1-ethylazepan-2-one

Endpoint summary

Description of key information

Skin sensitisation in vivo (OECD 429): not sensitising

Skin sensitisation in vitro (OECD 442D): negative

Skin sensitisation in chemico (OECD 442C): negative

Skin sensitisation in silico (QSAR prediction, DEREK NEXUS): WoE conclusion from available skin sensitisation data: predicted not skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 Mar - 04 Apr 2005

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: insufficient test design to conclude on endpoint (results not suited to calculate SI, no DMPs described, skin irritation cannot be concluded on, skin sensitisation might take place at high doses)

Remarks:

(cellular proliferation was not determined by incorporated radioactivity, thus no disintegrations per minute (DPM) and no SI values were calculated)

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted Apr 2002

Deviations:

yes

Remarks:

(cellular proliferation was not determined by incorporated radioactivity, thus no disintegrations per minute (DPM) and no SI values were calculated)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2004

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Pflanzenbau und Pflanzenschutz, Mainz, Germany

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approxiamtely 7 - 8 weeks
- Weight at study initiation: 17.0 - 20.6 g
- Housing: The animals were individually housed in Makrolon type I cages.
- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 29 Mar 2005 To: 04 Apr 2005

Vehicle:

other: acetone

Concentration:

3, 10 and 50% (w/w)

No. of animals per dose:

6

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Lymph node weight, cell count and ear weight were determined for the control and treatment animals.
- Criteria used to consider a positive response: In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous administration of several concentrations of the test material to the skin of the skin at the back of the ear is determined. The parameters used to characterize the response are lymph node cell count and to a certain extent weight. Because not only sensitization induction but also irritation of the ear skin by the test material may induce lymph node responses, the weight of ear punches taken from the area of test material application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test material.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the test material was applied to the entire dorsal surface of each ear of each mouse on Days 1, 2 and 3 in concentrations of 3, 10 and 50% in acetone. The irritation effects on the treatment site were assessed daily.
Three days after the last application the animals were sacrificed. Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear. The weight of the pooled punches was determined for each animal. Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal. After weight determination, the pooled lymph nodes of each animal were stored in phosphate
buffered saline in an ice-water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 6 mL of phosphate-buffered physiological saline. For determination of cell count the standard suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-Counter. Each suspension was measured in triplicate (3 dilutions of the standard suspension). The mean of the triplicate measurements was used for further evaluation.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The indices of Iymph node weight, cell count and ear weight were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group. Statistical analyses for lymph node weight, cell count and ear weight were performed with the Wilcoxon-Test.

Positive control results:

Studies using the positive control substance alpha-hexylcinnamaldehyde, techn. 85% are performed twice a year in the Iaboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen. In a study performed from 26 Apr to 10 May 2005 at the same testing facility, 1% alpha-hexylcinnamaldehyde in acetone showed skin sensitising properties (project # 45H0288/982059). Lymph node weight indeces were 1.12, 1.37 (p ≤ 0.05) and 1.96 (p ≤ 0.01), the cell count indeces were 1.28 (p ≤ 0.05), 1.63 (p ≤ 0.05) and 2.94 (p ≤ 0.01) and the ear weight indeces were 1.04, 1.06 (p ≤ 0.05) and 1.14 (p ≤ 0.01) in the animals treated with 1, 3 and 10% alpha-hexylcinnamaldehyde in acetone, respectively.

Parameter:

SI

Test group / Remarks:

3% application

Remarks on result:

not measured/tested

Parameter:

SI

Test group / Remarks:

10% application

Remarks on result:

not measured/tested

Parameter:

SI

Test group / Remarks:

50% application

Remarks on result:

not measured/tested

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The test material did not induce a statistically significant or biologically relevant response of the auricular lymph nodes when applied as 3% or 10% preparations in acetone. The minimal but statistically significant increase in mice treated with the 50% test substance preparation was too small to be considered biologically relevant. The statistically significant increases in ear weights indicate irritation of the ear skin at all concentrations. Thus, the minimal increase in cell count and lymph node weight index in test group 4 is considered to be related to ear skin irritation produced by the combination of vehicle and test substance, which starts already at the 3% concentration without relevant lymph node response. Higher concentrations were not tested, because the high concentration tested (50%) produced ear skin irritation.

CLINICAL OBSERVATIONS:
1/6 animals of the 50% dose group was found dead on study day 2. No macroscopic pathologic abnormalities were noted. The death was not considered to be test substance related.

BODY WEIGHTS
Body weights of the treatment and the control group were within the normal range.

Table 1 Results of LLNA

Test Group	Treatment	Lymph Node Weight [mg]			Cell Count [Counts/Lymph Node Pair]			Ear Weight [mg]
		Mean	S.D.	Index^1	Mean	S.D.	Index^1	Mean	S.D.	Index^1
1	vehicle acetone	5.5	0.6	1.00	9,588,333	1,972,130	1.00	26.9	0.3	1.00
2	3% in acetone	5.5	0.6	1.00	9,998,333	1,167,985	1.04	28.0	0.4	1.04 ##
3	10% in acetone	5.7	1.0	1.04	11,212,333	2,641,081	1.17	28.9	0.8	1.07 ##
4	50% in acetone	7.7	1.1	1.41 ##	15,870,000	4,998,814	1.66 ##	29.9	1.4	1.11 ##

S.D. = standard deviation

* test group x / test group 1 (vehicle control)

# = statistically significant for the value p ≤0.05

## = statistically significant for the value p ≤ 0.01

Interpretation of results:

other: non-sensitising according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

(Q)SAR prediiction

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
Lhasa Limited Derek Nexus

2. MODEL (incl. version number)
Derek Nexus: 6.0.1, Nexus: 2.2.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
SMILES code: C1CC(N(CCC1)CC)=O
CAS No.: 19797-08-1

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Structural alerts

5. APPLICABILITY DOMAIN
- Descriptor domain: The target chemical falls within the applicability domain of the prediction.

6. ADEQUACY OF THE RESULT
DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitization alerts.
The level of likelihood of a structure being sensitizing to skin is expressed in terms of:
Certain: There is proof that the proposition is true.
Probable: There is at least one strong argument that the proposition is true and there are no arguments against it.
Plausible: The weight of evidence supports the proposition.
Equivocal: There is an equal weight of evidence for and against the proposition.

The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment.

DEREK NEXUS contains an expert-derived functionality that can provide negative predictions for skin sensitization. This functionality further evaluates those compounds which do not fire any skin sensitization alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of skin sensitization data, producing the following outcomes:
• In compounds where all features in the molecule are found in accurately classified compounds from the reference set, a negative prediction is displayed: inactive.
• For those query compounds where features in the molecule are found in non-alerting skin sensitizers in the Lhasa reference set, the prediction remains negative and the misclassified (Misclassified features are those that have been derived from non-alerting substances in the Lhasa test reference set) features are highlighted to enable the negative prediction to be verified by expert assessment.
• In cases where features in the molecule are not found in the Lhasa reference set, the prediction remains negative and the unclassified (Unclassified features are those that have not been found in the Lhasa test reference set) features are highlighted to enable the negative prediction to be verified by expert assessment.

If a substance is predicted to be a skin sensitizer, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental data from the closest structurally-related substances (at least 3 substances should be present) using the following equation:

EC3Q = MWQ /(Σ ωNN / Σ TNN)

MW = molecular weight
T = Tanimoto similarity score
ω = weighting factor = (MWNN/EC3) * TNN
Q = query compound
NN = nearest neighbour

The EC3 is the estimated concentration needed to produce a stimulation index of 3.

Principles of method if other than guideline:

Calculation based on Derek Nexus: 6.0.1 and Nexus: 2.2.1. QSAR prediction of the skin sensitisation potential of the test substance.

GLP compliance:

no

Parameter:

other: QSAR prediction

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

other: the prediction results was negative for skin sensitising potential, based on QSAR prediction (DEREK Nexus). The results may only be used for classification purposes together with other data.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 - 25 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted Feb 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

TEST SYSTEM
- Supplier: JPT Peptide Technologies GmbH, Berlin, Germany
- Cysteine-containing peptide:
Alternative name: Ac-RFAACAA-COOH
Batch number: 260515HS_DW0718
Molecular weight: 751.5 g/mol
Purity: > 98%
- Lysine-containing peptide:
Alternative name: Ac-RFAAKAA-COOH
Batch number: 120514HS_DW_W0418
Molecular weight: 776.4 g/mol
Purity: > 98%

TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 and 258 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

VEHICLE CONTROL
- Substance: acetonitrile

CONTROL
- A co-elution control, test item at a concentration of 100 mM in acetonitril and the respective buffer solution (phosphate and ammonium acetate, respectively) without synthetic peptide, was prepared.
- Reference controls were prepared by mixing the respective synthetic peptide with acetonitril.
- Calibration solutions were prepared in order to generate a calibation curve.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
- Batch number: MKCB9970
- Purity: 99.1%
- Expiry date: Nov 2021

POSITIVE CONTROL PREPARATION
The positive control was prepared at a concentration of 100 mM.

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM preparation in acetonitrile.

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM; 10.3 mg synthetic peptide containing cysteine were dissolved in 20.56 mL phosphate buffer pH 7.5.
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM; 11.5 mg were dissolved in 22.20 mL ammonium acetate buffer pH 10.2.

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10; Lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 ± 2 °C
- Duration of treatment / exposure: 22.5 h

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and control

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 μm, 100 x 2.1 mm with guard column Phenomenex AJO4286
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in Mill-Q water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20 min
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 and 258 nm
- Injection volume: 3 μL
- Column temperature: 30 °C

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean peptide depletion of the positive control for the cysteine peptide was 70.7 %. The mean peptide depletion of the positive control for the lysine peptide was 53.7%.

Key result

Run / experiment:

other: Cysteine peptide

Parameter:

other: % depletion of cysteine-containing peptide (mean value of 3 replicates)

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Lysine peptide

Parameter:

other: % depletion of cysteine-containing peptide (mean value of 3 replicates)

Value:

0.3

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: cysteine and lysine peptide

Parameter:

other: % depletion of cysteine-containing peptide (mean value of 3 replicates)

Value:

0.9

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Table 1 Acceptability of the Direct Peptide Reactivity Assay (DPRA)

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r^2) standard calibration curve

>0.99

0.9995

>0.99

0.995

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.525 ± 0.012

0.50 ± 0.05

0.496 ± 0.005

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.509 ± 0.022

0.50 ± 0.05

0.499 ± 0.017

CV (%) for RC samples B and C

<15.0

3.7

<15.0

2.8

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

70.7

40.2-69.0

53.7

SD of peptide depletion cinnamic aldehyde (%)

<14.9

0.5

<11.6

1.4

SD of peptide depletion for the test item (%)

<14.9

2.0

<11.6

0.6

Table 2 SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

DPRA prediction and reactivity classification

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

N-ethylcaprolactam

1.4%

±2.0%

0.3%

±0.6%

0.9%

Negative: No or minimal reactivity

Interpretation of results:

other: no skin sensitising potential based on the key event "protein binding"

Conclusions:

The test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 Jul - 17 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted Feb 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance by addressing the second molecular key event of the Adverse Outcome Pathway (AOP), activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™.
The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Passage number: 7, 9 and 11

CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% fetal bovine calf serum and 500 µg/mL geneticin
Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% fetal bovine calf serum
- Temperature (°C): 37 ± 1.0 (actual range 35.9 - 37.2 °C)
- CO2 (%): 5.0 ± 0.5
- Humidity (%): 80 - 100% (actual range 81 - 99%)

TEST CONCENTRATIONS
0.98, 2.0, 3.9, 7.8, 16, 31, 63, 125, 250, 500, 1000 and 2000 µM

CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1%
Positive control
- Substance: ethylene dimethacrylate glycol
- Final concentration: 7.8 - 250 µM

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 1 h
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0

NUMBER OF REPLICATIONS: three replicates (luciferase activity) and one replicate (cell viability) each in three independent experiments, respectively

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- Incubation time: 3 - 4 h
- Device: plate reader (TECAN Infinite® M200 Pro Plate Reader)
- Wavelength: 570 nm

DETERMINATION OF LUMINESCENCE
- Luciferase Assay System: Steady-Glo Luciferase Assay Buffer and Steady-Glo Luciferase Assay Substrate
- Device: plate reader (TECAN Infinite® M200 Pro Plate Reader)

Positive control results:

The gene induction for the positive control ethylene dimethacrylate glycol was found to be > 2.0 at a concentration of 250 µM in all three experiments. The EC1.5 value for the positive control was found to be 67, 28 and 70 µM in all three experiments, respectively. The average gene induction for the positive control at 250 µM was found to be 3.32-, 3.63- and 2.44-fold in all three experiments, respectively.

Key result

Run / experiment:

other: Experiment I: at a dose level of 2000 µM

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.92

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment II: at the dose levels of 31 and 500 µM

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

0.84

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment III: at a dose level of 16 µM

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.17

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

The test item showed no toxicity (no IC30 and IC50 value) in all experiments. In the first experiment the test item showed a biologically relevant, induction of the luciferase activity (EC1.5 value of 381 μM) was measured. The maximum luciferase activity induction (Imax) was 1.92-fold, leading to an individual run conclusion of positive. In the second experiment no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 0.84-fold. This led to an individual run conclusion of negative. Since the first and second experiment were not concordant an additional third experiment was performed. In the third experiment no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.17-fold, leading to an individual run conclusion of negative. The test item is classified as negative in the KeratinoSens™ assay since negative results (<1.5-fold induction) were observed in two out of three experiments at test concentrations up to 2000 μM.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: The luciferase activity induction obtained with the positive control, ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
- Acceptance criteria met for negative control: The EC1.5 of the positive control was between 5 and 125 μM (67 μM, 28 μM and 70 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.32-fold, 3.63-fold and 2.44-fold in experiment 1, 2 and 3, respectively).
- Acceptance criteria met for variability between replicate measurements: The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (12%, 17% and 5.8% in experiment 1, 2 and 3, respectively).

Table 1: Overview Luminescence Induction and Cell Viability of the test item in Experiment 1, 2 and 3

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Exp. 1 luminescence	1.39	1.39	1.21	1.43	1.52	1.40	1.37	1.24	1.45	1.55***	1.53***	1.92***
Exp. 1 viability (%)	106.4	98.8	103.5	103.0	98.6	91.1	103.5	102.3	105.0	101.2	101.6	91.8
Exp. 2 luminescence	0.68	0.70	0.75	0.80	0.80	0.84	0.76	0.82	0.80	0.84	0.75	0.81
Exp. 2 viability (%)	124.0	111.0	102.6	97.8	93.7	87.6	87.3	90.4	88.6	91.2	96.5	101.1
Exp. 3 luminescence	1.07	0.96	1.09	1.11	1.17	1.12	1.13	1.09	1.01	1.00	1.05	1.06
Exp. 3 viability (%)	100.5	102.6	94.3	92.1	89.5	85.0	89.1	88.9	85.8	84.8	80.5	87.8

***p<0.001 Student’s t test

Table 2: Overview Luminescence Induction and Cell Viability Positive Control ethylene dimethacrylateglycol in Experiment 1, 2 and 3

Concentration (µM)	7.8	16	31	63	125	250
Exp. 1 luminescence	1.04	1.11	1.41	1.43	2.35***	3.32***
Exp. 1 viability (%)	100.9	106.0	100.7	102.1	101.3	91.1
Exp. 2 luminescence	1.22	1.30	1.54***	1.83***	1.99***	3.63***
Exp. 2 viability (%)	110.1	109.1	96.6	105.5	112.7	118.7
Exp. 3 luminescence	1.07	1.17	1.23	1.45	1.87***	2.44***
Exp. 3 viability (%)	101.2	103.4	104.9	113.4	115.0	119.8

***p<0.001 Student’s t test

Table 3: Overview EC1.5, Imax, IC30 and IC50 Values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test item Experiment 1	381	1.92	NA	NA
Test item Experiment 2	NA	0.84	NA	NA
Test item Experiment 3	NA	1.17	NA	NA
Positiv Control Experiment 1	67	3.32	NA	NA
Positiv Control Experiment 2	28	3.63	NA	NA
Positiv Control Experiment 3	70	2.44	NA	NA

Interpretation of results:

other: no skin sensitising potential based on the key event "activation of keratinocytes"

Conclusions:

Under the conditions of the test, it can be concluded, that the test substance is no sensitiser in KeratinoSens Assay. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Additional information:

Skin sensitisation in vivo:

The skin sensitisation potential of N-ethylcaprolactam was evaluated in a non-radioactive variant of the Local Lymph Node Assay (LLNA) following OECD guideline 429 under GLP conditions (BASF, 2005). The tests substance was applied at concentrations of 3, 10 and 50% (w/w) in acetone on three consecutive days to the dorsal surface of both ears of 6 female CBA/Ca mice each. The control group was treated with the vehicle alone. Three days after the last application, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content as indicator of cell proliferation and the weight of each animals pooled lymph nodes. Moreover, the weight of ear punches were determined in order to obtain an indication of possible skin irritation. A concurrent positive control with a known sensitizer was not included, but data from a recent study with alpha-hexylcinnamaldehyde (1, 3 and 10% in acetone) served as positive control data.

No treatment-related signs of systemic toxicity were noted. The test item did not induce a statistically significant or biologically relevant response of the auricular lymph nodes when applied as 3 or 10% preparation. 50% N-ethylcaprolactam induced a minimal, but statistically significant increase which was not considered biologically relevant. All test item concentrations significantly increased the ear weights, indicating skin irritation. Thus, the minimal increase in cell count and lymph node weight index at 50% test item concentration was considered to be related to ear skin irritation produced by the combination of vehicle and test substance, which starts already at the 3% concentration without relevant lymph node response. Thus, under the conditions of the test, the authors concluded, that N-ethylcaprolactam does not have a skin sensitising effect.

The reliability of this study is not assignable (Klimisch score 4). The results are not suited to calculate a stimulation index, therefore in principle the data cannot be used to conclude on this endpoint. DPMs were not described, skin irritation is possible but cannot be concluded on (skin irritation was not documented, and in the range of historical control values and lower than the positive control). It cannot be excluded that skin sensitization will take place at high doses. Thus, based on these considerations, the data is only used as supporting evidence within a weight-of-evidence approach.

Skin sensitisation in vitro:

1.      Inflammatory response in keratinocytes: KeratinoSens Assay

A KeratinoSens^TM assay was performed in accordance with OECD guideline 442D and in compliance with GLP (Charles River Laboratories, 2018) to evaluate the ability of N-ethylcaprolactam to activate the antioxidant/electrophilic responsive element (ARE)-dependent pathway. The tests uses a transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element (the KeratinoSens^TM cell line).

The test item, as well as vehicle and positive controls were incubated with the transgenic keratinocytes for 48 ± 1 h at 37 ± 1 °C, followed by determination of luciferase activity and assessment of cytotoxicity. Twelve concentrations of the test compound in the range of 0.98 – 2000 µM were included, as well as a concentration range of 7.8 – 250 µM of the positive control ethylene dimethacrylate glycol. DMSO (1% in cultivation medium) served as solvent control compound. Three independent experiments were performed, using each three replicates per condition. After 48 h of exposure, luciferase activity was measured and cytotoxicity was determined using the MTT assay. A dose-dependent, statistically significant luciferase induction > 1.5 fold was achieved with the positive control, while the luminescence reading of the negative control showed no luciferase induction (coefficient of variation < 20%), thus confirming the validity of the test system.

No precipitation was observed at the start and end of the incubation period in the plates for the test substance in any of the three experiments. N-ethylcaprolactam showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC₃₀ and IC₅₀ values could be calculated. In the first experiment, the maximum luciferase activity induction was 1.92 fold, leading to an individual run conclusion of positive. However, the second and third experiment revealed a maximum luciferase induction of 0.84 and 1.17, respectively. As two out of three experiments revealed negative results (< 1.5 fold induction of luciferase activity), N-ethylcaprolactam was classified as negative in the KeratinoSens^TM assay under the given conditions of the test.

 

2.      Skin sensitisation in chemico: Direct Peptide Reactivity Assay

The binding activity of N-ethylcaprolactam towards proteins in the skin was assessed in chemico using the Direct Peptide Reactivity Assay (DPRA). The study was performed in accordance with OECD guideline 442C and was compliant with GLP (Charles River Laboratories, 2018). The assay quantifies the depletion of both cysteine and lysine containing peptides following incubation with the test item and corresponding controls.

The test substance as well as the positive control (cinnamic aldehyde) were prepared at concentrations of 100 mM in acetonitrile and incubated with peptide concentrations of 0.5 ± 0.05 mM using three replicates per compound. In addition, a co-elution control using buffer instead of peptide was included. After 22.5 (cysteine peptide) and 25 h (lysine peptide) of incubation at 25 ± 2.5 °C the remaining cysteine and lysine concentration was determined via high performance liquid chromatography (HPLC) and photodiode array detection at 220 and 258 nm. Peak area responses at defined retention times were recorded and used to calculate the percentage of peptide depletion for cysteine-, as well as lysine containing peptide.

All validation parameters, i.e. calibration curve, mean concentration and coefficient of variation of reference controls and the mean percent peptide depletion if the positive control were within the acceptability criteria of the test. The test item itself revealed a cysteine peptide depletion of 1.4 ± 2.0% and a lysine peptide depletion of 0.30 ± 0.6% (0.9% mean total peptide depletion). As a result the test item gives a negative DPRA prediction and is assigned to the reactivity class “no or minimal reactivity” when using the Cysteine 1:10/Lysine 1:50 prediction model.

 

Skin sensitisation in silico:DEREK NEXUS

The skin sensitisation potential of N-ethylcaprolactam was further assessed with the in silico model DEREK NEXUS. DEREK NEXUS represents a knowledge-based system which contains 90 alerts for skin sensitisation based on the presence of molecular substructures and provides QSAR prediction of the skin sensitizing potential of a test compound.

DEREK NEXUS did not yield any alerts for skin sensitisation for N-ethylcaprolactam. Additionally, the query structure does not contain any unclassified or misclassified features. The test item is therefore predicted to be a non-sensitiser.

 

Conclusion:

Based on the results of one in vivo study, two in vitro studies and an in silico approach in a weight-of-evidence approach, the test item N-ethylcaprolactam is considered to have no skin sensitising potential.

Justification for classification or non-classification

The available data on skin sensitisation do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.