2-(2-aminoethoxy)ethanol

Endpoint summary

Description of key information

The registered substance was tested in a modern in vitro skin sensitization testing battery (BASF, 2022). The overall conclusion was that the substance was predicted not to be a skin sensitizer. The substance was additionally determined to be not skin sensitizing in a supporting Buehler test (Huntsman, 1991). 

No data available for the determination of the respiratory sensitisation potential.

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

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase LuSens test method

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:
000STD77L0
- Purity: 99.228% (analysis performed 27 Sep 2021)
- Physical state / color: liquid / colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: Because the test-substance preparation was incubated with the peptide shortly after preparation, no analysis of the test substance in the vehicle was performed.
- Solubility and stability of the test substance in the solvent/vehicle: The stability under storage conditions over the exposure period was guaranteed by the sponsor

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment 2-(2-aminoethoxy)ethanol will be dissolved in DMSO. For the MTT test (dose finding assay) twelve concentrations of the test item will be analysed, if possible. Therefore, dilutions will be prepared by 1:2 serial dilutions from the highest soluble/dispersible concentration. If the test item is not soluble/dispersible, sonication of the formulation for 5 min and/or heating up to 37 °C could be performed. The highest test concentration for the dose finding assay will be 2000 μM in accordance to the OECD guideline 442D.

OTHER SPECIFICS:
- Homogeneity: The test substance was homogeneous by visual inspection.
- Molecular weight: 105.14 g/mol
- Log KOW: -1.89 (calculated)

Details of test system:

Lusens transgenic cell line [442D]

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

STUDY CONTROLS
Medium control
Name: Treatment medium

Solvent control for the test item
Name: DMSO (CAS No. 67-68-5, purity: ≥99%), final concentration 1% (v/v) in treatment medium

Positive control
Name: EGDMA (CAS 97-90-5, purity: ≥97.5%), final concentration 120 μM
Solvent: Treatment medium including 1% (v/v) DMSO

Negative control
Name: Lactic acid (CAS 50-21-5, purity: ~ 90%), final concentration 5000 μM
Solvent: Treatment medium including 1% (v/v) DMSO

TEST SYSTEM AND SUPPORTING INFORMATION
Reasons for the choice of cells
The LuSens cell line is an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The plasmid contains a luciferase gene (reporter gene) which is under transcriptional control of an antioxidant response element (ARE) of the rat NQO1 gene. Genes dependent on the ARE such as NQO1 are known to be up-regulated by contact sensitisers (OECD 442D). The HaCaT human keratinocytes (provided by RWTH, Aachen, Germany) were genetically modified at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of CJ Wruck). The LuSens cells were obtained by BASF and in 2019 a contract services agreement was established between ICCR-Roßdorf GmbH (Licensee) and Promega.

LuSens cell cultures
Stocks of the LuSens cell line are stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH (aliquots of cells in freezing medium at 1.5-5 × 106 cells/cryovial) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. The stock cultures are thawed at 37 ± 1.5 °C in a water bath. The cells are sub-cultured twice weekly. The cell density should not exceed a cell density of 80-90%. The confluent cells will be incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % CO2 (standard cell culture conditions). The LuSens cells can be used up to one and a half month after thawing (passage number should not exceed passage 15 for the main experiment and passage 20 for the dose range finder).

Culture media
DMEM with supplements listed below will be used to culture the cells during the assay. The culture medium will be warmed to room temperature just before use.
Cultivation medium: DMEM culture medium with Penicillin/Streptomycin (1% v/v), FBS (10% v/v) and puromycin (0.005% v/v)
Seeding medium: DMEM culture medium with FBS (10% v/v)
Treatment medium: DMEM culture medium with FBS (1% v/v)

Preparation and seeding of the cells
Between 4 and 6 × 105 or 6 and 8 × 105 cells will be seeded in 15 mL cultivation medium and pre-cultured at least twice a week in culture flasks (80-90% confluent). The cell density between approximately 80 and 90% should not be exceeded.
After microscopic assessment of the cells, the cells will be washed at least twice with 10 mL Ca2+/Mg2+ free DPBS including EDTA. Thereafter, the cells will be trypsinated with 1-2 mL 0.05% Trypsin/EDTA for approximately 5 min at 37 ± 1.5 °C and 5.0 ± 0.5 % CO2. The cells will be resuspended in 10 mL cultivation medium to neutralise the trypsin.
For seeding of the cells, the cultivation medium will be removed, and the cells will be transferred into seeding medium. Each treatment well of a 96 well microtiter plate will be seeded with 100 μL cell suspension (9000-11000 LuSens cells/well), except the well of the blank control. The cells will be incubated for 24 h ± 30 min under standard cell culture conditions.

EXPERIMENTAL DESIGN AND STUDY CONDUCT
Dose finding assay (MTT test)
The doses investigated in the main experiment (LuSens) will be determined with a MTT test.
The MTT test is based on the cleavage of the yellow tetrazolium salt MTT [= 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to form a blue-violet formazan dye by MTT reduction.
At least one cytotoxicity experiment (dose finding assay) will be performed to obtain a CV75, if possible.

MTT labelling mixture
The MTT working solution consists of two components, the MTT stock solution (5 mg/mL MTT in Ca2+/Mg2+ free DPBS) and treatment medium. Both components will be mixed right before application at a ratio of 1:10.

Treatment and microscopic evaluation
For the treatment of the cells in the dose finding assay, a stock solution of the test item and the controls will be prepared. The test item will be dissolved in the solvent and 1:2 serially diluted in the solvent to obtain the desired test item concentrations (twelve concentrations).
The solvent control DMSO (twelve replicates), the positive control (two replicates) and the negative control (three replicates), as well as the test item concentrations (each three replicates) will be subsequently diluted 1:25 in treatment medium. The treatment medium control (six replicates) will be used undiluted.
24 h ± 30 min after seeding of the cells, the seeding medium will be removed from the wells. Thereafter, 150 μL treatment medium will be added per well and 50 μL of the solvent control, the negative and positive control, the medium control and the test item dilutions will be added to the wells, respectively. At the end of the incubation period of 48 ± 1 h under standard cell culture conditions, the cell cultures will be microscopically evaluated for morphological alterations, precipitation or phase separation.

MTT Labelling and Measurement
At the end of the incubation period, treatment medium will be removed from the wells and the cells will be washed at least twice with 200 μL DPBS including Ca2+/Mg2+. Thereafter, 200 μL of the MTT working solution will be added to each treatment well and the cells will be incubated for 3 h ± 30 min under standard cell culture conditions. After rinsing the MTT working solution, the cells of each well will be treated with 100 μL MTT lysis agent (isopropanol with 0.04 N HCl) for at least 30 min, while gently shaking. Thereafter, the microplate will be transferred to a microplate reader equipped with a 570 nm filter to measure the absorbance (reference wavelength 690 nm).

Acceptability of the MTT assay
The MTT assay is considered to be acceptable if it meets the following criteria:
• mean absorbance of the solvent control is ≥0.2

Calculation of the test doses for the main experiments
Six test item concentrations will be tested in the main experiments. The highest concentration used will be CV75 × 1.2. Five further test item dilutions will be prepared by serial dilution with a dilution factor of 1.2. If no CV75 can be determined, the maximum guideline recommended concentration of 2000 μM will be used as highest test item concentration and five further test item dilutions will be prepared by serial dilution with a dilution factor of 1.2.
Alternative concentrations may be used upon justification (e.g. in case of too low or too high cytotoxicity or poor solubility.

Main experiments
The test item will be tested in at least two independent main experiments. The main experiments will be performed with independent cell cultures (cells are collected from different culture flasks). The test item will be prepared separately for each run. For each main experiment one 96-well microtiter plate will be prepared for MTT assay and one for the luciferase activity measurement.

Treatment of the cells
For the treatment of the cells in the main experiments, a stock solution of the test item and the controls will be prepared.
The solvent control DMSO (24 replicates), the positive control (five replicates) and the negative control (six replicates), as well as the test item concentrations (each three replicates) will be subsequently diluted 1:25 in treatment medium. The treatment medium control (twelve replicates) will be used undiluted.
After incubation of the LuSens cells, seeding medium will be removed and 150 μL of treatment medium will be distributed in each well. Thereafter, 50 μL of the test item and control dilutions and the medium control will be added into the corresponding wells. At the end of the incubation period of 48 ± 1 h under standard cell culture conditions, the cell cultures will be microscopically evaluated for morphological alterations or precipitation.

Measurement of the cell viability (MTT assay)
The treatment of the cells with the MTT labelling mixture and the measurement of the cell viability for each main experiment will be performed as described as above.

Measurement of the luciferase activity
The Steady-Glo® mix will be prepared by adding Steady-Glo® buffer to one bottle of Steady-Glo® substrate and mixing by inversion until the substrate will be dissolved. The Steady-Glo® working solution will be prepared by mixing one part of DPBS (without Ca2+/Mg2+) with one part of Steady-Glo® mix.
At the end of the incubation period (48 ± 1 h) the Treatment medium will be removed from the wells and the cells will be washed at least twice with 200 μL DPBS including Ca2+/Mg2+. Thereafter, 200 μL of the Steady-Glo® working solution will be added in each well. After slowly shaking of the microtiter plate for at least 10 min in the dark, the plate will be transferred to a microplate reader and the luminescence will be measured for 2 sec per well.

Statistics
If the luciferase induction of the test item is ≥1.5-fold in at least two consecutive non-cytotoxic tested concentrations, significance levels will be determined. In case of normally distributed data, a T-test will be performed for the statistical evaluation. For the statistical evaluation of non-normally distributed data, a Wilcoxon rank sum test will be used. The statistical evaluation will be performed with a validated test script of “R”, a language and environment for statistical computing and graphics. Statistical significance will be set at the five per cent level (p <0.05).

Acceptance criteria
The following acceptance criteria should be met in the LuSens test method (OECD 442D):
• The average luciferase activity induction obtained with the positive control, 120 μM EGDMA should be ≥2.5, and the positive control should have a relative cell viability ≥70% as compared to the solvent control.
The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells should be <1.5-fold as compared to the average solvent control.
• The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) should be below 20% in each main experiment.
• At least three test item concentrations should have cell viability of at least 70% relative to the solvent controls. Moreover, in case a result is to be considered negative, at least one concentration should be cytotoxic, i.e. have a cell viability <70%, or the maximum concentration of 2000 μM (or 2000 μg/ mL for substances with no defined MW) should have been tested.
If a test item is tested with a concentration <2000 μM (or 2000 μg/ mL for substances with no defined MW) and no luciferase induction as well as no cytotoxicity are observed, a second main experiment should be performed using e.g. a 1.44 serial dilution factor based on the CV75 (i.e. starting with 1.44 x CV75) instead of the 1.2 serial dilution factor. If in the second main experiment cytotoxicity and luciferase induction are still not observed, a third main experiment should be conducted with the maximum concentration of 2000 μM (or 2000 μg/ mL for substances with undefined MW). This main experiment should then be confirmed by performing a fourth main experiment (OECD 442D).

Interpretation of the results and prediction model
At least two independent main experiments will be performed.
If the luciferase induction is ≥1.5-fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥70%) and at least three tested concentrations are non-cytotoxic, the main experiment of the LuSens prediction is considered positive.
If these conditions are met in 2 of 2 or in 2 of 3 main experiments, the LuSens prediction is considered positive, otherwise the LuSens prediction is considered negative.
A negative result obtained with a test item that does not form a stable dispersion and was not tested up to 2000 μM (or 2000 μg/mL for test items with no defined MW) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive (OECD 442D).
If no clear dose-response curve or a biphasic dose-response curve is observed, the experiment should be repeated to verify whether this is specific to the test item or due to an experimental artefact. If the biphasic response (i.e. when the threshold of 1.5 is crossed twice) is reproducible in an independent verification experiment, the lower concentration of a ≥ 1.5 induction should be reported (i.e. when the threshold of 1.5 is crossed the first time).
Mono constituent test items with a Log Pow > 7 may be insoluble in the culture medium. However, if the test item is soluble or can be stably dispersed/suspended, the LuSens test may be performed.
Accordingly, to the OECD Guideline 497, the “2 out of 3” Defined Approach for in vitro skin sensitization (OECD 442C-E) has been extended by borderline ranges to define areas with lower confidence. In addition to the evaluation criteria cited above the following borderline range applies: luciferase induction between the 1.28-fold and 1.76-fold compared to solvent control. Results in this range are evaluated “borderline”. The final outcome (negative / borderline / positive) is based on the two congruent outcomes.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

EGDMA (120 M) [442D]

Positive control results:

Positive control: EGDMA ongoing
mean (fold induction): 8.03 (1st experiment) / 8.76 (3rd experiment) / 6.76 (5th experiment)

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Remarks:

@ 229 µM: highest concentration tested

Value:

1.85

Cell viability:

74.80%

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

borderline: After treatment with the test item for 48 ± 1 h the luciferase induction was between 1.28-fold and 1.76-fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥70%) and at least 3 tested concentrations are non-cytotoxic. The LuSens prediction is considered borderline in this experiment; however no relevant cytotoxicity was observed.

Key result

Group:

test chemical

Run / experiment:

other: experiment 3

Parameter:

EC 1.5 [442D]

Remarks:

@ 396 µM: highest concentration tested

Value:

1.43

Cell viability:

66.24%

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

In the third and fifth experiment, after treatment with the test item for 48 ± 1 h, the luciferase induction is below the borderline range (≥1.28-fold≤1.76-fold) compared to the solvent control in all concentrations between 159 and 330 μM. Since the acceptance criterion regarding testing at least one cytotoxic concentration is met, the test item is considered negative in the third and fifth main experiment.

Key result

Group:

test chemical

Run / experiment:

other: experiment 5

Parameter:

EC 1.5 [442D]

Value:

0.74

Cell viability:

49.07%

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

In the third and fifth experiment, after treatment with the test item for 48 ± 1 h, the luciferase induction is below the borderline range (≥1.28-fold≤1.76-fold) compared to the solvent control in all concentrations between 159 and 330 μM. Since the acceptance criterion regarding testing at least one cytotoxic concentration is met, the test item is considered negative in the third and fifth main experiment.

Other effects / acceptance of results:

RESULTS OF THE PRE-TEST (for selection of concentrations for the LuSens):
In the dose finding assay, cytotoxic effects were observed following incubation with the test item starting with the concentration of 250 μM up to the highest tested concentration of 2000 μM (threshold of cytotoxicity: <70% cell viability). The CV75 value of the cytotoxicity test was calculated as 190.8 μM.

The following concentrations of the test item were tested in the first main experiment:
92.0, 110, 133, 159, 191, 229 μM

Since no relevant cytotoxicity was present in the first main experiment, the following concentrations of the test item were tested in main experiments 3 and 5:
159, 191, 229, 275, 330, 396 μM

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

other: No keratinocyte activating potential.

Conclusions:

2-(2-aminoethoxy)ethanol did not activate LuSens cells under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP.
No predictions can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

 

LuSens:

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of the skin sensitization Adverse Outcome Pathway (AOP)) of 2-(2-aminoethoxy)ethanol.
In the dose finding assay, cytotoxic effects were observed following incubation with the test item starting with the concentration of 250 μM up to the highest tested concentration of 2000 μM (threshold of cytotoxicity: <70% cell viability). The CV75 value of the cytotoxicity test was calculated as 190.8 μM.
The test item was tested in 5 independent main experiments, of which 2 main experiments yielded in inconclusive results. Only the results of valid main experiments (1, 3 and 5) were reported and the original numbering is kept in the report.
The following concentrations of the test item were tested in the first main experiment:
92.0, 110, 133, 159, 191, 229 μM
After treatment with the test item for 48 ± 1 h the luciferase induction was between 1.28-fold and 1.76-fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥70%) and at least 3 tested concentrations are non-cytotoxic. The LuSens prediction is considered borderline in this experiment; however no relevant cytotoxicity was observed.
Since no relevant cytotoxicity was present in the first main experiment, the following concentrations of the test item were tested in main experiments 3 and 5:
159, 191, 229, 275, 330, 396 μM
In the third and fifth experiment, after treatment with the test item for 48 ± 1 h, the luciferase induction is below the borderline range (≥1.28-fold≤1.76-fold) compared to the solvent control in all concentrations between 159 and 330 μM. Since the acceptance criterion regarding testing at least one cytotoxic concentration is met, the test item is considered negative in the third and fifth main experiment.
Since these conditions are met in 2 of 3 main experiments, the LuSens prediction is considered negative.
The acceptance criteria were met:
- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 8.03; ME 3: 8.76; ME 5: 6.76) and statistically significant.
- The positive control had a relative cell viability ≥70% as compared to the solvent control (ME 1: 103.13%; ME 3: 96.27%; ME 5: 94.10%).

The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 0.93; ME 3: 1.02; ME 5: 1.21), as well as the basal expression of untreated cells was <1.5-fold as compared to the average solvent control (ME 1: 1.31; ME 3: 1.38; ME 5: 1.17).
- The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (ME 1: 8.7%; ME 3: 10.1%; ME 5: 11.7%).
- At least three test item concentrations had a cell viability of at least 70% relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability <70%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD442E "In vitro skin sensitisation assays addressing the key event on activation of dendritic cells on the adverse outcome pathway for skin sensitisation"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:
000STD77L0
- Purity: 99.228% (analysis performed 27 Sep 2021)
- Physical state / color: liquid / colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: Because the test-substance preparation was incubated with the peptide shortly after preparation, no analysis of the test substance in the vehicle was performed.
- Solubility and stability of the test substance in the solvent/vehicle: The stability under storage conditions over the exposure period was guaranteed by the sponsor

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
On the day of the experiment, 2-(2-aminoethoxy)ethanol will be dissolved in culture medium to prepare a stock solution.
For the cytotoxicity tests (dose finding assay) eight concentrations of the test item will be analysed. Therefore, dilutions will be prepared by 1:2 serial or individual dilutions. For the preparation of the test item, sonication of the formulation for 5 min and/or heating up to 37 °C could be performed.
The highest test item concentration for the first dose finding assay will be 5000 μg/mL in accordance to the OECD guideline 442E.
Cytotoxicity tests (dose finding assay) are performed to determine the CV75, being the test chemical concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The mean of at least two CV75 values or the highest soluble test item concentration will be used to determine the dose-range for the main experiment.

OTHER SPECIFICS:
- Homogeneity: The test substance was homogeneous by visual inspection.
- Molecular weight: 105.14 g/mol
- Log KOW: -1.89 (calculated)

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

CONTROLS FOR CYTOTOXICITY TEST AND h-CLAT
If possible, concurrent controls will be used for several ICCR-Roßdorf GmbH studies performed simultaneously.
Medium control
Name: Culture medium

Solvent control for the test item
Name: Culture medium

Positive control (h-CLAT)
Name: DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7, Purity ≥99%) final concentration: 3 and 4 μg/mL
Solvent: DMSO (final concentration 0.2%)

Solvent control for the positive control (h-CLAT)
Name: DMSO (dimethyl sulfoxide, CAS No. 67-68-5, Purity ≥99%) final concentration 0.2% in culture medium.

TEST SYSTEM AND SUPPORTING INFORMATION
Reasons for the choice of THP-1 cells
THP-1 cells (human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.

THP-1 cell cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH (aliquots of cells in freezing medium at 1-2 × 106 cells/mL) allowing the repeated use of the same working cell stock in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures will be propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured at least twice a week. The cell density should not exceed 1 × 106 cells/mL. The THP-1 cell suspension will be incubated at 37 ± 1.5 °C and 5.0 ± 0.5% CO2 atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).

Culture medium
RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1 mM sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells and during the assay. Medium with supplements has to be stored at 2 – 8 °C and used within one month.

Preparation and seeding of THP-1 cells
Directly before the treatment of the cells for the cytotoxicity or main experiments (h-CLAT), a volume of 500 μL with a cell density of 1.8-2.0 × 106 THP-1 cells/mL will be seeded in each corresponding well of a 24-well flat bottom plate.

Experimental design and procedures of the cytotoxicity test
Dose finding assay (flow cytometer)
The doses investigated in the main experiment (h-CLAT) will be selected with at least two cytotoxicity tests. The tests will be performed with independent cell cultures (cells are collected from different culture flasks) or on different days. If more than one run of the test item will be performed on the same day, the working solutions of the test item will be prepared separately for each run and the cells will be used from different cell cultures.

Treatment of the cells
The test item dilutions will be prepared freshly before each experiment.
Each volume (500 μL) of the dilutions of the test item and culture medium will be added to the cells. The treated THP-1 cells will be incubated for 24 ± 0.5 h. All dose groups will be tested in at least one replicate for each cytotoxicity test. At the end of the incubation period, the cell cultures will be microscopically evaluated for morphological alterations. Each concentration of the test item and culture medium will be prepared for the 7-AAD staining.

Staining of the cells
Each well of test item-treated and untreated cells will be collected in sample tubes centrifuged, washed twice (2-8 °C) with 2 mL FACS buffer (PBS with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 min before the flow cytometry acquisition, 5 μL of a 7-AAD solution will be added in each sample tube.

Flow cytometry acquisition (cytotoxicity test)
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH, software FACSComp 6.0), the device will be calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The cytotoxicity will be analysed by flow cytometry using the software Cellquest Pro 6.0. The 7-AAD acquisition channel (FL-3) should be set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
Preparation of the acquisition (cytotoxicity test)
The following acquisition plots will be prepared:
• 2D plot consisting of FSC (forward scatter) versus SSC (side scatter)
• Histogram plot of the FL-3 channel
The voltage of FSC and SSC will be set to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot should be checked to make sure that a single population appears without contamination or excessive debris. The FL-3 voltage will be set and compensate to appropriate position (FACSCalibur, Becton Dickinson GmbH).
The cell viability will be measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells will be analysed. If the cell viability will be low, then up to 30,000 cells including dead cells should be acquired. Alternatively, the acquisition can be finished 1 minute after the acquisition start.
The maintenance of the flow cytometer will be in accordance with the manufacturer’s instructions. The process of washing should be conducted very carefully since insoluble chemicals could flow in the flow line.

Acceptability of the cytotoxicity assay
The cytotoxicity test is considered to be acceptable if it meets the following criteria:
• The cell viability of the medium and solvent control (if the test item is solved in DMSO) should be more than 90%.
6.5.7 Calculation of the test doses for the main experiment (h-CLAT)
The mean of at least two CV75 values or the highest soluble test item concentration will be used to determine the dose-range for the main experiment (h-CLAT).
Eight final concentrations (μg/mL) will be used for the test item in the main experiment (h-CLAT). The highest concentration used will be 1.2 × mean CV75 with a serial or individual dilution factor of 1.2.
If CV75 cannot be determined, a stock solution will be prepared by serial or individual 1:1.2 dilution from 5000 μg/mL in culture medium.

Experimental design and procedures of h-CLAT
The test item will be tested in at least two independent runs. The tests will be performed with independent cell cultures (cells are collected from different culture flasks) or on different days. If more than one run of the test item will be performed on the same day, the working solutions of the test item will be prepared separately for each run and the cells will be used from different cell cultures.

Treatment of the cells
The test item dilutions will be prepared freshly before each experiment.
Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control will be added to the cells. All dose groups will be tested in triplicates. The treated THP-1 cells will be incubated for 24 ± 0.5 h. At the end of the incubation period, the cell cultures will be microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control will be prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the cells
The triplicates of each well of test item treated and untreated cells will be pooled and equally distributed into three sample tubes, collected by centrifugation and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells will be centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2-8 °C (on ice) for approx. 15 min. After blocking, the cells will be centrifuged and the cell pellets will be re-suspended in 100 μL FACS buffer. The cells will be stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions must be kept light protected at 2-8 °C or on ice during the staining and analysis procedure.
The cells with the different antibodies or the IgG1 will be mixed and incubated light protected for 30 ± 5 min at 2-8 °C (on ice).
6.6.3 Sample preparation for measurement
After staining with the antibodies, the cells will be washed twice (2-8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution will be added.

Flow cytometry acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device will be calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) will be analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) should be set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) should be set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
Preparation of the acquisition
The following acquisition plots will be prepared:
• 2D plot consisting of FSC (forward scatter) versus SSC (side scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC will be set to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot should be checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage will be set and compensate to appropriate position. The FL-1 voltage will be set using the FITC labelled-mouse IgG1 medium-treated cells tube, such that the MFI of control cells will be set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACS Calibur, Becton Dickinson GmbH).
The cell viability will be detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD). Therefore, the R1 gate will be set approximately at the middle position between the peak of the negative fraction and the positive fraction in the FL-3 histogram using DNCB-treated cells. The negative fraction corresponds to the living cells and will be kept for the subsequent analyses. For each control and all test item concentrations, the cell viability will be recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the CD54 and CD86 cell tube, where the mean of the three samples is used for the cell viability evaluation.
If the peak in the FL-3 histogram shows a shift to the right side (due to possible interference of the test item) and in case no clear cytotoxicity is observed in the 2D plot consisting of FSC (forward scatter) versus SSC (side scatter), the R1-gate can be shifted to the right side to obtain a reliable result for the cell viability. If this shift is also observed in the dose finding experiment, the R1-gate setting of the dose finding experiment can be used as setting for the h-CLAT run. The evaluation of these data will be discussed in the final report.
The maintenance of the flow cytometer will be in accordance with the manufacturer’s instructions. The process of washing should be conducted very carefully since insoluble chemicals could flow in the flow line.

Acquisition
Dead cells will be gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) will not be done. A total of 10,000 living cells will be analysed. If the cell viability will be low, then up to 30,000 cells including dead cells should be acquired. Alternatively, the acquisition can be finished 1 minute after the acquisition start. Mean fluorescence intensity (MFI) of viable cells and viability for each sample will be used for analysis. The other tubes will be acquired without changing the settings of the cytometer. The MFI will be recorded for each condition. The relative fluorescence intensity (RFI) will be calculated but excluded from the evaluation, if the cell viability is less than 50% (due to diffuse labelling of cytoplasmic structures that are generated due to cell membrane destruction).

Data analysis and interpretation

Calculation of EC150 and EC200
For the test items predicted as positive with the h-CLAT, optionally, two effective concentrations (EC) values, the EC150 for CD86 and EC200 for CD54, i.e. the concentration at which the test chemicals induced a RFI of 150 or 200, may be determined if possible. These EC values potentially could contribute to the assessment of sensitising potency when used in integrated approaches such as IATA (OECD 442E guideline). They can be calculated by the following equations:
EC150 (for CD86) = Bconcentration + [(150- BRFI) / (ARFI - BRFI) x (Aconcentration-Bconcentration)]
EC200 (for CD54) = Bconcentration + [(200- BRFI) / (ARFI - BRFI) x (Aconcentration-Bconcentration)]
where
Aconcentration is the lowest concentration in μg/mL with RFI >150 (CD86) or 200 (CD54)
Bconcentration is the highest concentration in μg/mL with RFI <150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI >150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI <150 (CD86) or 200 (CD54)

Acceptance criteria
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90%.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥150% and CD54 ≥200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be >105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥150% and CD54 ≥200%) and the cell viability should be >50% in at least one concentration of the two tested positive control concentrations.
• For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of <90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximum test concentration of a test chemical, a negative result is acceptable even if the cell viability is >90% (OECD 442E guideline).

Prediction model
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). A 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 (OECD 442E guideline):
− The RFI of CD86 is ≥150% at any tested concentration (with cell viability ≥50%)
− The RFI of CD54 is ≥200% at any tested concentration (with cell viability ≥50%)
Otherwise, the h-CLAT prediction is considered NEGATIVE (see chapter 6.6.8.1).
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. A h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline). Accordingly, to the OECD Guideline 497, the “2 out of 3” Defined Approach for in vitro skin sensitization (OECD 442C-E) has been extended by borderline ranges to define areas with lower confidence. In addition to the evaluation criteria cited above the following borderline range applies: RFI CD54 157% - 255% and RFI CD86 122% - 184%. Results in this range are evaluated “borderline”. The final outcome (negative / borderline / positive) is based on the two congruent outcomes.

Positive control results:

Positive control: DNCB 4 µg/mL:
- RFI CD86 mean [%]: ongoing (1st experiment) / ongoing (2nd experiment)
- RFI CD54 mean [%]: ongoing (1st experiment) / ongoing (2nd experiment)
- Rel. Viability [%]: ongoing (1st experiment) / ongoing (2nd experiment)

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC200, CD54 [442E]

Value:

856.1 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC200, CD54 [442E]

Value:

762.5 µg/mL

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:

RESULTS OF PRELIMINARY CYTOTOXICITY ASSESSMENT: The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be ongoing [μg/mL].

Interpretation of results:

other: ongoing

Conclusions:

2-(2-aminoethoxy)ethanol with a calculated log Pow of -1.89 activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation AOP.
No prediction can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

 

This in vitro human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization Adverse Outcome Pathway (AOP)) of 2-(2-aminoethoxy)ethanol dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 h.
This h-CLAT can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 (luciferase test method)) based on the OECD AOP for the assessment of the skin sensitisation potential of chemicals.
The highest concentration of the test item in both dose finding assays was 5000 μg/mL in accordance to the OECD Guideline 442E.
Cytotoxic effects (threshold of cytotoxicity: <75%) were observed following incubation with the test item starting with the concentration of 2500 μg/mL up to the highest tested concentration (5000 μg/mL) in both cytotoxicity tests. The mean CV75 value of both cytotoxicity tests was calculated as 1446.1 μg/mL.
The following concentrations of the test item were tested in two main experiments (h-CLAT):
484, 581, 697, 837, 1004, 1205, 1446, 1735 μg/mL
The test item with a calculated log Pow of -1.89 was tested in 2 independent runs. The relative fluorescence intensity (RFI) of CD86 and CD54 was greater than 150% and 200%, respectively, in at least one concentration of both runs. In the first main experiment the treatment with the test item concentrations 581 μg/mL (151.7%) and 837 μg/mL (179.2%) resulted in borderline values in CD86 and the concentrations of 697 μg/mL (186.6%) and 837 μg/mL (183.6%) resulted in borderline values for CD54. In the second main experiment, borderline values for CD86 were observed at 697 μg/mL (132.8%), 837 μg/mL (151.5%), 1004 μg/mL (166.0%) and 1205 μg/mL (175.9%), and for CD54 at 1004 μg/mL (238.8%). However, because at least one test item concentration of both main experiments resulted in CD86 and CD54 RFI above the borderline range, the h-CLAT prediction is considered positive for the test item in this h-CLAT.
All acceptance criteria were met. The cell viability of the medium and DMSO control was >90%. For both medium and solvent control, the MFI ratio of CD86 and CD54 to isotype control was >105%. In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥200% and CD86 ≥150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥200% and CD86 ≥150%) and the cell viability was >50%. For the test item, the cell viability was more than 50% in at least four tested concentrations in each run.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

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))

Deviations:

no

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
- batch No.of test material:
000STD77L0
- Purity: 99.228% (analysis performed 27 Sep 2021)
- Physical state / color: liquid / colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: Because the test-substance preparation was incubated with the peptide shortly after preparation, no analysis of the test substance in the vehicle was performed.
- Solubility and stability of the test substance in the solvent/vehicle: The stability under storage conditions over the exposure period was guaranteed by the sponsor

TEST SUBSTANCE SOLUBILITY
Prior to the assay the solubility of the test substance at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test-substance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as a 100 mM preparation in acetonitrile considering a molecular weight of 105.14 g/mol and a purity/contents of 99.228%. After short stirring the test substance was soluble in the vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material): 100 mM preparation in acetonitrile

OTHER SPECIFICS:
- Homogeneity: The test substance was homogeneous by visual inspection.
- Molecular weight: 105.14 g/mol
- Log KOW: -1.89 (calculated)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

EXPERIMENTAL PROCEDURE
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) and positive control (=PC) 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.

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.

CONTROLS
-Negative control (NC): vehicle control = deionized water (triplicates)
-Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as 50 mM emulsion in deionized water
-Co-elution control (SK): Sample prepared of the respective peptide buffer and the test substance but without peptide.

SELECTION OF CONCENTRATIONS
The test substance was prepared as a 100 mM preparation in acetonitrile considering a molecular weight of 105.14 g/mol and a purity/contents of 99.228%. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the Kcontaining peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).

PREPARATION OF PEPTIDE STOCK SOLUTIONS
Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide).

PREPARATION OF CALIBRATION SAMPLES
The calibration samples were prepared from the peptide stock solutions in 20% acetonitrile in the respective buffer (= dilution buffer). The analysis of the calibration samples was started before analysis of the test-substance samples.

TEST SUBSTANCE PREPARATION
The test-substance preparations were prepared on a weight per volume basis within 4 hours of preparation of test-substance samples.
The test substance was prepared as a 100 mM preparation in acetonitrile considering a molecular weight of 105.14 g/mol and a purity/contents of 99.228%. After short stirring the test substance was soluble in the vehicle.
Vehicle: acetonitrile
Reason for the vehicle: The test substance was soluble in acetonitrile.

PREPARATION OF THE TEST-SUBSTANCE SAMPLES
The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in the dark for 24 ± 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

PREPARATION OF THE VEHICLE CONTROLS
Several vehicle controls were prepared in triplicates in the same way as the test-substance samples described above but with the vehicle (acetonitrile) instead of the test substance:
One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

PREPARATION OF THE CO-ELUTION CONTROL
One sample per peptide was prepared in the same way as the test-substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

DATA EVALUATION
The integrated peak areas were transferred electronically into EXCEL data spreadsheets to carry out the necessary calculations. Some test substances or reaction products may co-eluate with the peptides. In these cases where proper integration and calculation of peptide depletion was not possible, the result for the respective peptide is reported as interference. For evaluation of peptide depletions peak areas at 220 nm are used. When samples were additionally analyzed by measuring UV absorbance at 258 nm, the area ratio 220 nm/ 258 nm may be calculated and serve as a measure of peak purity. The ratio of a pure peptide peak should be consistent over all samples (100% ± 10% of the mean of the vehicle controls). However, due to small peak areas calculation of the area ratio may not be possible for all samples.

STATISTICAL ANALYSES
For the statistical evaluation of luciferase fold induction the EXCEL-function T.TEST is used.

ACCEPTANCE CRITERIA
- 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.

Positive control results:

Positive control (EGDMA):
Mean depletion (Cysteine-Peptide): 53.38% (+/- 1.07)
Mean depletion (Lysine-Peptide): 6.99% (+/- 0.11)
Mean of both depletions: 30.18%

Key result

Group:

test chemical

Parameter:

other: Mean depletion (Cysteine-Peptide) [%]

Value:

4.58

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Parameter:

other: Mean depletion (Lysine-Peptide) [%]

Value:

-0.25

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Parameter:

other: Mean of both depletions [%]

Value:

2.29

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 6: Mean peptide depletions of Cysteine, Lysine and both peptides.

	Cysteine-Peptide   mean depletion [%]      SD  [%]		Lysine-Peptide   mean depletion [%]        SD [%]		mean of both depletions [%]
test substance	4.58	0.47	-0.25	0.11	2.29
PC:EGDMA	53.38	1.07	6.99	0.11	30.18

 

Table 7: Peptide depletion of NC, PC and the test substance for cysteine-peptide.

Reaction with cysteine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean           SD
NC: H2O	-0.15	0.66	-0.52	0.00           0.60
test substance	4.57	4.12	5.06	4.58           0.47
PC:EGDMA	52.21	53.62	54.31	53.38         1.07

 

Table 8: Peptide depletion of NC, PC and the test substance for lysine-peptide.

Reaction with lysine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean SD
NC: H2O	0.05	-0.09	0.04	0.00 0.08
test substance	-0.13	-0.27	-0.35	-0.25 0.11
PC:EGDMA	7.09	6.88	6.98	6.99 0.11

 

Table 9: Historic control data of negative control / vehicle control (not including present study). De-ionized water (Historic period: Jan 2019 - Jan 2021)

	C-peptide concentration [mM]	K-peptide concentration [mM]
Min	0.432	0.457
Max	0.564	0.545
Mean	0.501	0.508
SD	0.027	0.016
n	20	23

Table 10: Historic control data of positive control (not including present study). EGDMA, 50 mM in de-ionized water (Historic period: Jan 2019 - Jan 2021)   C-peptide concentration [mM]   C-peptide depletion [%] K-peptide concentration [mM]   K-peptide depletion [%] Min 0.138 48.13 0.420 5.51 Max 0.265 69.49 0.513 14.56 Mean 0.231 54.11 0.452 11.39 SD 0.025 4.04 0.019 1.98 n 18 18 19 19

Interpretation of results:

other: Non peptide binding/peptide binding

Conclusions:

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model, it was concluded that 2-(2-aminoethoxy)ethanol shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.
However, no predictions can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

 

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 deionized water. 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 coelution 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 following results were obtained in the DPRA:

The test substance was dissolved in deionized water at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present.

The mean C-peptide depletion, caused by the test substance was determined to be 4.58%.

The mean K-peptide depletion, caused by the test substance was determined to be -0.25%.

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 2.29%.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Jan - March 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Purity not given. Study was performed according to OECD 406 guideline and in compliance with the GLP Regulations. It is not clear whether 8 or 10 animals were used in the dose-range finding study. It is not clear whether challenge was performed 13 or 14 days after the last induction period. However, it is unlikely that this will have an impact on the outcome of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Test has been performed 1991, before the regulation has been revised with respect to deviation from the LLNA as "gold standard"

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Buckberg Lab Animals, Tomkins Cove, New York
- Weight at study initiation: 300-700g
- Housing: individually in stainless steel wire mesh cages
- Diet (e.g. ad libitum): purina guinea pig diet, ad libitum
- Water (e.g. ad libitum): fresh tap water, ad libitum
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: Jan 1991 To: March 1991

Route:

epicutaneous, semiocclusive

Vehicle:

other: ethanol

Concentration / amount:

10 %

Day(s)/duration:

0

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, semiocclusive

Vehicle:

other: ethanol

Concentration / amount:

10 %

Day(s)/duration:

7

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

other: no data

Vehicle:

other: ethanol

Concentration / amount:

10 %

Day(s)/duration:

14

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

other: ethanol

Concentration / amount:

10 %

Day(s)/duration:

29

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

epicutaneous, semiocclusive

Vehicle:

other: ethanol

Concentration / amount:

10 %

Day(s)/duration:

36

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

dose range: 8 or 10 (unclear)
test article: 20 (10 male, 10 female)
positive control: 5
negative control: 10

Details on study design:

RANGE FINDING TESTS:
10 unexposed animals (5male/5female) are exposed to 4 different concentrations of the test material: 80% ethanol as the vehicle.
primary challenge responses were graded
Highest non-irritatting concentration = concentration that induced responses not exceeding 2 + and 2 0 grades in the group of 4 animals.
the dose chosen for induction, challenge and rechallenge : 10%


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 5 (3 inductions, 1challenge, 1 rechallenge)
- Exposure period: -
- Test groups: test substance in vehicle (80% ethanol)
- Control group: vehicle only (80% ethanol)
- Site: L shoulder
- Frequency of applications: once a week
- Duration: 6 h
- Concentrations: 10%


B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: day 28 (or 29) and 35 (or 36) (unclear)
- Exposure period: -
- Test groups: test substance in vehicle
- Control group: vehicle only (left flank), test article (right flank)
- Site: naive site on left side
- Concentrations: 10%
- Evaluation (hr after challenge): 24 and 48h


OTHER: 24h after challenge, all animals were depilated with Neet Cream Hair Remover (Whitehall Laboratories, Inc., New York). A minimum of 2h after depilation test sites were graded. The grading was repeated 24h later (48h grade).

Positive control substance(s):

yes

Remarks:

1-chloro-2,4-dinitrobenzene (DNCB)

Positive control results:

Sensitising effects are observed in all 5 animals of the positive control group.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10 %

No. with + reactions:

1

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Remarks:

Severity: 0.3

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10 %

No. with + reactions:

1

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Remarks:

Severity: 0.2

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

10 %

No. with + reactions:

2

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Remarks:

Severity: 0.3

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

10 %

No. with + reactions:

2

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Remarks:

Severity: 0.2

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.3 % DNCB

No. with + reactions:

5

Total no. in group:

5

Remarks on result:

positive indication of skin sensitisation

Remarks:

Severity: 3.0

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.3 % DNCB

No. with + reactions:

5

Total no. in group:

5

Remarks on result:

positive indication of skin sensitisation

Remarks:

Severity: 2.6

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100 % acetone

No. with + reactions:

0

Total no. in group:

0

Remarks on result:

no indication of skin sensitisation

Remarks:

Severity: 0.0

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100 % acetone

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Remarks:

Severity: 0.0

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

challenged with 10% test substance

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Remarks:

Severity: 0.2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

challenged with 10% test substance

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Remarks:

Severity: 0.2

Primary challenges were carried out after 14 days, 1 positive response was observed after 24 and 48 hours in the 10% DGA group. 7 days after the primary challenge, all test article treated animals were rechallenged at 10% concentration. 2 positive responses were observed after 24 and 48 hours after this rechallenge. After initial challenge a positive response was observed in all animals receiving the DNCB positive control. Erythema observed at 24 and 48 hours in 3 of negative control group at initial challenge.

Under EU criteria, the product is not classed as a sensitizer as it indicates a maximum (at rechallenge) of 10% positive (2/20) responses. A minimum figure of 15% under any study would be necessary for classification as a sensitizer with R43 under EU 18th ATP of the DSD. Practical experience over 20 years of manufacturing this product adds weight to this conclusion.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined to be not skin sensitizing in a Buehler test.

Executive summary:

In two preliminary dose-range-finding studies, ten animals (five males and five females) were each exposed to four different concentrations of the test substance in either 80% ethanol or acetone, to determine the highest non-irritating dose.

Based upon the results of the dose-range-finding studies, the test article was dosed at a 10% concentration. In order to determine if the test article is capable of causing delayed contact hypersensitivity, the test substance, was dermally applied to twenty guinea pigs (ten males and ten females) for a total of three six hour insult periods. Another group of five guinea pigs (three males and two females) was treated with 1-chloro-2,4-dinitrobenzene at a 0.3% concentration for a total of three six-hour insult periods. An additional group of ten guinea pigs (five males and five females) was treated with vehicle (80% ethanol) for a total of three six-hour insult periods. Fourteen days after the last induction period, all animals were challenged at a naive site.

A positive response was elicited in the animals receiving the positive control article, 1-chloro- 2, 4-dinitrobenzene (DNCB). Slight patchy erythema was observed at 24 and 48 hours in three of the negative control animals challenged with the test article at a 10% concentration. One positive response was observed at 24 and 48 hours after challenge in the test article-treated group. No responses were observed in the negative control animals challenged with the vehicle (acetone).

Seven days following the primary challenge, all test article treated animals were rechallenged at a naive site at a 10% concentration. Two positive responses were observed at 24 and 48 hours after rechallenge.

Based upon the observations made in the Delayed Contact Hypersensitivity Study in Guinea Pigs, the test substance induced, challenged and rechallenged at a 10% concentration did not cause delayed contact hypersensitivity in guinea pigs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

in vitro skin sensitization test battery

The objective was to assess the skin sensitization potential of 2-(2-aminoethoxy)ethanol (SoR, OECD 497, 67V0392//09B032; not part of RSS, BASF 2022). Due to the complexity of the skin sensitization process a single in vitro assay is not sufficient to adequately assess this toxicological endpoint. Therefore, a combination of several methods addressing key events of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD (OECD 2012) were used (test battery, OECD 497):
• OECD TG 442C: protein reactivity (DPRA),
• OECD TG 442D: activation of keratinocytes (LuSens), and
• OECD TG 442E: activation of dendritic cells (h-CLA T).

Therefore, the results of these tests were submitted in this technical dossier as weight of evidence.

The assessment in this study applies the Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification, which was included as case study 1 in the OECD guidance document 256: Any two of the three tests determine the overall results, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauch et al. 2012, OECD 497).

Testing 2-(2-aminoethoxy)ethanol in three different non-animal methods addressing different key events of the skin sensitization Adverse Outcome Pathway resulted in one positive result in the h-CLAT and two negative results in DPRA and LuSens. The three tests generally used in the "2 out of 3" DEFINED APPROACH assess protein binding in chemico (DPRA), triggering an antioxidant response in keratinocytes (LuSens) and activation of dendritic cells (h-CLAT). Of note, the molecular initiating event of protein binding also occurs in the cell-based assays (Lu Sens, h-CLAT) and is not solely detected via the DPRA.

The borderline range depicts the variance of the test method, including technical and biological variability. lt addresses the uncertainty around their respective classification thresholds and represents a range in which the likelihood to obtain a positive or negative result just below or above the classification threshold is equal. This range was determined statistically based on "log pooled median absolute deviations" using the data of the respective validation study of each assay (Kalle et al., 2021 ). In the present study the first main experiment of the LuSens met the "borderline"-criteria. However, main experiments two and three were unambiguously  negative. Further, the results of the DPRA and h-CLAT were unambiguous. Hence, in the present study, none of the performed single assay met the  "borderline" criteria and the results are therefore considered to be unambiguous.

The test substance activated dendritic cells in the h-CLAT, but neither indicated protein binding in the DPRA nor activation of keratinocytes in the LuSens assay. The activation of the dendritic cells in the h-CLAT may be unrelated to the skin sensitization process (Gallucci et al., 1999). There is no indication that the predictions of the DPRA and the LuSens assays might be false negative ones. Further, the lack of peptide reactivity is consistent with the lack of structural alerts. With antigen formation and keratinocyte activation missing, the test substance is not expected to drive the AOP towards a skin sensitization.

Test Method	Test Result	Test Evaluation
Direct Peptide Reactivity Assay (DPRA)	2.29% mean peptide depletion (4.58% cysteine-peptide depletion; -0.25% lysine-peptide depletion) a)	Negative a)
Keratinocyte Activation Assay - LuSens	In two of three independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed.	Negative
Dendritic Cell Line Activation Assay Human Cell Line Activation Test (h-CLAT)	In two independent experiments an induction of the expression of CD86 (above 150%) and CD54 (above 200%) were observed at sufficiently noncytotoxic (cell viability 2: 50%) concentration. The EC200 for CD54 was calculated to be 856.1 μg/ml and 762.5 μg/ml; the EC150 for CD86 was calculated to be 575.1 µg/ml and 825.8 µg/ml.	Positive

a) Negative depletions were considered to be "zero" for calculation of mean peptide depletion.

Based on the results summarized above, 2-(2-aminoethoxy)ethanol is not peptide reactive, does not activate keratinocytes and activates dendritic cells. In conclusion, 2-(2-aminoethoxy)ethanol is predicted not to be a skin sensitizer.

 

 

Buehler Test

The test substance was evaluated at 10% concentration for dermal sensitization in 20 guinea pigs (10 f, 10 m) for a 3 x 6 hour induction period. Animals were challenged after 14 days and one positive response was observed after 24 and 48 hours relative to 100% response (5/5) in the positive control group. In a rechallenge after 7 days 2/20 positive responses were observed after 24 and 48 hours, equivalent to a reponse rate of 10 %. A minimum response rate of 15% would be necessary for classification as a sensitizer. Therefore, the test substance, was determined to be no skin szensitizer. Practical experience over 20 years of manufacturing this product adds weight to this conclusion.

 

 

Skin sensitising effects in humans (MAK, 2014)

See also IUCLID chapter 7.10 – Sensitization data (humans).

A 39-year-old metalworker developed workplace-related dermatitis of the hands, which initially healed after a week of work-free time, but recurred after resuming work and finally extended to the arms within three months. After another four-week break from work, the eczema was almost healed, but recurred again within two weeks of continuing the activity. The patient developed an infiltrated vesicular erythema in the epicutaneous test after three days after application of a 1% preparation of the test substance in petrolatum. The cooling lubricant used at his workplace contained 10% of the test substance, but was not available for an epicutaneous test or an application test (Geier et al. 2002).

About twelve months after the introduction of a new cooling lubricant, a 26-year-old metal worker developed vesicular eczema on his hands and forearms. The epicutaneous test showed concentration-dependent pronounced reactions to the 0.6%, 3% and 6% tested cooling lubricant, which also contained 10% 2-(2-aminoethoxy)ethanol. Moreover, a positive result was obtained after three days with a 6% preparation in the "Repeated Open Application Test" (ROAT). An 1% pereparation of 2-(2-aminoethoxy)ethanol in petrolatum led to a clear papular reaction in the patient in the epicutaneous test. No reaction to this preparation was observed in 80 control subjects (Frosch et al. 2002).

A worker reacted strongly positive to the professionally used cooling lubricant, which had been tested both fresh and used. According to the safety data sheet, the cooling lubricant contained a formaldehyde separator, (ethylene dioxy)dimethanol, CAS 3586-55-8, which was not tested separately. When testing the standardized test allergens, the patient showed positive reactions to formaldehyde and the formaldehyde separator methylene bis(methyloxazolidine), as well as to 2-aminoethanol and 2-(2-aminoethoxy)ethanol. Whether the professionally used cooling lubricant contained 2-aminoethanol or 2-(2-aminoethoxy)ethanol remained unclear, as only "alkanolamines" were listed as ingredients in the safety data sheet (Geier et al. 2011).

Between April 2000 and July 2002, 2-(2-aminoethoxy)ethanol was tested as a 1% preparation in petrolatum in a total of 228 employees of the metal industry in the epicutaneous test as part of a study in five centers of the IVDK (Information Network of Dermatological Clinics). Four of the 228 employees tested (1.8%) showed a onefold positive reaction on the third day, and the fourth positively tested person (0.4%) showed a twofold positive reaction (Geier et al. 2002). For three tested individuals (1.3%) a questionable reaction was observed (Geier et al. 2003 a). In 2003, 108 patients were epicutaneously tested with 1% of 2-(2 aminoethoxy)ethanol in petrolatum as a new component of the DKG (German Contact Allergy Group) test series "Cooling lubricants currently" in the clinics of the IVDK. Two of them (1.9%) showed one positive and six tested (5.5%) showed a questionable reaction (Geier et al. 2004).

From June 2004 to June 2005, 144 metalworkers were tested with an extended series of cooling lubricants as part of a study in seven clinics of the IVDK and the occupational dermatology department of Lund University in Malmö. In 137 of them, 2-(2-aminoethoxy)ethanol was also tested, in five cases (3.7%) with a positive result (Geier et al. 2006).

From July 2003 to August 2010, a total of 2972 patients were tested in the IVDK clinics with 2-(2 aminoethoxy)ethanol as part of the DKG test series "Kühlschmierstoffe aktuell". In addition to 35 onefold and ten double positive reactions (a total of 1.5% positive reactions), there were 44 questionable reactions, three follicular reactions and eight classified as irritative. With a positivity ratio (PR)¹⁾ of 78% and a reaction index (RI)²⁾ of -0.1, 2-(2-aminoethoxy)ethanol proves to be a rather problematic test substance. Between September 2010 and June 2012, 2-(2 aminoethoxy)ethanol was tested in 864 patients as part of the DKG test series "Cooling Lubricants". Twelve onefold positive reactions occurred (1.4%), ten reactions rated as questionable and three as irritative. In the two periods, 197 out of 2995 tested (6.6%; 156 single, 36 double and 5 triple positive reactions; 135 questionable, 12 follicular and 37 irritant reactions; PR: 79%; RI: 0.0) and in 41 out of 876 tested (4.7%; 38 simple, 3 double positive reactions; PR: 93%; RI: 0.2) showed a positive reaction (IVDK 2013).

Conclusion: There have been reports of positive, clinically relevant epicutaneous test responses to 2-(2-aminoethoxy)ethanol. The negative result of an animal study without the use of adjuvant indicates a low sensitization potential. However, the irritant effect of the substance appears to be an important cofactor which, together with other factors damaging the skin barrier, especially in metal working with often large-scale and regular contact with cooling lubricants, can lead to sensitization, as the positive epicutaneous tests in these occupational groups show. Whether some of the reactions may be due to cross-reactions after sensitization by 2-aminoethanol or other ethanolamines cannot be assessed on the basis of the available data. Although the number of observed cases is relatively small so far, 2-(2-aminoethoxy)ethanol is marked with "Sh" due to the risk of sensitization due to the use of the substance in the metalworking industry, a marking with "Sa" is still not carried out.

 

¹⁾ The positivity ratio is defined as the percentage of simple positive reactions in the totality of positive reactions (Geier et al. 2003 b).

²⁾ The reaction index is defined as the quotient: (a – d – i) / (a + d + i); with: a = number of allergic reactions, d = number of questionable reactions, i = number of irritative reactions (Brasch and Henseler 1992).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data on 2-(2-aminoethoxy)ethanol are reliable and suitable for classification purposes under Regulation 1272/2008. In a Buehler Test (OECD 406), 10% of all animals showed mild local reactions after an epicutaneous induction with a 10% preparation of the test substance. This data support that the substance does not require classification as a skin sensitizer. However, based on available data on humans (see also IUCLID chapter 7.10 – Sensitization data (humans)) and in accordance with the recommendations of the recent MAK opinion, the test substance was re-evaluated. A modern in vitro skin sensitization test battery was conducted and gave no indication of a skin sensitizing potential. Based on these results and in combination with the negative results of the Buehler Test, the substance does not need to be classified for skin sensitisation under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.