Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol

Endpoint summary

Description of key information

A complete lack of cytotoxicity is already indicative for a lack of irritation potential.

In the EpiDerm the substance showed a60 min viability 80.6%.This value is above the threshold for irritancy of ≤ 50%and thereforethe test item is not considered to possess an irritant potential.

In the Bovine Corneal Opacity test (BCOP), the calculated mean in vitro irritancy score was 0.37. This value is below the threshold of 3, and therefore the test item is not considered to irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 24 November 2016 and 09 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other:

Source strain:

not specified

Species:

other: EPIKSIN in vitro Reconstructed Human Epidermis (RHE) Model

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Epi-200 SIT kits
Supplier : MatTek Corporation, 82105 Bratislava, Slovakia
Date received : Not reported

Epi-200 SIT Kit lot number : 23383
Maintenance Medium lot number :23383
Assay Medium lot number :23383

Type of coverage:

other:

Preparation of test site:

other:

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: Dissiminated (delete when editing): Positive control can be added here.

Amount / concentration applied:

Test material
- Applied volume: 30 ul
- Concentration (if solution): undiluted

Positive and negative control: 30 µL each, used as supplied

Duration of treatment / exposure:

60-Minute exposure period and 43 hours post-exposure incubation period.

Number of animals:

A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Details on study design:

Test for Direct MTT Reduction and Colour Interference
A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test item is present in the tissues when the MTT viability test is performed.
Some non-coloured test items may change into coloured test items in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility has to be performed (step 1).
Step 1
30 µL of the test item were added to 0.3 mL of deionised water (transparent glass test-tube) The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.
Since the test item did not dye water or did not change colour in the presence of water, an additional test (step 2) with viable tissues (but without MTT addition) was not necessary to be performed.
Step 3
All test items (including those already evaluated in step 1 and step 2) were further evaluated for their potential to interfere with MTT assay. To test if a test item directly reduces MTT, 30 µL of the test item were added to 1 mL of the MTT-solution (1mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.
Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

EXPERIMENTAL PERFORMANCE
Pre-warming of EpiDerm™ Tissues
The plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that
1. air bubbles between agarose and insert were not > 30% of the total surface,
2. liquid on top of the insert was removed with sterile cotton tips,
3. if again moisture is observed on top of the inserts after the pre-incubation or in
case of visible defects the respective skin models were discarded.
0.9 mL of the assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further approximately 21 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

Treatment
After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative and positive control, and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The treatment time was 60 minutes in total. Within this period the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate. Tissues were incubated for nearly 25 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation the inserts were transferred into new 6-wells plates containing fresh medium. Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation period was approximately 43 hours.

MTT Assay
On the day of testing the MTT concentrate was diluted with the MTT diluent (1 mg/mL). The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1 °C, 5 ± 0.5 % CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the tissues were rinsed three times with DPBS, and carefully dried with blotting paper. The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues are completely covered. The 24-well plate was sealed to inhibit the isopropanol evaporation.
The formazan salt was extracted for about 2 hours while shaking (~120 rpm) at room temperature.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3  200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 nm filter. Mean values were calculated from the 3 wells per tissue

Irritation / corrosion parameter:

other: other: Mean relative Absorbance (% Negative Control)

Value:

> 80.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results after treatment with the test item and the controls

Treatment	Tissue No.	Mean Absorbance in 3 wells after Blank correction	Mean Absorbance of 3 tissues after blank correction *	Rel. Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]***
Blank		0.000		-	-	-
Negative Contol	1	1.469	1.336	1.110	8.7	100.0
	2	1.254		93.9
	3	1.283		96.1
Test Item	1	1.121	1.076	83.9	7.4	80.6
	2	0.984		73.6
	3	1.124		84.1
Positive Control	1	0.079	0.072	5.9	14.8	5.4
	2	0.076		5.7
	3	0.059		4.5

*       Mean of three replicate wells after blank correction

**     relative absorbance per tissue [rounded values]

***   relative absorbance per treatment group [rounded values]

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 80.6% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: UN GHS and EU CLP regulation

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, ElfaMoist AC is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol by means of the Human Skin Model Test.

The test was performed under monochromatic yellow light.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS), or the positive control (5% SLS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD  0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item ElfaMoist AC the mean relative absorbance value decreased to 80.6% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, ElfaMoist AC is not irritant to skin.

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:

This study was conducted on 25 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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:

July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Abbatoir at Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): adult >9 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: none reported

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

Duration of treatment / exposure:

10 minutes in a horizontal position followed by 2 h in a vertical position, i.e. 130 minutes total

Duration of post- treatment incubation (in vitro):

90 MInutes

Number of animals or in vitro replicates:

3

Details on study design:

Experimental Design and Study Conduct

Collection of Bovine Eyes
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in HBSS. The corneae were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.

Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

Exposure of the Corneae to the Test Groups
The corneae were distributed as follows:
Groups Number of Corneae
1 Negative Control 3
2 Positive Control 3
3 Test Item 3

The anterior compartment received the test item or negative or positive control at a volume of 0.75 mL on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted ten minutes.
After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). The opacity measurement is described inbelow.
In the second step of the assay, permeability of the corneae was determined. The permeability measurement is described below.

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of three eyes

Value:

> 0.37

Negative controls validity:

valid

Remarks:

Not categorized

Positive controls validity:

valid

Remarks:

Category 1

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

This in vitro study was performed to assess the corneal damage potential of ElfaMoist AC by means of the BCOP assay using fresh bovine corneae.
After a first opacity measurement of the fresh bovine corneae (t0), the neat test item ElfaMoist AC, the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time (t130).
After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.00).
The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 94.25) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
The test item ElfaMoist AC was tested undiluted. Relative to the negative control, the test item ElfaMoist AC did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.37 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437, the test item is not categorized

Results after 10 Minutes incubation time

Test Group	Opacity value = Difference (t130-t0) of Opacity	Permeability at 490 nm (OD490)	IVIS	Mean IVIS	Proposed in vitro Irritancy Score
Negative Control	0	0.054	0.81
	0	0.068	1.02	1.00	Not categorized
	0	0.078	1.17
Positive Control	70.00	1.350	90.26
	76.00	1.144	93.17	94.25	Category 1
	84.00	1.222	99.34
ElfaMoist AC (Test Item)	0.00	0.004	0.06
	0.00	0.009	0.14	0.37	Not categorized
	0.00	0.060	0.91

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, The test item is not categorized (GHS)).

Executive summary:

This in vitro study was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t₁₃₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.37. According to OECD 437 the test item is not categorized (GHS).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol has shown to be devoid of cytotoxicity when tested in in-vitro genotoxicity studies for 4 and 24 hours up to the maximum concentration of 10 mM. This lack of cytotoxicity is indicative for a lack of irritation potential, which is confirmed by specific testing for skin and eye irritation.

 

Dermal irritation:

The skin irritation potential of Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol is assessed in the Human Skin Model Test EpiDerm™ under GLP according to OECD 439.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference).

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS), or the positive control (5% SLS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol the mean relative absorbance value decreased to 80.6% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

 

Eye irritation:

The eye irritation potential of Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol was assessed in the Bovine Corneal Opacity and Permeability (BCOP) test under GLP according to OECD 437 guideline.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

The negative and positive controls showed appropriate responses.

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.37.

 

Respiratory irritation:

Exposures to humans via the inhalation route are not likely considering the low vp of 0.028 Pa at 25 °C, high boiling point of 332°C, and use applications not leading to dusts or aerosols.

Considering the lack of irritation potential on skin and eyes, respiratory irritation cannot be expected.

Justification for classification or non-classification

Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol has been shown to be devoid of cytotoxicity in in-vitro cell-based systems. Further, lack of irritating potential to skin and eyes has been demonstrated in appropriate in-vitro test systems. Consequently, the product does not need to be classified for skin or eye irritation.