2-piperazin-1-ylethanol

Endpoint summary

Description of key information

Based on the results of the DPRA and LuSENS assays, 2-Piperazin-1-ylethanol is not peptide reactive and does not activate keratinocytes in vitro. Applying the evaluation criteria 2-Piperazin-1 -ylethanol is predicted not to be a skin sensitizer based on the in vitro testing strategy.

Further, and in addition to this, Leung et al., 1997, performed a Guinea Pig Maximization Test according to Magnusson with 2 -Piperazin-1 -ylethanol. Based on the results of this study and applying the current CLP criteria, 2 -Piperazin-1 -ylethanol does not meet criteria for classification, and is therefore not classified as a skin sensitizer in vivo.

Note, a guinea pig maximization test of Union Carbide Corp. is available as well. But because a mixture of amines and piperazine derivatives containing only 0.5 to 5% of the test item 2 -Piperazin-1 -ylethanol was tested, these results are not used for evaluation and classification of 2 -Piperazin-1 -ylethanol.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

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:

other: Direct Protein Reactivity Assay

Specific details on test material used for the study:

Batch B1057

Details on the study design:

In the DPRA the reactivity of a test item (=test substance) towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated. For this purpose, the test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm.
The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.

Synthetic peptides: Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and JPT Peptide Technologies GmbH, Berlin, Germany) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

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

EXPERIMENTAL PROCEDURE
The test substance was dissolved in a suitable vehicle, here acetonitrile. Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was
determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

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-susbtance preparation) should be used. The preferred solvent was acetonitrile.

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). The peptide stock solution was used for preparing the calibration samples and the test-substance and control samples.

Preparation of calibration samples
The following calibration samples were prepared from the peptide stock solutions in 20% acetonitrile in the respective buffer (= dilution buffer) using serial dilution:
Calib. 1 0.534 mM peptide
Calib. 2 0.267 mM peptide
Calib. 3 0.134 mM peptide
Calib. 4 0.067 mM peptide
Calib. 5 0.033 mM peptide
Calib. 6 0.017 mM peptide
Dilution buffer 0.000 mM peptide

Preparation of the test-substance samples
The samples were prepared in triplicates for each peptide.
The test substance was prepared at a 100 mM concentration (considering a purity/contents of 99.7%) in acetonitrile.
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 K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).

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 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 or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

Positive control results:

62.92 +- 2.55 % cysteine-peptide depletion
10.25 +- 0.59 % lysine-peptide depletion
mean: 36.58 % peptide depletion

Key result

Run / experiment:

other: triplicates for each peptide

Parameter:

other: peptide depletion [%]

Value:

0.65

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results 2-Piperazin-1-ylethanol is not peptide reactive.

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:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Batch B1057

Details on the study design:

Preparation of the cells
LuSens cells (Human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen, Germany) from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) for at least passage ≥5 but not longer than 15 passages prior to testing.
Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 10^5 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. Two independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.

Test-substance application for MTT and luciferase assay
After cell adaption for 24 hours cell culture medium 2 was aspirated and replaced with 150 μL medium 3. Each test substance preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%).
After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability.

Visual inspections
Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates.

Luciferase assay
After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Subsequently 200 μL of One-Glo-preparation (= 100 μL One-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

Cell viability assay MTT
Cell culture medium was aspirated from all wells. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and medium 3) was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

Negative control (NC): DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 μM (= 450 μg/mL) in 1% DMSO in culture medium 3
Positive control (PC): ethylene glycol dimethacrylate (EGDMA, CAS no.: 97-90-5), 90.8 μM (= 18 μg/mL) in 1% DMSO in culture medium 3
Vehicle control (VC): 1% DMSO in culture medium 3
Blank control: Culture medium 3 without cells
Basal control: Culture medium 3 with cells

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

Positive control results:

fold induction 1st experiment: 4.31 +- 0.42
fold induction 2nd experiment: 4.80 +- 0.35

Key result

Run / experiment:

other: 8 concentrations/ experiment; two independent experiments performed

Parameter:

other: fold induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Concentration (test substance) [μM]	1st experiment
	fold induction		rel. viability [%]		t-test
	mean	SD	mean	SD	p-value	markers
560	1.38	0.12	96	7	0.014	*
672	1.43	0.16	84	6	0.020	*
806	1.43	0.20	83	7	0.031	*
967	1.35	0.08	84	3	0.005	**
1161	1.29	0.04	75	16	0.000	**
1393	1.60	0.09	62	1	0.002	**
1672	1.38	0.05	53	9	0.000	**
2006	1.17	0.11	61	10	0.059	n.s.
VC	1.00	0.06	100	10	-	-
EGDMA 90.8 μM	4.31	0.42	75	4	0.000	**
LA 5000 μM	0.87	0.09	97	6	0.016	*

Concentration (test substance) [μM]	2nd experiment
	fold induction		rel. viability [%]		t-test
	mean	SD	mean	SD	p-value	markers
560	1.40	0.17	77	8	0.025	*
672	1.26	0.14	81	12	0.038	*
806	1.35	0.10	68	5	0.009	**
967	1.30	0.19	71	5	0.053	n.s.
1161	1.46	0.11	72	26	0.007	**
1393	1.56	0.10	53	3	0.003	**
1672	1.34	0.11	47	7	0.013	*
2006	1.38	0.17	50	4	0.027	*
VC	1.00	0.08	100	7	-	-
EGDMA 90.8 μM	4.80	0.35	84	9	0.000	**
LA 5000 μM	0.91	0.11	120	8	0.074	n.s.

Interpretation of results:

GHS criteria not met

Conclusions:

2-Piperazin-1-ylethanol does not have a keratinocyte activating potential

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This study was performed in 1997 when LLNA and in vitro tests were not available yet. At this time the GPMT was the standard test method to assess a skin sensitizing potential of a test chemical.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Dunkin Hartley Haz:(DH)fBR albino guinea pigs (5-7 weeks old, 278-444 gr) were obtained from HRP Inc. (Denver, PA).
In the publication referred to no further information on housing conditions is given.

Route:

intradermal and epicutaneous

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100% HEP
1st 0.1 ml intradermal injections
2nd epicutaneous application of filter paper soaked with test substance (100% HEP), secured with tape

Day(s)/duration:

7

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

2 x 2 cm filter paper squares soaked in 100 % HEP

Day(s)/duration:

14 days after epicutaneous induction (i.e., 21 days from the start of the study). Patches were left in place for 24 h, and the sites inspected for signs of irritation 24-48 h after removal of the occlusive dressings.

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

test groups: 10 male and 10 female guinea pigs
control group: five male and five female guinea pigs

Details on study design:

Guinea Pig Maximization Test
The method deseribed by Magnusson'6"7 was used to evaluate the allergie contact sensitization potential. Dunkin Hartley Haz:(DH)fBR albino guinea pigs (5-7 weeks old, 278-444 gr) were obtained from HRP Inc. (Denver, PA). The teehnique used, a maximization proeedure, involved the induetion of an immunologically based sensitization process by injection of the test material into the skin (intradermal induetion) followed by applieation of test material on the skin (epicutaneous induction). After a rest period, the animais were given an epicutaneous challenge to determine if an exaggerated immunologic response (compared with a primary irritant response) was obtained (i.e., to determine if a delayed hypersensitivity reaction had been elicited).
Range-finding studies were conducted to select appropriate concentrations for the intradermal and epicutaneous procedures. Animais were inspected 24 and 48 h after dosing for signs of necrosis and ulceration. The concentration that produced only local necrosis (i.e., no extensive necrosis or ulceration) was used for the intradermal induction. The highest concentration that produced oniy mild irritation was appropriate for the epidutaneous induction, and the highest concentration that did not produce irritation was used for the epicutaneous challenge.
In the definitive sensitization test, groups of 10 male and 10 female guinea pigs each received 0.1 ml intradermal induction injections into 2 sites each of the clipped shoulder skin as follows: 50% (v/v) Freund~s complete adjuvant (FCA) water emulsion, the test material or vehicle, and the test material in FCAlwater emulsion or FCA/water emulsion. Epicutaneous inductions were conducted 7 days later. The test material was applied to saturation (0.2 ml) to a 2 x 4 cm filter paper, which was then placed 011 the test site and secured with tape. The patches were left in place for 48 h, after which they were removed and the skin wiped free of any excess test material. Epicutaneous challenge was undertaken by applying 2 x 2 cm filter paper squares soaked in the appropriate concentration of the test material to a previously untreated site (right flank) 14 days after epicutaneous induction (i.e., 21 days from the start of the study). Patches were left in place for 24 h, and the sites inspected for signs of irritation 24-48 h after removal of the occlusive dressings.
Irritation control animals, five male and five female guinea pigs, received the same challenge procedures as in the definitive sensitization study, but did not have preceding intradermal and/or epicutaneous induction procedures. This allowed differentiation between primary skin irritation due to the test material, and those produced by a hypersensitivity reaction.
Seven days after the challenge exposure (i.e., 28 days from the start of the study), the cross-challenge treatment was administered. Test materials were administered to the clipped skin in a similar manner as in the challenge phase but at previously untreated sites (left flank). Smaller patches (0.825 in2 adbesive bandages) were used in order to allow all of the test materials to fit on the test site. Materials were applied to saturation (0.03 ml per patch). Patches were left in place for 24 h and the sites inspected for signs of irritation 24-48 h after removal of the occlusive dressings.

Grading of Irritation
Skin responses were evaluated according to:

Skin Response = Score
No reaction = 0
Very slight (barely perceptible) eiythema, usually nonconfluent = 0.5
Slight (well-defined) erythema, usually confluent = 1
Moderate erythema = 2
Severe erythema, with or without edema, necrosis, or eschar formation = 3

Edema and necrosis or eschar formation were noted when present. Erythema or edema at the challenge site of any of the observations greater than that seen in the irritation control animais was considered an allergic response. In general, scores of 1 or greater were considered clearly indicative of sensitization. Scores of 0.5 were considered equivocal, although a high percentage of 0.5 scores with no response in irritation control animals would be considered suggestive of sensitization. The percentage of animais reacting, rather than the intensity of reactions, was the criterion for assessing sensitization potency.

Challenge controls:

Irritation control animals, five male and five female guinea pigs, received
the same challenge procedures as in the definitive sensitization study, but did not
have preceding intradermal and/or epicutaneous induction procedures. This allowed
differentiation between primary skin irritation due to the test material, and
those produced by a hypersensitivity reaction.

Positive control substance(s):

not specified

Positive control results:

no positive control indicated

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100 % undiluted test substance

No. with + reactions:

2

Total no. in group:

20

Group:

positive control

Remarks on result:

other: not indicated

Group:

negative control

Remarks on result:

other: primary irritation control

Table 2. Skin Sensitization Responses of Nine Alkyleneamines Tested with the Guinea Pig Maximization Protocol

	Concentration (%) Used for			Skin Sensitization Response^a
	Intradermal	Epicutaneous	Epicutaneous
	Induction	Induction	Challenge	Percentage	Normalized
EDA	5	10	5	45	0.900
DETA	5	50	25	80	0.064
HEEDA	5	50	25	40	0.032
AEP	5	50	25	25	0.020
TETA	5	95	50	74	0.0.16
PEHA	5	100	100	100	0.010
PIP	5	50	25	5	0.004
TEPA	5	60	50	5	0.002
HEP	5	100	100	10	0.001

a Percentage of animal in the group (n = 20) showing a positive skin response with score >=1.

Normalized response = response/(epicutaneous induction conc.)(challenge conc.).

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the GPMT performed by Leung et al., 1997 and applying current CLP criteria the substance 2-Piperazin-1-ylethanol (HEP; CAS 103-76-4) is considered to be non sensitizing.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

2 -Piperazin-1 -ylethanol is not peptide reactive and does not activate keratinocytes. Each individual assay was performed under GLP conditions and the cell based assay LuSens consisted of at least two independent experiments. Positive and negative controls were included in each experiment and confirmed the functionality and validity of the individual assays. The DPRA and the keratinocyte activation assay follow the procedures of the respective OECD testing guidelines (OECD 442C and D). In accordance with the published evaluation scheme (Bauchet al., 2012: Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, 2 -Piperazin-1 -ylethanol is judged not to be a skin sensitizer.

Further a Guinea Pig Maximization Test as described by Magnusson was used by Leung et al. (1997) to evaluate the allergic contact sensitization potential of 2 -Piperazin-1 -ylethanol (HEP).

Dunkin Hartley Haz:(DH)fBR albino guinea pigs were used in a maximization proeedure, which involves the induction of an immunologically based sensitization process by injeetion of the test material into the skin (intradermal induction) followed by applieation of test material on the skin (epicutaneous induction). After a rest period, the animals were given an epicutaneous challenge to determine if an exaggerated immunologic response (compared with a primary irritant response) was obtained (i.e., to determine if a delayed hypersensitivity reaction had been elicited). Based on the evaluation of the skin responses 2 -Piperazin-1 -ylethanol is not considered to be skin sensitizing.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data (in vitro an in vivo) are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered not to be classified for skin sensitization under the Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.