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EC number: 213-195-4
CAS number: 929-06-6
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.
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 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 μMAfter 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 μMIn 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%.
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/mLThe 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.
Table 6: Mean peptide depletions of Cysteine, Lysine and both peptides.
[%] SD [%]
[%] SD [%]
mean of both depletions [%]
Table 7: Peptide depletion of NC, PC and the test substance for cysteine-peptide.
Reaction with cysteine- peptide
peptide depletion [%]
Table 8: Peptide depletion of NC, PC and the test substance for lysine-peptide.
Reaction with lysine- peptide
Table 9: Historic control data of negative control / vehicle control (not including present study). De-ionized water (Historic period: Jan 2019 - Jan 2021)
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 depletion [%]
K-peptide depletion [%]
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.
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.
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
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.
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.
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.
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.
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).
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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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