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Description of key information

Skin irritation (OECD 439): not irritating

Skin corrosion (OECD 431): non corrosive

Eye irritation/corrosion (OECD 437): irreversible damage

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Mar - 30 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
GLP compliance:
yes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempearture, protected from light
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek Corporation, Ashland MA, USA
Source strain:
other: EpiDerm Skin Model (EPI-200)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200) (MatTek Corporation, Ashland MA, USA)
- Tissue batch number: 28307, kit E and F

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 ± 0.5 min exposure); 37.0 ± 1.0 °C (60 ± 5 min exposure)
- Temperature of post-treatment incubation: 37.0 ± 1.0 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed with phosphate buffered saline to remove residual test item; not further specified
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (1 mg/mL)
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.851 ± 0.085 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 1% Triton X-100. The ET-50 value was determined to be 7.24 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi. None of these were detected.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable as the test item did not show reducing capacity 1 h after MTT incubation.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
Step 1
- The test item is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% and the viability after 1 hour exposure is less than 15%.
- The test item is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Step 2 (for test items identified as corrosive in Step 1)
- Sub-category 1A: if the viability after 3 minutes exposure is less than 25%
- A combination of sub-categories 1B and 1C : if the viability after 3 minutes exposure is greater than or equal to 25%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
- Concentration : 8 N

NEGATIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
3 min and 60 ± 5 min
Number of replicates:
duplicates for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean values of 2 tissues
Run / experiment:
3 min exposure
Value:
97
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean values of 2 tissues
Run / experiment:
60 min exposure
Value:
25
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The test item did not show reducing capacity after 1 h incubation with MTT
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water.

ACCEPTANCE OF RESULTS:
- The mean OD of the tissue replicates treated with the negative control are within the laboratory historical control data range, i.e. ≥ 1.258.and ≤ 2.615 for every exposure time (range: 1.871 to 2.120).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (6.2%).
- Acceptance criteria met for variability between replicate measurements: The Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 2.7% to 15%).

Table 2. Mean absorption values

 

3-minute application

1-hour application

A (OD570)

B (OD570)

Mean  ±

(OD570)

SD

A (OD570)

B (OD570)

Mean ±

(OD570)

SD

Negative control

1.871

1.923

1.897

±

0.037

2.120

2.112

2.116

±

0.006

N-ethylcaprolactam

1.918

1.751

1.834

±

0.118

0.532

0.518

0.525

±

0.010

Positive control

0.212

0.244

0.228

±

0.023

0.143

0.122

0.132

±

0.015

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0414). Isopropanol was used to measure the background absorption.

Table 3. Mean tissue viability

 

3-minute application

viability

(percentage of control)

1-hour application

viability

(percentage of control)

Negative control

100

100

N-ethylcaprolactam

97

25

Positive control

12

6.2

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Conclusions:
CLP: not classified
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 - 17 Dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 Jul 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted 20 Jul 2002
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN Small ModelTM reconstructed three-dimensional human epidermis (EPISKIN-SMTM)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModellTM (SkinEthic Laboratories, Lyon, France)
- Tissue batch number: 18-EKIN-050

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature for 15 ± 0.5 min
- Temperature of post-treatment incubation: 37 °C for 42 h

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with phosphate buffered saline (PBS) to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h at 37 °C
- Spectrophotometer: TECAN Infinite # M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA:
- Viability: The quality of the EpiSkin Small Model tissue was assessed via MTT test (IC50 determination): The determined IC50 value was 2.0 mg/mL and fits to the specification 1.5 mg/mL ≤ IC50 ≥ 3 mg/mL.
- Morphology: The histology of tissues revealed a well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and thick stratum corneum and was thus considered satisfactory.
- Contamination: On blood of the donors, the absence of HIV1 and 2 antibodies, hepatitis C antibodies, hepatitis B antigen HBs were verified. On epidermal cells of the donors, the absence of bacteria, fungus and mycoplasma were verified.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item has been tested previously for possible MTT reduction and colour interference in the skin corrosion test using EpiDerm as skin model (Charles River, 2018). The test item turned out to be not-interfering and not directly reducing MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be irritant to the skin if the relative mean tissue viability of three individual tissues after 15 min of exposure to the test item and 42 h of post-incubation is less or equal to 50% of the mean viability of the negative controls.
- The test substance is considered non-irritant to the skin if the relative mean tissue viability of three individual tissues after 15 min of exposure to the test item and 42 h of post-incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 µL

NEGATIVE CONTROL
- Amount applied: 25 µL

POSITIVE CONTROL
- Amount applied: 25 µL
- Concentration: 5% in water
Duration of treatment / exposure:
15 ± 0.5 min at room temperature
Duration of post-treatment incubation (if applicable):
42 h at 37 °C
Number of replicates:
3 replicates for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
15 min exposure
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: Based on results of a preliminary skin corrosion study (Charles River, 2018) with additional freeze killed tissues, the test substance was not considered to be a MTT reducer.
- Colour interference with MTT: The test substance was not colour-interfering with water, as shown in a preliminary skin corrosion study (Charles River, 2018). Thus, an additional test with viable tissues was not performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean absolute OD570 nm of the three negative control tissues was within the historical control data range and the standard deviation vale of the % viability was ≤ 18%.
- Acceptance criteria met for positive control: Yes, the mean relative tissue viability of the three positive control tissues was ≤ 40% (11%) relative to the negative control and the standard deviation value of the % viability was ≤ 18%.
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation calculated from individual % tissue viabilities of the three identically treated replicates is ≤ 18% (< 7%).
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted Jul 2013
Deviations:
no
GLP compliance:
yes
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Vitelco, 's Hertogenbosch, The Netherlands
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: The eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
Duration of treatment / exposure:
10 ± 1 min
Duration of post- treatment incubation (in vitro):
120 ± 10 min
Number of animals or in vitro replicates:
Three corneas were used for each treatment group.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine
(Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 h at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

TREATMENT METHOD
After treatment, the holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 min at 32 ± 1 °C. Possible pH effects of the test item on the corneas were recorded.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 min at 32 ± 1 °C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490) .

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as Category 1.
Test substance with an IVIS ≤ 3 was regarded as No Category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value of 3 corneas
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (physiological saline) the calculated IVIS ranged from 0.8 to 1.2, and therefore within the standard deviations of the current historical mean of the negative control (IVIS: -2.8 to 3.0) (please refer to Table 1 and 2 under "any other information on results incl. tables").
- Acceptance criteria met for positive control: After treatment with the positive control (ethanol) the calculated IVIS ranged from 56 to 63, and therefore within the standard deviations of the current historical mean of the positive control (IVIS: 28.0 - 110.9) (please refer to Table 1 and 2 under "any other information on results incl. tables").

Table 1: Results

Treatment Permeability score
Final OD*
Final Opacity* In vitro Irritancy Score
Negative control -0.001 1.2 1.2
0.013 0.7 0.9
0.002 0.8 0.8
Positive control 2.615 16 56
5.439 21 103
3.195 15 63
Test item 4.453 44 111
4.343 23 89
4.153 50 112

* Positive control and test item are corrected for the negative control.

Table 2 Historical control data

  Negative control Positive control
  Opacity Permeability In vitro Irritancy Score In vitro Irritancy Score
Range -2.9 - 3.0 -0.034 - 0.100 -2.8 - 3.0 28.0 - 110.9
Mean 0.18 0.00 0.23 55.28
SD 1.10 0.01 1.13 15.14
n 113 113 113 88
Interpretation of results:
other: corrosive according to Regulation (EC) No. 1272/2008
Conclusions:
Under the conditions of the BCOP assay, the test substance showed corrosive properties towards eyes.
CLP: Eye Damage 1, H318 according to Regulation(EC) No. 1272/2008
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion in vitro:

The human Skin Model Test with EpiDermTM tissue models was conducted to assess the corrosive potential of the test substance N-etyhlcaprolactam in human keratinocytes according to OECD guideline 431 and in compliance with GLP (Charles River Laboratories, 2018).

The test item passed the colour interference and MTT reduction test and turned out to be non-interfering and non-reducing. Duplicate tissues of the human skin model EpiDermTMwere each exposed to the test item, the negative control (deionized water) or the positive control (8.0 N KOH) for 3 min (room temperature) and for 60 min (37 ± 1.0 °C). Each 50 µL of the test item, of the negative or of the positive control were applied on the top of the skin tissues. After exposure, the tissues were washed and cytotoxicity was evaluated by MTT reduction.

Negative control absorbance values were within the range of historical control data and the positive control induced a decrease in viability as compared to the negative control to 6.2%, thus confirming the quality of the tissues and matching the acceptability criteria for the study. The relative mean tissue viability obtained after 3 min and 1 h treatments with the test substance compared to the negative control tissues was 97% and 25%, respectively.

In conclusion, under the conditions of the study the test item is not considered to be corrosive.

 

Skin irritation in vitro:

In a next step, the skin irritation potential of N-ethylcaprolactam was determined in epidermal keratinocytes in vitro using reconstructed human epidermis. The test was conducted with EpiSkin Small ModelTM tissues (EpiSkin SMTM) according to OECD guideline 439 and in compliance with GLP (Charles River Laboratories, 2019).

25 µL of the undiluted test item, the negative control (phosphate buffered saline, PBS) and the positive control (5% sodium dodecyl sulfate, SDS) were applied topically to the surface of the EpiSkin SMTM tissues and incubated for 15 ± 0.5 min at room temperature. After the exposure and 42 h post-incubation period at 37 °C, cytotoxicity was evaluated by MTT reduction. The test item was known to not colour-interfer with water and not directly reduce MTT.

Test acceptability criteria were met, as the negative control values were within the range of historical control data, and the positive control reduced the mean tissue viability to 11% and the standard deviation value of the percentage viability of three tissues treated identically was < 7%, thus demonstrating the functionality of the test.

Compared to the mean relative tissue viability of the negative control, the mean relative tissue viability of N-ethylcaprolactam was 104% after 15 ± 0.5 min of exposure. This value is above the threshold for irritancy of≤50%. Therefore, under the conditions of the test, the test item is considered to be non-irritant to the skin.

 

Eye irritation in vitro:

The eye irritating potential of N-ethylcaprolactam was investigated using a bovine corneal opacity and permeability (BCOP) test in vitro. The BCOP test was conducted according to OECD guideline 437 and in compliance with GLP (Charles River Laboratories, 2018). After an initial opacity reading, 750 µL of undiluted test substance, negative control (physiological saline) or positive control (ethanol) were introduced onto the epithelium of each three corneas. After 10 ± 1 min of incubation at 32 ± 1 °C the substances were rinsed off and the corneas were post-incubated for 120 ± 10 min at 32 ± 1 °C. After completion of the incubation period a second opacity reading was performed. In addition, permeability of the corneas was evaluated by measuring the transfer of sodium fluorescein after incubation for 90 ± 5 min at 32 ± 1 °C.

The negative control responses for opacity and permeability were less than the upper limits of the laboratories historical control range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control was 74 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The corneas were turbid after the 10 minutes of treatment with the test substance. Relative to the negative control, the calculated mean in vitro irritancy score (IVIS) of the test substance was 104. Thus, under the conditions of the test, the test item is considered to be corrosive to the eye since it induced an IVIS ≥ 55.

Justification for classification or non-classification

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

The available data on eye irritation meet the criteria for classification as Eye Damaging Category 1 (H318) according to Regulation (EC) No. 1272/2008.