Hexamethylene diisocyanate, oligomers, reaction products with Bis-(Trimethoxysilylpropyl)amine

Endpoint summary

Description of key information

- WoE: in vitro Skin Sensitization Turnkey Testing Strategy: sensitizing

- Supporting (RA): LLNA (2012): sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

- Principle of test: The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer.

GLP compliance:

yes (incl. QA statement)

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: 17/0241-1, Batch 294-296

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient

Details on the study design:

Skin sensitisation (In vitro test system)
Details on study design:
LuSens cells from the working cell bank were thawed and cultured using culture medium, under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) for at least passage ≥5 but not longer than 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 105 cells/mL cell suspension), using culture medium 2 for incubation for 24 hours. Two independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested. After cell adaption for 24 hours culture medium 2 was aspirated and replaced with 150 μL culture medium 3. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%). After test-substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability. Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates. After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Subsequently 200 μL of One-Glo-preparation (= 100 μL One-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer. Cell culture medium was aspirated from all wells. Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and culture medium 3) was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

- Cell line: LuSens
Human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen, Germany.

Run / experiment:

mean

Parameter:

other: CV75 value (µM)

Remarks:

estimated concentration that affords 75% cell viability

Value:

56

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2nd experiment

Parameter:

other: EC1.50 (µM)

Remarks:

Luciferase fold induction

Value:

21

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 3rd experiment

Parameter:

other: EC1.50 (µM)

Remarks:

Luciferase fold induction

Value:

15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Acceptance criteria:
A tested concentration is not further evaluated when relative viability is less than 70%.
The cell viability of VC cells must yield at least 85%.
The mean of the positive control EGDMA should achieve ≥2.50 fold-induction and the mean of the LA <1.50 and the mean of the viability must be ≥70%.
The CV [%] of the luminescence in the vehicle control wells for each plate should be below 20%.
The mean of the basal expression of the cells must be <1.50 fold-induction as compared to the solvent control.
In addition, positive, negative and vehicle control data should lie within the range of the historic data.

Evaluation criteria:
A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold-induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least two consecutive concentrations of two independent experiments.
A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 2000 μM if molecular weight is applicable or 2000 μg/mL if molecular weight is not applicable) or up to the cytotoxicity limit (at least one concentration displaying viability below 70%).
To be relevant for evaluation, the cell viability must be more than 70% in at least three tested concentrations of an experiment.

Interpretation of results:

other: keratinocyte activating potential

Conclusions:

Under the conditions of the study, the test substance has a keratinocyte activating potential.

Executive summary:

The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 56 μM (corresponding to test substance as provided by the sponsor).

In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. The following results were observed:

At concentrations used in the main experiment the test substance was soluble in DMSO (100 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

The EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was calculated to be 21 μM (experiment 2) and 15 μM (experiment 3), respectively.

In summary, after 48 hours of exposure to test substance, luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance has a keratinocyte activating potential.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Principles of method if other than guideline:

- Principle of test: Chemical reactivity has been shown to be well associated with allergenic potency (Gerberick et al., 2007) and has been described as the molecular initiating event in the OECD adverse outcome pathway (OECD Publication No.168; ENV/JM/MONO(2012)10). Within this context measuring the amount of proteins with nucleophilic side chains such as cysteine or lysine residues after incubation with putative allergens serves as surrogate markers.
In the DPRA the reactivity of a test item (=test substance) towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated. For this purpose, the test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm.
The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 17/0241-1, Batch 294-296
- Expiration date of the lot/batch:
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient

Details on the study design:

Skin sensitisation (In chemico test system)
- Details on study design: Chemical reactivity has been shown to be well associated with allergenic potency (Gerberick et al., 2007) and has been described as the molecular initiating event in the OECD adverse outcome pathway (OECD Publication No.168; ENV/JM/MONO(2012)10). Within this context measuring the amount of proteins with nucleophilic side chains such as cysteine or lysine residues after incubation with putative allergens serves as surrogate markers.
In the DPRA the reactivity of a test item (=test substance) towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated. For this purpose, the test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm.
The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.
- Synthetic peptides:
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: JPT Peptide Technologies GmbH, Berlin, Germany) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.
- The test substance was dissolved in a suitable vehicle. Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm.
In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

Run / experiment:

mean

Parameter:

other: C-Peptide depletion (%)

Value:

93.23

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

mean

Parameter:

other: K-peptide depletion (%)

Value:

3.64

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

mean

Parameter:

other: Peptide depletion

Remarks:

%

Value:

48.43

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Acceptance criteria of the DPRA:
The standard calibration curve should have an r² >0.99.
The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
The CV of the nine vehicle controls B and C should be < 15%.
Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).
In addition the positive control should cause depletion of both peptides comparable to historic data.

Interpretation of results:

other: Study is part of the in-vitro skin sensitization battery and cannot be used as stand-alone method for classification.

Conclusions:

Under the conditions of the study, the test substance is peptide reactive.

Executive summary:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at 100 mM in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides.

Further, in order to detect possible interference of the test substance with the peptides, a co-elution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.

The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were emulsions at the time of preparation. Visual observation after the 24-hour incubation revealed precipitates in all samples of the test substance with the peptides.

No co-elution of test substance and peptides was present. However, for the C-peptide reaction an alternative analysis method was used.

The mean C-peptide depletion, caused by the test substance was determined to be 93.23%.

The mean K-peptide depletion, caused by the test substance was determined to be 3.64%.

Thus, the mean peptide depletion was calculated to be 48.43%.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model, it was concluded that the test item shows high chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance in the samples with both peptides the values for depletion could be under-predictive.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of the test substance was assessed using a combination of in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization as defined by the OECD.

DPRA:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at 100 mM in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides.

Further, in order to detect possible interference of the test substance with the peptides, a co-elution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.

The mean C-peptide depletion, caused by the test substance was determined to be 93.23%. The mean K-peptide depletion, caused by the test substance was determined to be 3.64%. Thus, the mean peptide depletion was calculated to be 48.43%.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test item shows high chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance in the samples with both peptides the values for depletion could be under-predictive.

LuSens:

The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 56 μM (corresponding to test substance as provided by the sponsor).

At concentrations used in the main experiment the test substance was soluble in DMSO (100 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

The EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was calculated to be 21µM (2nd Run) and 15µM (3rd Run), respectively.

In summary, after 48 hours of exposure to test substance luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance has a keratinocyte activating potential.

For a final assessment of the test substance, the h-CLAT is not necessary and is therefore waived.

Based on thes results of the DPRA and LuSens, the test substance is peptide reactive and activates keratinocytes and is therefore predicted to be a skin sensitizer.

Read-across substance:

In a GLP compliant study according to OECD guideline 429 the possible skin sensitization potential of the read-across substance was studied (Harlan CCR, 2012). Three groups each of five female mice were treated daily with the test item at concentrations of 2.5, 5, and 10% (w/w) in acetone:olive oil (4+1 v/v) by topical application to the dorsum of each ear once daily for three consecutive days. The test item could be dissolved in the vehicle. The appropriateness of the used concentrations was previously assessed by three pre-experiments. Two further groups each of five mice were treated with the vehicle only or with the positive control at 25% (w/v). Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes were excised, pooled per animal and immediately weighed. Furthermore, after excision of the lymph nodes, both ears of the mice were punched at the apical area using a biopsy punch and were immediately weighed pooled per animal using an analytical balance. Afterwards, single cell suspensions of lymph node cells were prepared from lymph nodes pooled per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. Subsequently the suspensions were washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of³H-methyl thymidine measured in a ß-scintillation counter. All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. On day 2, an erythema of the ear skin (Score 1) was observed in all animals treated with 10% test item concentration. A statistically significant increase in ear weights was observed in the low and the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, for BALB/c mice, a cutoff-value of 1.1 was reported for a positive response of the ear weight index regarding ear skin irritation. The indices determined for the low and high dose group exceeded this threshold (indices of 1.2). A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 2.81, 5.96, and 10.36 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in acetone:olive oil (4+1 v/v) and an EC3 value of 2.7% (w/w) was calculated. A statistically significant increase in mean DPM value, lymph node weight, and lymph node cell count was observed in all test item treated groups in comparison to the vehicle control group (p<0.05). A clear dose response was observed in all parameters. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count were exceed in the mid and high dose group (index of 2.1 and 2.8, respectively) and therefore considered to be biologically relevant. The S.I. determined with the positive control item at 25% was 10.77, demonstrating the validity of the study. Furthermore, a statistically significant increase in DPM value and also in lymph node weight and -cell count (p<0.05) was observed here in comparison to the acetone:olive oil (4+1 v/v) vehicle control group, corroborating this result. Also, the cutoff value for a positive response of the lymph node cell count index of 1.55 was exceeded in this group (index: 3.44). Although a certain amount of skin irritation was observed, it was unlikely that irritation had an influence on lymphocyte proliferation, as a Stimulation Index above the threshold value of 3 was also obtained in the mid dose, where no statistically significant or biologically relevant amount of irritation had been noted. The test item was a skin sensitizer under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the findings of the available studies, the test substance has to be classified R43 (Directive 67/548/EEC) and Skin sensitisation Cat. 1B (CLP).