female guinea pigs (Dunkin Hartley) were used. The study was performed with 2 test groups: 1 negative control group (10 animals) and 1 treated group (20 animals). 4 additional animals were used in a pilot study to assess the irritation potential via intradermal and topical applications and to determine the test concentrations for induction and challenge phase of the main study. A parallel positive control group was tested with α-hexylcinnamaldehyde on 5 animals in a concurrent study and served as a reliability check.The animals were inspected daily during the study. All skin reactions and any unusual findings, including systemic reactions, results from induction and challenge procedures were observed and recorded. Individual weights of animals were determined shortly before the test substance was applied and at the end of the study. At the end of the observation period animals were sacrificed. For the induction phase, the test substance was administered at a concentration of 10% through intradermal injection and at 25% through topical application. For the challenge phase, 5% of the test substance was used. No clinical signs and mortality were observed in the treated groups. Slight signs of erythema were observed in 7/20 animals of the treated group 24 h after removal of the patches. This represents skin sensitisation reaction in 35% of the tested animals. Very slight erythema was observed in 4/20 animals 48 h after removal of the patches. Edema was not observed in any of the tested animals. Well-defined erythema was observed 24 h after removal of the patches in 2/5 animals in the positive control group and slight erythema in the remaining 3/5 animals of this group. This represents skin sensitisation reaction in 100% of the tested animals. Under the study conditions, the substance produced skin sensitisation reactions in 35% of the tested animals (Hozova, 2017).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of in vitro and in vivo studies, the substance warrants a classification as Skin Sens. 1B - H317 (may casuse an allergic reaction) according to the EU CLP (EC 1272/2008) criteria.