N,N-Dimethyl-9-dodecenamide

Endpoint summary

Description of key information

Skin irritation:

In vitro skin corrosivity

The test item was considered to be non-corrosive to the skin in a study performed according to OECD TG 431.

In vitro skin irritation

The test item was considered to be a Category 2 skin irritant in a study performed according to OECD TG 439.

Eye irritation:

In vitro eye irritation

No stand-alone prediction of eye irritation can be made under the conditions of the test for studies performed according to OECD TG 437 and OECD TG 492.

In vivo eye irritation

A supporting study is available for an analogue substance, which is included to assist with determination of classification and labelling at the Annex VII tonnage band. The substance is classified as Eye Irritation Category 2 on the basis of a study performed to OECD TG 405.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 - 27 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The test system utilises reconstructed human epidermis (RhE) (obtained from human derived nontransformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 36119
- Delivery date: 25 January 2022
- Assay Medium lot number: 012022MPC
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.
CoA (Annex 2) attached.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for the 3 min exposure period, 37 °C for the 60 min exposure period

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue (20x) under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader and LT-com analysis software
- Wavelength: 570 nm
- Filter bandwidth: 10 nm
- Linear OD range of spectrophotometer: see Annex 3 attached

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540-570 nm) = 1.54 ± 0.049 (Acceptance criteria: 1.0-3.0)
- Barrier function: ET-50 = 6.06 h (Acceptance criteria: 4.77 - 8.72)
- Morphology: tissue viabilty and barrier function test are within the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers
- Contamination: no contamination
CoA attached (Annex 2)

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8N in water

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item, 3 minute exposure, mean value

Value:

104.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item, 60 minute exposure, mean value

Value:

81.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.
- Colour interference with MTT: The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 2.094 for the 3-Minute exposure period and 2.421 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 2.2% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Tissue	Exposure Period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	2.127	2.094	0.047	2.2	100*
		2.061
	60 Minutes	2.601	2.421	0.255	10.5
		2.240
Positive control	3 Minutes	0.063	0.060	0.005	na	2.9
		0.056
	60 Minutes	0.054	0.054	0.001	na	2.2
		0.053
Test Item	3 Minutes	2.445	2.194	0.356	16.2	104.8
		1.942
	60 Minutes	2.054	1.967	0.123	6.3	81.2
		1.880

OD = Optical density

* = The mean percentage viability of the negative control tissue is set at 100%

na = Not applicable

Interpretation of results:

GHS criteria not met

Executive summary:

Introduction

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

Methods

Duplicate tissues were treated with the negative control, positive control and test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the control items and test item were rinsed from the tissues before each tissue was taken for MTT-loading. After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 -well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	2.9	104.8
60 minute	100*	2.2	81.2

*The mean viability of the negative control tissues is set at 100%

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

Conclusion

In this study and under the experimental conditions reported:

The test item was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 - 04 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDermTM SIT (EPI-200) is a three-dimensional human skin model, comprising of a reconstructed epidermis with a functional stratum corneum. The individual tissues are formed from normal human-derived epidermal keratinocytes, cultured to form a multilayered highly differentiated model of the human epidermis. The tissues consist of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 36120
- Delivery date: 01 February 2022
- Assay Medium lot number: 012722LHC
CoA (Annex 2) attached

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: the plates were incubated (37 °C, 5% CO2) for 35 ± 1 minutes then removed from the incubator and kept in the biological safety cabinet at room temperature for the remainder of the 60-minute exposure period.
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2 in air for a total of 42 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS without Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert approximately fifteen times using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were then submerged three times in 150 ml of DPBS without Ca++ and Mg++ before a final rinse inside and outside of the tissue insert and drying with a cotton swab.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
MTT concentration: 1.0 mg/mL
- Incubation time: 1 hours
- Spectrophotometer:
Labtech LT-4500 microplate reader and LT-com analysis software
- Wavelength: 570 nm
- Filter bandwidth: 10 nm
- Linear OD range of spectrophotometer: see Annex 3 attached

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540-570 nm) = 1.6 ± 0.088 (Acceptance criteria: 1.0-3.0)
- Barrier function: ET-50 = 5.99 h (Acceptance criteria: 4.77 - 8.72)
- Morphology: tissue viabilty and barrier function test are within the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers
- Contamination: no contamination
CoA attached (Annex 2)

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean relative tissue viability of three individual tissues is reduced to ≤ 50% of the negative control.
- The test substance is considered to be non-irritant to skin if the mean relative tissue viability of three individual tissues is > 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% w/v aqueous solution

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item, mean of 3 replicates

Value:

3.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: relative mean viability after 60 min exposure period and 42-Hour post-exposure incubation period.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.
- Colour interference with MTT: The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.940. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 3.5% relative to the negative control treated tissues. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation between the three tissue replicates of each treatment group did not exceed 18%. The acceptance criterion was therefore satisfied.

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	1.974	1.940	0.161	101.8	100*	8.3
	1.765			91.0
	2.082			107.3
Positive Control Item	0.064	0.067	0.003	3.3	3.5	0.2
	0.067			3.5
	0.069			3.6
Test Item	0.055	0.062	0.007	2.8	3.2	0.4
	0.069			3.6
	0.062			3.2

OD = Optical Density

SD = Standard deviation

*= The mean viability of the negative control tissues is set at 100%

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EpiDerm™ Skin Irritation Test (SIT) according to the OECD Test Guideline 439 In Vitro Skin Irritation Reconstructed Human Epidermis (RHE) and the synonymous EU B.46. test method.

The test item was applied topically for a treatment period of 60 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in RHE cultures following topical exposure to the test item by means of the colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue/purple formazan dye in the test item treated tissues relative to the negative control.

Method

Triplicate tissues were treated with the test item for an exposure period of 60 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction. At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 3.2% after the 60-Minute exposure period and 42-Hours post-exposure incubation period.

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

Conclusion

This study alone does not allow the conclusion on whether the test item is a corrosive or an irritant (EU CLP/UN GHS Category 1 or Category 2). However, per the results of an OECD 431 In Vitro Skin Corrosion study (Reconstructed Human EpiDermis (RHE) Test Method; Labcorp Study Number: 8484301), the test item was considered to be non-corrosive. Thus, based on the results of this study and the results of the OECD 431 In Vitro Skin Corrosion study, the test item was considered to be an irritant and the following classification criteria apply:

EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 February 2022

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:

26 June 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: not stated

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

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): neat

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three replicates per treatment

Details on study design:

NUMBER OF REPLICATES : 3 per treatment

NEGATIVE CONTROL USED :
Identification: Sodium chloride 0.9% w/v
Lot: 19J07BB1C (7634)
Purity: 0.9%
Supplier: Baxter Healthcare SA
Expiry Date: 01 September 2022
Storage Conditions: Room temperature

POSITIVE CONTROL USED :
Identification: Ethanol
Batch: STBJ5270
Purity: >99.8%
Supplier: Sigma Aldrich
Expiry Date: 16 March 2025
Storage Conditions: Room temperature

APPLICATION DOSE AND EXPOSURE TIME : 0.75 mL, 10 minutes

TREATMENT METHOD: closed chamber
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost (horizontally), at 32 ± 1 ºC for 10 minutes.

POST-INCUBATION PERIOD: yes, 120 minutes

REMOVAL OF TEST SUBSTANCE
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
Data evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
Visual Observation
The condition of the cornea was visually assessed post treatment and post incubation.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
The test item is classified according to the prediction model as follows:
 IVIS/ UN GHS
 ≤ 3/ No Category
>3; ≤ 55/ No stand-alone prediction can be made
 > 55/ Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

28.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Corneal Opacity and Permeability Measurement
Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.
Corneal Epithelium Condition
The condition of each cornea is given in Table 2.
The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.
The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.
The negative control values were within accepted limits. The negative control acceptance criteria were therefore satisfied.
Historical control data are shown in Appendix 3, attached.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD492)		In vitro irritancy score
		Pre-treatment	Post- treatment	Post- incubation	Post- incubation – Pre- treatment	Corrected value		Corrected value
Negative control	2	4	5	5	1		0.000
	3	3	4	4	1		0.000
	6	4	4	3	0		0.000
					0.7*		0.000^+		0.7
Positive control	9	4	45	42	38	37.3	1.131	1.131
	11	4	39	37	33	32.3	1.052	1.052
	12	4	37	36	32	31.3	1.331	1.331
						33.7^		1.171^	51.2
Test item	14	5	10	18	13	12.3	1.063	1.063
	15	4	10	16	12	11.3	1.152	1.152
	16	4	9	19	15	14.3	0.984	0.984
						12.7^		1.066^	28.7

OD = Optical density * = Mean of the post-incubation − pre-treatment values + = Mean permeability ^ = Mean corrected value

Table 2: Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment	Cornea number	Observation
		Post treatment	Post incubation
Negative Control	2	Clear	Clear
	3	Clear	Clear
	6	Clear	Clear
Positive Control	9	Cloudy	Cloudy
	11	Cloudy	Cloudy
	12	Cloudy	Cloudy
Test Item	14	Clear	Clear
	15	Clear	Clear
	16	Clear	Clear

Interpretation of results:

other: No stand-alone prediction of eye irritation can be made under the conditions of the test.

Executive summary:

Method

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS	UN GHS
≤ 3	No Category
>3; ≤ 55	No stand-alone prediction can be made
> 55	Category 1

Results

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	28.7
Negative Control	0.7
Positive Control	51.2

Conclusion

According to UN GHS Classification for the test item N,N-dimethyl dodec-9-enamide, No stand-alone prediction of eye irritation can be made under the conditions of the test.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 - 17 November 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ EIT was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. The EpiOcular™ EIT was endorsed as an in vitro test that can be used to identify those chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS (No Category).
- Test system:
EpiOcularTM Human Corneal Model (0.6 cm2)
A Certificate of Analysis supplied by the supplier is given in Annex 3 (Attached).
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava - Slovakia
Date received: 15 November 2022
EpiOcularTM Tissues Lot Number: 34993
Assay Medium Lot Number: 111422ISA

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Test item: 50 μL
- Positive & negative controls: 50 μL

Duration of treatment / exposure:

30 ±2 minutes

Duration of post- treatment incubation (in vitro):

120 ±15 minutes

Number of animals or in vitro replicates:

2

Details on study design:

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. Therefore, it was necessary to assess this ability of the test item to directly reduce MTT prior to conducting the assay. This property of the test item is only a problem, if at the time of the MTT test (after the test item has been rinsed off) there is still a sufficient amount of the test item present on (or in) the tissues. In this case the (true) metabolic MTT reduction and the (false) direct MTT reduction can be differentiated and quantified by the procedure described as follows:
50 μL of the test item was added to 1 mL of MTT solution and incubated at 37 °C, 5% CO2 for 3 hours. A control (50 μL sterile water in MTT solution) was run concurrently. If the MTT solution turned blue/purple, the test item was presumed to have directly reduced the MTT.
The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of eye irritation potential was performed in parallel on viable and non-viable, freeze-killed, tissues.
This step employs freeze-killed tissues that possess negligible metabolic activity but absorb and bind the test item similar to viable tissues.
Freeze-killed tissues were prepared in-house (outside of the confines of the study) by placing untreated EpiOcularTM tissues in a freezer (-35 to -10 °C) overnight, thawing to room temperature, and then refreezing (two freeze-thaw cycles). Once killed, the tissues may be stored indefinitely in the freezer. Freeze-killed tissues were thawed for approximately 60 minutes at 37 ±2 °C, 5 ±1% CO2 in air before use.
Each MTT reducing test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues remained untreated (the untreated controls show a small amount of MTT reduction due to residual reducing enzymes within the killed tissue). The entire assay was performed on the frozen tissues in parallel to the viable tissues.
If the interference by the test item was ≤ 60% of the negative control value, the net OD of the test item treated killed control may be subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
Data were corrected as follows: True viability = Viability of treated tissue – direct MTT interference from test item = OD tvt – (mean OD tkt – mean OD ukt)
Key:
OD = optical Density at 570 nm
tvt = treated viable tissues
tkt = treated killed tissues
ukt = untreated killed tissues
If the interference by the test item was greater than 60% of the negative control value the test item may be considered incompatible with this test system.

Assessment of Color Interference with the MTT endpoint
Colored test items or those which become colored after application to the tissues may interfere with the quantitative photometric MTT measurement if the colorant binds to the tissue and is extracted together with MTT. Therefore, the test item was checked for its colorant properties.
Test items which absorb light and appear red, yellow, green or blue should be considered as intrinsic colorants. A test item which appears black may absorb light and should be considered as a colorant. Blue, purple and black test items may be directly tested on colorant controls without further tests because it is obvious that they can interfere with the blue/purple MTT product. Such test items should also be tested on killed controls because it may not be possible to assess their potential to directly reduce MTT.
For non-colored test items, tests have to be performed to assess if they become colorants after contact with water or isopropanol. For this purpose 50 μL of the test item was added to 1.0 mL of water in a 6-well plate and the mixture was incubated in the dark at 37 ±1 °C in a humidified atmosphere of 5 ±1% CO2 in air for at least 1 hour. Furthermore, 50 μL of the liquid test item was added to 2 mL of isopropanol, the same amount as used for MTT extraction, incubated in 6 well plates, and placed on an orbital plate shaker for 3 hours at room temperature.

Preparation and Pre-Incubation of EpiOcular Tissues
Upon receipt of the EpiOcularTM tissues, the sealed 24-well plate and the assay medium were placed into the refrigerator (2 to 10 °C) until the equilibration step. The vial containing the MTT concentrate was placed in the freezer (-35 to -10 °C) and the MTT diluent placed in the refrigerator (2 to 10 °C). The positive control, Methyl Acetate, was stored at room temperature, in the dark.
On the day of receipt the equilibration step (15 minutes at room temperature in the 24-well shipping container) was started. An appropriate volume of EpiOcular™ Assay medium was warmed to approximately 37 °C and 1 mL of the medium aliquoted into the appropriate wells of pre-labeled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with ethanol soaked tissue paper. The sterile gauze was removed and each tissue inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping container using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for 1 hour in Assay Medium. After 1 hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 to 24 hours).

Main Test
Application of Test Item and Rinsing
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++ Mg++ free DPBS to mimic the wet condition of the human eye. If the Ca++ Mg++ free DPBS was not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at 37 °C, 5% CO2 for 30 ±2 minutes.
50 μL of test item was applied atop duplicate cultures for an exposure period of 30 ±2 minutes at 37 °C, 5% CO2 followed by rinsing, a post-treatment immersion and a post-treatment incubation (described below). 50 μL of the negative and positive controls were similarly applied.
At the end of the test item exposure period, the test item was removed by extensively rinsing the tissues with Ca++ Mg++ free DPBS at room temperature. Three clean beakers (glass or plastic with minimum 150 mL capacity), containing a minimum of 100 mL each of Ca++ Mg++ free DPBS were used per test item or control with each test item or control item utilizing a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. The tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent paper towel and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The inserts were then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent paper. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent paper (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of assay medium at room temperature in a pre-labeled 12-well plate for a 12 ±2 minutes immersion incubation (post‐treatment immersion) at room temperature. This incubation in assay medium was intended to remove any test item absorbed into the tissue.
At the end of the post‐treatment immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, the insert was blotted on absorbent paper, and transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium at approximately 37 °C. The tissues were incubated for a period of 120 ±15 minutes at 37 °C, 5% CO2 (post-treatment incubation).

MTT Assay
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were placed into the 24-well plate containing 0.3 mL of 1.0 mg/mL MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated at 37 °C, 5% CO2 in air for 3 hours.
Each insert was removed from the 24‐well plate after approximately 3 hours. The bottom of the insert was blotted on absorbent paper and transferred to a pre-labeled 24‐well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol flowed into the insert on the tissue surface. The plates were sealed with a film sealer (between the plate cover and upper edge of the wells) or a standard plate sealer and stored overnight at 2 to 10 °C in the dark. At the end of the extraction period, each tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.

Absorbance/Optical Density Measurements
At the end of the extraction period, using a pipette fitted with a 1000 μL tip, the extraction solution was forced vigorously up and down to thoroughly mix. The tissues and empty inserts were discarded.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. 200 μL of isopropanol alone was added to eight wells designated as ‘blanks’. All wells were examined and any air bubbles were removed. The absorbance at 570 nm (OD570) of each well was measured using the LabTech LT-4500 microplate reader and LT-com analysis software. Servicing, calibration, pass band width and linearity range of the microplate reader is given in Annex 2 (attached).
The plate reader LT-com analysis software was set to correct for blanks and calculate the mean OD570 values of the duplicate wells representing each tissue. The mean OD570 values of the duplicate tissues were manually calculated.

Data Evaluation
The relative mean tissue viabilities were compared to the mean of the negative control (n=2) treated tissues. The relative mean tissue viabilities were calculated as follows:
Relative mean tissue viability = 100x Mean OD570 of test item/Mean OD570 of negative control.

Classification of eye irritancy potential is based on relative viability according to the following table:
Viability Measured after Exposure Time Points/Prediction to be considered according to EU CLP Regulation (EC) No 1272/2008 UN GHS
Mean Tissue Viability >60%/ No Category
Mean Tissue Viability ≤60%/ No prediction can be made*
*If the relative mean tissue viability is ≤60%, differentiation between EU CLP/UN GHS Category 1 and Category 2 is not possible. This is because, in the case of a true positive, this method cannot resolve between UN GHS Categories 1 and 2. Thus further information on serious eye damage/irritation will be required to decide on the final classification. This would be conducted outside the confines of this study.

Acceptability Criteria
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
1) The negative control OD is > 0.8 and < 2.8.
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single group of duplicate tissues is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (test items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: Relative mean viability (%)

Run / experiment:

Test item

Value:

11.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- The relative mean tissue viability for the positive control treated tissues was 37.4% relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.
- The mean OD570 for the negative control treated tissues was 2.210. The negative control acceptance criterion was therefore satisfied.
- The difference in viability between the two relating tissues in each treatment group was <20%. This acceptance criterion was therefore satisfied.

Direct MTT Reduction

The MTT solution containing the test item turned blue. Therefore, an assessment found the test item was able to directly reduce MTT and an additional procedure using non-viable, freeze-killed, tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred in the main test. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes. The results generated from the freeze-killed tissues are given below:

Mean of test item treated killed tissues (tkt) = 0.045 OD₅₇₀

Mean of untreated killed tissues (ukt) = 0.050 OD₅₇₀

The direct reduction by the test item relative to the negative control value:

(0.045 (tkt) – 0.050 (ukt)) / 2.210 (mean of negative control) x 100 = 0.0 %

Assessment of Color Interference with the MTT endpoint

The test item did not become colored in either the water or isopropanol solutions. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD₅₇₀ values and tissue viabilities and the difference in viability for the test item, negative control item and positive control item are given in the table below. The mean viabilities of the test item and positive control, relative to the negative control are also given in the table below.

The relative mean viability of the test item treated tissues was 11.5% after a 30-Minute exposure period and 2-Hour post-exposure incubation period.

Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of duplicate tissues	Individual tissue viability (%)	Relative mean viability (%)	Difference in viability (%)
Negative Control Item	2.171	2.210	98.2	100*	3.6
	2.249		101.8
Positive Control Item	0.741	0.827	33.5	37.4	7.8
	0.913		41.3
Test Item	0.189	0.254	8.6	11.5	5.8
	0.319		14.4

OD = Optical Density

∗ = The mean viability of the negative control tissues is set at 100%

 

Interpretation of results:

study cannot be used for classification

Executive summary:

Introduction

The purpose of this study was to determine if the test item does not require classification and labelling for eye irritation or serious eye damage using the EpiOcular™ Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.

Method

Duplicate tissues were treated with the test item for an exposure period of 30 minutes. At the end of the exposure period each tissue was rinsed before incubating for 120 minutes. As the test item had been shown to directly reduce MTT additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading the tissues was placed into 2 mL of isopropanol for formazan extraction.

At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD₅₇₀).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 11.5%.

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

Conclusion

Mean Tissue Viability was ≤ 60% and therefore no prediction can be made.

In the case of a true positive, this method cannot resolve between UN GHS Categories 1 and 2.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

other: Mol: Russian

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Mollegaard Breeding and Research Centre A/S
- Weight: 2.4-2.6kg
- Housing: animal room 2, filtered Air, ppo cages
- Diet (e.g. ad libitum): ad libitum / Altromin 2123 , Altromin , Lage , Germany
- Water (e.g. ad libitum): ad libitum (acified, HCl)
- Acclimation period: at least one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3°C
- Humidity (%): 55±15%
- Air changes (per hr): 10times/h
- Photoperiod (hrs dark / hrs light):12h each

Vehicle:

unchanged (no vehicle)

Controls:

other: right eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1ml

Observation period (in vivo):

1h, 24h, 48h, 72h, 7d, 14d, 21d

Number of animals or in vitro replicates:

3

Irritation parameter:

cornea opacity score

Remarks:

corne opacity

Basis:

mean

Remarks:

three animals

Time point:

other: mean from 24h, 48h and 72h reading

Score:

<= 1

Max. score:

4

Reversibility:

fully reversible within: 21 days in 2 of 3 animals

Remarks on result:

other: When Fluorescein has been applied only the highest score for degree of corneal opacity from three readings are included

Irritation parameter:

iris score

Remarks:

iris lesion

Basis:

mean

Remarks:

three animals

Time point:

other: mean from 24h, 48h and 72h reading

Score:

<= 0

Max. score:

2

Remarks on result:

other: normal reaction observed

Irritation parameter:

conjunctivae score

Remarks:

redness of conjunctiva

Basis:

mean

Remarks:

three animals

Time point:

other: mean from 24h, 48h and 72h reading

Score:

<= 2.6

Max. score:

3

Reversibility:

fully reversible within: 21 days in 2 of 3 animals

Irritation parameter:

chemosis score

Remarks:

oedema of conjunctiva (chemosis)

Basis:

mean

Remarks:

three animals

Time point:

other: mean from 24h, 48h and 72h reading

Score:

<= 2.8

Max. score:

4

Reversibility:

fully reversible within: 21 days in 2 of 3 animals

Interpretation of results:

other: Category 2

Remarks:

Criteria used for interpretation of results: other: EU GHS EC 1272/2008

Conclusions:

Eye irritant potential

Executive summary:

The eye irritant effect of SAT 970 419 (N,N-Dimethyldecan-1-amide) was investigated according to the method recommended in the OECD Guideline No. 405, "Acute Eye Irritation/Corrosion", Feb. 1987, and EEC Guideline B.5 "Acute Toxicity (Eye Irritation)", 29.12.1992.

Three female albino rabbits were exposed to 0.1 ml of the test article in the left eye. The eyes were examined and the changes were graded according to a numerical scale 1, 24, 48 and 72 hours as well as 7, 14 and in two animals 21 days after dosing.

Moderate to severe signs of eye irritation were observed among the rabbits.

The following mean values, based on the results from the 24, 48 and 72 hour readings, were calculated:

Cornea opacity 1.0

Iris lesion 0.0

Redness of conjunctiva 2.6

Oedema of conjunctiva 2.8

After 14 days all animals showed hairless areas around the eye. Treatment related adverse eye reactions were fully reverse within 14 (1 animal) and 21 days.

Based on the result of this study the test substance is classified as eye irritant

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin corrosivity:

The skin corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

Duplicate tissues were treated with the negative control, positive control and test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the control items and test item were rinsed from the tissues before each tissue was taken for MTT-loading. After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200µL samples were transferred to the appropriate wells of a pre-labeled 96 -well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	2.9	104.8
60 minute	100*	2.2	81.2

*The mean viability of the negative control tissues is set at 100%

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

In vitro skin irritation:

The skin irritation potential of the test item was evaluated using the EpiDerm™ Skin Irritation Test (SIT) according to the OECD Test Guideline 439 In Vitro Skin Irritation Reconstructed Human Epidermis (RHE) and the synonymous EU B.46. test method.

The test item was applied topically for a treatment period of 60 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in RHE cultures following topical exposure to the test item by means of the colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue/purple formazan dye in the test item treated tissues relative to the negative control.

Triplicate tissues were treated with the test item for an exposure period of 60 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction. At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 3.2% after the 60-Minute exposure period and 42-Hours post-exposure incubation period.

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

This study alone does not allow the conclusion on whether the test item is a corrosive or an irritant (EU CLP/UN GHS Category 1 or Category 2). However, per the results of an OECD 431 In Vitro Skin Corrosion study (Reconstructed Human EpiDermis (RHE) Test Method; Labcorp Study Number: 8484301), the test item was considered to be non-corrosive. Thus, based on the results of this study and the results of the OECD 431 In Vitro Skin Corrosion study, the test item was considered to be an irritant and the following classification criteria apply: EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2.

In vitro eye irritation:

The eye irritation potential of the substance was evaluated in an in vitro study performed according to OECD TG 437.

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	28.7
Negative Control	0.7
Positive Control	51.2

According to UN GHS Classification for the test item N,N-dimethyl dodec-9-enamide, No stand-alone prediction of eye irritation can be made under the conditions of the test.

A second study, using the EpiOcular™ Eye Irritation Test (EIT) according to the OECD TG 492 Reconstructed human Cornea-like Epithelium (RhCE) test method, is also available.

In this study duplicate tissues were treated with the test item for an exposure period of 30 minutes. At the end of the exposure period each tissue was rinsed before incubating for 120 minutes. As the test item had been shown to directly reduce MTT additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading the tissues was placed into 2 mL of isopropanol for formazan extraction.

At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD₅₇₀).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 11.5%.

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

Mean Tissue Viability was ≤ 60% and therefore no prediction can be made.

In the case of a true positive, this method cannot resolve between UN GHS Categories 1 and 2.

In vivo eye irritation:

A supporting study is available for an analogue substance, N,N-Dimethyldecan-1-amide (CAS No. 14433-76-2) which is included to assist with determination of classification and labelling at the Annex VII tonnage band.

In this study, performed according to OECD TG 405 and EC Method B.5, three female albino rabbits were exposed to 0.1 ml of the test article in the left eye. The eyes were examined and the changes were graded according to a numerical scale 1, 24, 48 and 72 hours as well as 7, 14 and in two animals 21 days after dosing. Moderate to severe signs of eye irritation were observed among the rabbits. The following mean values, based on the results from the 24, 48 and 72 hour readings, were calculated: Cornea opacity 1.0; Iris lesion 0.0; Redness of conjunctiva 2.6; Oedema of conjunctiva 2.8.After 14 days all animals showed hairless areas around the eye. Treatment related adverse eye reactions were fully reverse within 14 (1 animal) and 21 days.

Justification for classification or non-classification

Skin irritation/corrosion:

Based on the results of an OECD TG 431 study (not corrosive) and an OECD 439 study, the substance is considered to be an irritant and the following classification criteria apply:

EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2.

Eye irritation:

The results of the in vitro eye irritation tests (OECD TG 437, OECD TG 492) indicate that no prediction on classification can be made on the basis of these studies alone. As the substance is classified as a Skin irritant, Category 2 and the pH in solution is reported as 5 (Log Pow study) there is no automatic requirement to consider a Category 1 classification. Taking into account the result of the supporting in vivo study on N,N-Dimethyldecan-1-amide (CAS No. 14433-76-2) a classification of 'Category 2 eye irritant' (DSD Xi R36) is appropriate based on available information. This will be reviewed at the next tonnage band.