Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

Endpoint summary

Description of key information

The available information obtained in chemico and in vitro does not point to a skin sensitizing activity in vivo. The substance was tested in chemico for the first key event of skin sensitization (peptide reactivity, OECD 442C) and in vitro for the second (keratinocyte activation, OECD 442D) and third key event of skin sensitization (dendritic cell activation, OECD 442E). All tests revealed negative results.

The above listed tests and procedures are adequate for non-classification regarding skin sensitization and risk assessment according to point 8.3 of Annex VII of Regulation (EC) 1907/2006.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD TG 442C and in compliance with GLP without deviation.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In vitro methods are applicable for this substance and are adequate for classification and risk assessment according to REACH Regulation.

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The Direct Peptide Reactivity Assay (DPRA) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.
For comparison, tests were performed with the test item, the vehicle (solvent control = negative control) and the known sensitizer Cinnamic aldehyde (positive control).

The DPRA quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 +/-2.5ºC. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. The synthetic peptides contain phenylalanine to aid in the detection.

Preparation of the cysteine or lysine containing peptides:
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item. The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.667 mM in pH 10.2 ammonium acetate buffer.

Preparation of the test item:
Solubility of the test item in an appropriate solvent was assessed before performing the assay. 92.80 mg test item were dissolved in 3 mL acetonitrile immediately before testing to prepare a 100 mM solution. A factor of 1.21 was used to correct for the purity of the test item. The test item solution was then tested as such without any further dilution by incubating at 1:10 or 1:50 ratio with the cysteine peptides and lysine peptides, respectively.

Positive control, reference controls and co-elution control:
Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile. In addition reference controls (i.e. samples containing only the peptide and added acetonitrile) were also included in the HPLC run sequence and these were used to verify the HPLC system suitability prior to the analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. In addition a co-elution control constituted by the test item alone for the test item analysed was included in the run sequence to detect possible co-elution of the test item with either the lysine or the cysteine peptide.

Incubation of the test item with the cysteine and lysine peptide solutions:
Cysteine and lysine peptide solutions were incubated in glass autosampler vials with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5°C for 24 ± 2 hours before running the HPLC analysis. The test item assay was analysed in triplicate for both peptides. Samples were visually inspected prior to HPLC analysis.
If a precipitate would be observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item, in this case one cannot be sure how much test item remained in the solution to react with the peptide. Therefore, in such a case, a positive result could still be used, but a negative result is uncertain and would be interpreted with due care. No precipitate or phase separation was observed.

Preparation of the HPLC standard calibration curve:
A standard calibration curve was generated for both the cysteine and the lysine peptides. Peptide standards were prepared in a solution of 20% acetonitrile : buffer using 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide and 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide. Using serial dilution standards of the peptide stock solution (nominal concentrations: 0.666 mM of cysteine peptide in sodium phosphate or 0.666 mM lysine peptide in ammonium acetate), 6 calibration standards were prepared to cover the range from 0.534 to 0.0167 mM. A blank of the dilution buffer was also included in the standard calibration curve. Suitable calibration curves should have an r2 > 0.99.
If a test item promotes the oxidation of the cysteine peptide, the peak of the dimerised cysteine peptide would have been visually monitored. If dimerisation appears to have occurred, this would have been noted as percent peptide depletion which would have been over-estimated leading to false positive predictions and/or assignment to a higher reactivity class. No dimerisation of the cysteine peptide occurred.
HPLC analysis for the cysteine and lysine peptides was performed on one day. All test item solutions were freshly prepared for both assays on one day. The analysis was timed to assure that the injection of the first sample (reference control C) started 22 to 26 hours after the test item had been mixed with the peptide solution. The HPLC run sequences were set up in order to keep the HPLC analysis time to less than 30 hours.

Data evaluation:
The concentrations of cysteine or lysine peptide were photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The percent peptide depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference control C.

Acceptance criteria:
The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.

The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
b) The mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 68.65% for cysteine and 68.56% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

0

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

0.84

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The acceptance criteria for a DPRA test to be considered valid were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes

The test item was dissolved at a concentration of 100 mmol/L in acetonitrile. No precipitation occured before or after incubation.
Absorbance at 220 nm was not observed. Retention time similar to peptide was not observed. Co-elution of the test item with the peptides was not observed.
No relevant depletion of cysteine and lysine peptides became obvious in the DPRA for the test item (0.42%). According to the cysteine 1:10/lysine 1:50 prediction model “no or minimal reactivity” was derived for the test item in acetonitrile, leading to a DPRA prediction of “negative“. The test item is thus predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Results of the DPRA:

Test item	Mean % Cysteine peptide depletion	Mean % Lysine peptide depletion	Mean % Cysteine/Lysine peptide depletion	reactivity class	DPRA prediction
positive control (cinnamic aldehyde)	68.65	68.56		high	positive
test item	0.0	0.84	0.42	no or minimal	negative

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is predicted to be a non-sensitizer in the DPRA.

Executive summary:

The Direct Peptide Reactivity Assay (DPRA; OECD 442C) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

The test item was dissolved at a concentration of 100 mmol/L in acetonitrile. No precipitaton of the test item occured before or after incubation. The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. No relevant depletion of cysteine and lysine peptides became obvious in the DPRA (mean of 0.42%). According to the prediction model “no or minimal reactivity” was derived for the test item in acetonitrile, leading to a DPRA prediction of “negative“. The test item is thus predicted to be a non-sensitizer in the DPRA.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD TG 442D and in compliance with GLP without deviation.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted Feb 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

In vitro methods are applicable for this substance and are adequate for classification and risk assessment according to REACH Regulation.

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

Test chemicals are considered positive in the KeratinoSens™ if they induce a statisticallysignificant induction of the luciferase activity above a given threshold (i.e. > 1.5 fold or 50% increase), below a defined concentration which does not significantly affect cell viability (i.e. below 1000 μM and at a concentration at which the cellular viability is above 70%). For this purpose, the maximal fold induction of the luciferase activity over solvent (negative) control (Imax) is determined. Furthermore, since cells are exposed to series of concentrations of the test chemicals, the concentration needed for a statistically significant induction of luciferase activity above the threshold (i.e. EC1.5 value) is interpolated from the dose-response curve. Finally, parallel cytotoxicity measurements are conducted to assess whether luciferase activity induction levels occur at subcytotoxic concentrations.

Preparation of the keratinocyte cultures:
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland as specified in OECD Test Guideline 442D (batch number 15042016).Cells were propagated and stored frozen as a homogeneous stock. Cells from this original stock were propagated up to a maximum passage number of 25 and were employed for routine testing using the appropriate maintenance medium.
For testing, cells were 80-90% confluent and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested and distributed into 96-well plates (10 000 cells/well). Attention was paid to avoid sedimentation of the cells during seeding to ensure homogeneous cell number distribution across wells. For each repetition, three technical replicates were used for the luciferase activity measurements, and three parallel technical replicates used for the cell viability assay.

Preparation of the test and control items:
Due to information on the hydrolytic property of the test item given by the Sponsor the test item was completely dissolved in dimethyl sulfoxide (DMSO) free from water. DMSO was used as solvent control. 61.77 mg Levagard 4090 N were dissolved in 1 mL dimethyl sulfoxide to a concentration of 200 mM. A correction factor of 1.21 was used to correct for the content of 82.6%. Fresh preparations of the test and control items were used for the treatment. The final concentration of the vehicle in the culture system did not affect cell viability or growth rate.
Based on the stock solution of the test item, serial dilutions were made using solvent to obtain 12 master concentrations to be tested (from 0.098 to 200 mM). The master concentrations were then further diluted in treatment culture medium containing 1% serum3, so that the final concentrations of the test item ranged from 0.98 to 2000 μM. The vehicle DMSO was used as the negative control. Six wells per plate were prepared. It was diluted following the same dilution scheme as described for the master concentrations, so that the final negative control concentration is 1%, which is known to not affect cell viability and corresponds to the same concentration of DMSO found in the test item and in the positive control.
Cinnamic aldehyde4 was used as the positive control. A series of 5 master concentrations ranging from 0.4 to 6.4 mM was prepared in DMSO and diluted as described for the master concentrations, so that the final concentration of the positive control range from 4 to 64 μM.

Luciferase activity measurements:
After the 48 hour exposure time with the test and control items, cells were washed with a phosphate buffered saline, and the relevant lysis buffer (One GlowTM Luciferase Assay System)5 for luminescence readings added to each well for an adequate time at room temperature. Plates with the cell lysate will then be placed in the luminometer (Tecan6 Infinite 200Pro) for reading.

Cytotoxicity assessment:
For the KeratinoSensTM cell viability assay, the 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)7 and cells incubated for 4 hours at 37°C in the presence of 5% CO2. The MTT medium was removed and cells were lysed by adding 10% aqueous SDS8 solution to each well overnight or for up to 3 days at 37°C. After shaking, the absorption was measured at i.e. 620 nm with a photometer (TecanSunrise Magellan Version 7.2)

A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing 9% foetal bovine serum and 1% Geneticin.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The positive control cinnamic aldehyde was run in both repetitions. Cinnamic aldehyde was shown to be positive in both repetitions (i.e. induction of > 1.5). The induction at 64 μM and the EC1.5 for cinnamic aldehyde were also calculated. The targets are:
• the average induction in the two replicates for cinnamic aldehyde at 64 μM should be between 2 and 8,
• the EC1.5 value should be within two standard deviations of the historical mean value
At least one of these two numerical criteria must be met in order to accept a repetition. In the experiments performed both criteria were fulfilled in both repetitions. Thus both repetitions were valid for the positive control.
In addition, the EC1.5 average value of the positive control of 24.15 μM is within two standard deviations of the historical mean

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

2 000 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

1 500.56 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: proven

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
As further performance criterion the variability of the luminescence reading for the negative control DMSO must be below 20% in each repetition which consists of 6 wells tested in triplicate. The average coefficients of variation (CV) for the negative control were 6.81% or 8.05% for the first or second repetition, respectively.
- Acceptance criteria met for positive control: yes (see above)
- Acceptance criteria met for variability between replicate measurements: yes; the EC1.5 average value of the positive control of 24.15 μM is within two standard deviations of the historical mean

The maximum average fold induction of the luciferase activity (Imax) value observed at any concentration of the test item was 1.12 ± 0.06 and no EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) could be calculated.

The calculated IC50 value was > 2000 μM test item and the IC30 was 1500.56 μM for 30% reduction of cellular viability, respectively.

The KeratinoSensTM prediction of the test item is considered negative as the luciferase induction value was < 1.5 compared to the solvent control.

Results table:

Criteria	Experiment 1	Experiment 2	mean
I max	1.16	1.07	1.12
IC50	> 2000µM	> 2000 µM
EC1.5	not calculated	not calculated

Interpretation of results:

GHS criteria not met

Conclusions:

The substance revealed no sensitising properties in the ARE-Nrf2 Luciferase test method.

Executive summary:

The substance was examined for sensitising properties in the ARE-Nrf2 luciferase test method following OECD 442D. The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

Two endpoints were measured: luciferase induction after a 48 hour treatment with the test item and cytotoxicity determined with the MTT assay with the same cell batch and employing the same dilutions of the test item. Dimethyl sulfoxide (water-free DMSO) was used as solvent control. For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration needed to reach an 1.5 fold induction (EC1.5) were calculated. For cytotoxicity the IC50 and IC30 values were interpolated.

The test item was completely dissolved in DMSO free from water and tested at 12 concentrations in the range from 0.98 to 2000 μM. Cinnamic aldehyde, tested at five concentrations from 4 – 64 μM, was used as the positive control. In the experiments performed with the positive and the vehicle control all quality cirteria were fulfilled in both repetitions.

The maximum average fold induction of the luciferase activity (Imax) value observed at any concentration of the test item was 1.12 ± 0.06 and no EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) could be calculated. The calculated IC50 value was > 2000 μM test item and the IC30 was 1500.56 μM for 30% reduction of cellular viability, respectively.

The KeratinoSensTM prediction of the test item is considered negative as the luciferase induction value was < 1.5 compared to the solvent control.

In conclusion, the substance revealed no sensitising properties in the ARE-Nrf2 Luciferase test method.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD TG 442E and in compliance with GLP without deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

adopted 09 October 2017

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

In vitro methods are applicable for this substance and are adequate for classification and risk assessment according to REACH Regulation.

Details of test system:

THP-1 cell line [442E]

Details on the study design:

The test item was examined for sensitising properties in the human Cell Line Activation test (h-CLAT). The h-CLAT is an in vitro assay proposed to address the third key event of the skin sensitisation AOP (Adverse Outcome Pathway) by quantifying changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression were measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control were calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.
The h-CLAT has been recommended to be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The human monocytic leukaemia cell line, THP-1 (TIB-202™, ATCC) was used for performing the h-CLAT assay. THP-1 cells were incubated at 37°C under 5% CO2 and humidified atmosphere in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.1% 2-mercaptoethanol, 100 units/mL penicillin, and 100 μg/mL streptomycin. THP-1 cells were routinely seeded every 2-3 days at the density of 0.1E06 to 0.2E06 cells/mL. They were maintained at densities from 0.1E06 to 1.0E06 cells/mL. Prior to using them for testing, the cells were qualified by conducting a reactivity check. The reactivity check of the cells was performed using the positive controls (2,4-dinitrochlorobenzene (DNCB) and nickel sulfate (NiSO4)) and the negative control (lactic acid (LA)) at the earliest two weeks after thawing. Both DNCB and NiSO4 produce a positive response of both CD86 and CD54 cell surface markers; LA produces a negative response of both CD86 and CD54 cell surface markers. Only the cells which have passed the reactivity check were used for the assay. Cells can be propagated up to two months after thawing. Cells should not exceed 30 passages after thawing.
For testing, THP-1 cells were seeded at a density of either 0.1E06 cells/mL or 0.2E06 cells/mL, and pre-cultured in culture flasks for 72 or 48 hours, respectively. It is important that the cell density in the culture flask just after the pre-culture period is as consistent as possible in each experiment, because the cell density in the culture flask just after pre-culture could affect the CD86/CD54 expression induced by allergens. On the day of testing, cells harvested from the culture flask were resuspended with fresh culture medium at 2E06 cells/mL. Then, cells were distributed into a 24-well flat-bottom plate with 500 μL per well (1E06 cells/well).

A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. Ten dilutions (Ten concentrations) were prepared, by two-fold serial dilution with medium and a final range of concentrations in the plate of 7.8 - 5000 μg/mL culture medium. Culture medium was used as solvent control in the preliminary experiment.
The CV75 value was used to determine the concentration of test item for the CD86/CD54 expression measurement. In this preliminary experiment (consisting of two independent runs) no cytotoxicity was noted and thus no CV75 could be determined (CV75 of > 5000 μg/mL). Due to information on the hydrolytic property of the test item given by the Sponsor dimethyl sulfoxide (DMSO) free from water was used as solvent for the main experiment. According to the guideline the final concentration in the plate should not exceed 1000 μg/mL in case of DMSO used as solvent. Hence, the test item was completely dissolved in water-free dimethyl sulfoxide (DMSO) to a concentration of 500 mg/mL and was tested at 8 concentrations in the range from 279.1 to 1000 μg/mL. A correction factor of 1.21 was used to correct for the content of 82.6%. Also in water-free DMSO no cytotoxicity was noted for the test item. DNCB (2,4- dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 μg/mL in the plate. Each experiment consisted of two independent runs for CD86/CD54 expression measurement.

The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1 (525 nm). Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 of the medium, the positive control cells and the test item-treated cells were calculated compared to the solvent control. The cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibody) was also calculated.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 μg/mL in the plate. Each experiment consisted of two independent runs for CD86/CD54 expression measurement. The RFI for CD54 expression was 270 and 502 and for CD86 expression 234 and 483 in Experiments 1 and 2, respectively. Cell viability was above 50% in both experiments. Thus, the acceptance criteria for the positive control were met.

Key result

Group:

test chemical

Run / experiment:

other: Maximum RFI (experiment 1 &2)

Parameter:

RFI CD54>150 [442E]

Value:

111 %

Cell viability:

> 90%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Range from 64 to 111%

Key result

Group:

test chemical

Run / experiment:

other: Maximum RFI (experiment 1 &2)

Parameter:

RFI CD86>200 [442E]

Value:

106 %

Cell viability:

> 90%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Range from 82 to 106%

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The cell viability of the solvent controls (medium and waterfree DMSO) was higher than 90% in each independent run.
In the solvent control (medium), RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
For solvent control (medium), the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
For the positive control in Experiment 1, the RFI for CD86 was just below 150% (i.e. 139%). However, as cell viability was greater than 90% at each concentration of positive control and there were no border line responses with the test article, it was considered that the experiment was valid
For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.
Thus, all acceptance critera were met.

Relative fluorescence intensity (RFI) values:

Concentration (µg/mL)	RFI CD54 - 1st Exp.	RFI CD86 - 1st Exp.	RFI CD54 - 2nd Exp.	RFI CD86 - 2nd Exp.
279.1	64	91	53	82
334.9	78	90	89	101
401.9	89	99	86	98
482.3	91	93	84	96
578.7	86	89	84	97
694.4	96	105	79	97
833.3	103	100	92	103
1000.0	111	106	92	102
medium control	99	96	106	106
vehicle control (0.2% DMSO)	100	100	100	100
positive control (DNCB)	270	234	502	483

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be negative in the human Cell Line Activation Test at up to and including the maximum attainable concentration.

Executive summary:

The study was conducted to investigate the potential of the test item to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression were measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control were calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers. The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E.

A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. Ten dilutions (Ten concentrations) were prepared, by two-fold serial dilution with medium and a final range of concentrations in the plate of 7.8 - 5000 μg/mL culture medium. The CV75 value was used to determine the concentration of test item for the CD86/CD54 expression measurement. In this preliminary experiment (consisting of two independent runs) no cytotoxicity was noted and thus no CV75 could be determined (CV75 of > 5000 μg/mL). Due to information on the hydrolytic property of the test item dimethyl sulfoxide (DMSO) free from water was used as solvent for the main experiment. According to the guideline the final concentration in the plate should not exceed 1000 μg/mL in case of DMSO used as solvent. Hence, the test item was completely dissolved in water-free dimethyl sulfoxide (DMSO) to a concentration of 500 mg/mL and was tested at 8 concentrations in the range from 279.1 to 1000 μg/mL.

The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1 (525 nm). Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 of the medium, the positive control cells and the test item-treated cells were calculated compared to the solvent control.

In the CD86/CD54 expression experiments 1 and 2 the relative fluorescence intensity (RFI) values of the test article were <150% for CD86 and <200% for CD54 at all tested concentrations compared to the solvent controls. The test article therefore gave a negative prediction in the assay at up to and including the maximum attainable concentration. All acceptance and evaluation criteria were met in both experiments.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

An assessment of potential skin sensitization properties based on defined approaches and individual sources was conducted for the substance within an IATA. The outcome of this assessment is given below:

No animal studies are available for the substance to assess the endpoint skin sensitization.

No human data are available for the substance to assess the endpoint skin sensitization.

Information on dermal bioavailability:

No information is available on dermal bioavailability of the substance. The substance is a liquid with a molecular weight of 255 g/mole. The substance highly soluble in water (900 g/L) and a log Kow (octanol/water) of -1.9 was calculated. Based on these physical-chemical parameters dermal absorption can be expected to be low (ECHA Guidance on Information Requirements, Chapter R7c, 2017).

In silico information - Structure Activity Relationship (QSAR via OECD Toolbox; Schlecker 2018):

As in silico tool for the prediction of skin sensitization the QSAR OECD Toolbox 4.2.1 was used. The outcomes of the mechanistic/endpoint relevant profilers for skin sensitization potential are:

Protein binding alerts: As protein binding alert SN2 (nucleophilic substitution type 2) was identified by OASIS and OECD for the substance.

Protein binding potency: Not possible to classify

DPRA Cysteine/Lysine peptide depletion: Out of mechanistic domain of the Direct Peptide Reactivity Assay (DPRA)

Keatinocyte gene expression: not possible to classify

Protein binding potency h-CLAT: no alert found

Protein binding alerts for skin sensitization according to GHS: Skin sensitization Category 1B

In conclusion, the OECD Toolbox revealed an alert for protein binding of the substance identified as nucleophilic substitution type 2 (Skin Sens 1B).

In chemico testing - Direct Peptide Reactivity Assay (DPRA following OECD TG 442C, Rehders D 2018):

The substance was tested in the Direct Peptide Reactivity Assay (DPRA) following OECD 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)). This test addresses the first key event of skin sensitization, the covalent binding to skin proteins (haptenation). Protein binding is measured in chemico by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a cysteine/lysine prediction model to categorize a substance in one of four classes of reactivity:

• ‘no or minimal reactivity’ (DPRA prediction negative): ≤ 6.38% depletion

• ‘low reactivity’ (DPRA prediction positive): > 6.38 to ≤ 22.62% mean depletion

• ‘moderate reactivity’ (DPRA prediction positive): > 22.62 to ≤ 42.47% mean depletion and

• ‘high reactivity’ (DPRA prediction positive): > 42.47 to ≤ 100% mean depletion

The test item showed no relevant depletion of peptides in the DPRA with a mean cysteine/lysine depletion rate of 0.42%. The obtained value counts for a DPRA prediction of ‘no or minimal reactivity – negative’.

In vitro testing in a first cell-based test – KeratinoSens test (following OECD TG 442D, Spruth B 2018):

The substance was tested in the KeratinoSens assay, an in vitro skin sensitization test (ARE-Nrf2 Luciferase Test Method) following OECD TG 442D (In vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method). This test addresses the second key event of skin sensitization, the activation of keratinocytes in cell culture. Skin sensitizers are reported to induce genes that are regulated by the antioxidant response element (ARE). Small electrophilic items such as skin sensitisers can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g. covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes.

The ARE-Nrf2 luciferase test method utilizes an immortalized adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

A prediction in the KeratinoSens is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions (Imax >1,5-fold; cell viability > 70%, EC1.5 value < 1000 µM, apparent dose response curve).

Since the Imax in both experiments was below 1.5-fold, the test item was considered to be negative in the ARE-Nrf2 Luciferase Test.

In vitro testing in a second cell-based test - h-CLAT (following OECD TG 442E, Spruth B 2018):

The substance was tested in the human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test following OECD TG 442E (In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT)). This test addresses the third key event of skin sensitization, the activation of dendritic cells in culture. In principle, changes in the expression of cell surface markers (i.e. CD86 and CD54) on a human monocytic leukaemia cell line are quantified following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic dendritic cell (DC) activation, which plays a critical role in T-cell priming. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control is calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (positive or negative). An h-CLAT prediction is considered positive if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered negative:

• The relative fluorescence intensity (RFI) of CD86 is equal to or greater than 150% at any tested concentration (with cell viability ≥ 50%);

• The RFI of CD54 is equal to or greater than 200% at any tested concentration (with cell viability ≥ 50%).

Based on the above conditions, if the first two runs give concordant result for CD86 and/or CD54, the h-CLAT prediction is clear (positive or negative) and a third run does not need to be conducted.

A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. In this preliminary experiment no cytotoxicity was noted and thus no CV75 could be determined (CV75 of > 5000 μg/mL). Levagard 4090 N was tested at 8 concentrations in the range from 279.1 to 1000 μg/mL in the main experiment . DNCB (2,4 -dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement.

In the CD86/CD54 expression experiments the relative fluorescence intensity (RFI) values of thetest item were <150% for CD86 and <200% for CD54 at all tested concentrations compared to the solvent controls. The test article therefore gave a negative prediction in the assay at up to and including the maximum attainable concentration. In conclusion, the test item did no reveal any sensitizing properties in the h-CLAT method.

Discussion and conclusion:

This assessment of potential skin sensitizing properties is based on defined approaches and individual sources to be used within an Integrated Approach to Testing and Assessment (IATA).

An alert for protein binding was identified in silico by the OECD QSAR Toolbox 4.2.1 as nucleophilic substitution type 2. The Toolbox categories the substance according to GHS with Skin Sens 1B. This protein binding alert could not be confirmed in chemico and/or in vitro.

As in chemico tool the Direct Peptide Reactivity Assay (DPRA, OECD 442C) was used (Rehders, 2018). The DPRA addresses the first key event of skin sensitization, the covalent binding to skin proteins (haptenation). The test substance showed a negative result (‘no or minimal reactivity’) in the DPRA with a cysteine/lysine peptide depletion of 0.42%. Thus, no binding affinity to proteins became obvious in chemico.

The substance was tested for the second key event of skin sensitization, the activation of keratinocytes in vitro, in the KeratinoSens assay (ARE-Nrf2 Luciferase Test, OECD 442D). The test substance was considered negative in this test (Spruth, 2018). With regard to the third key event of skin sensitization (activation of dendritic cells) the human Cell Line Activation Test (h-CLAT, OECD 442E) was performed.  The test substance gave a negative prediction in the assay at up to and including the maximum attainable concentration (Spruth, 2018).

In conclusion and in applying the Weight of Evidence approach, the available data in this evaluation do not point to a skin sensitization potential in vivo. Additionally, based on the physico-chemical characteristics of the substance dermal absorption in vivo can be expected to be low.

Although a protein binding alert was obtained via in silico assessment with the OECD Toolbox, no such effects could be observed via testing. The substance was tested in chemico for the first key event of skin sensitization (peptide reactivity) and in vitro for the second (keratinocyte activation) and third key event of skin sensitization (dendritic cell activation). In all tests the acceptance criteria were met and throughout consistently negative results were obtained.

The above listed tests and procedures are adequate for non-classification regarding skin sensitization and risk assessment according to point 8.3 of Annex VII of Regulation (EC) 1907/2006.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No data available.

Justification for classification or non-classification

Harmonised classification: 

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self classification: 

Based on the available information no additional self-classification is proposed according to the CLP and the GHS.

No information is available regarding respiratory sensitisation.