1-methylheptyl acrylate

Endpoint summary

Description of key information

The substance was found to be non-irritant when tested in in-vitro human skin and rabbit eyes models according to OECD guidelines 439 and 437 respectively.

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:

16 August 2017 - 06 September 2017

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)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

other: human skin model reconstructed human epidermis

Source species:

human

Cell type:

other: EpiskinTM Model Kit (0.38 cm2 tissues)

Cell source:

other: EpiSkin, Lyon, France

Details on animal used as source of test system:

not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: several
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: no data
- Wavelength: 570 nm
- Filter: no data
- Filter bandwidth: no data

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
SCORING SYSTEM:
- Optical density (OD) was measured at 570 nm:
Relative mean viability (%) = 100 x mean cOD(test item or positive control) / mean cOD(negative control)
where:
- mean cOD Negative Control = mean ODNC – mean ODblank
- mean cOD Test Item = mean ODTI – mean ODblank
- mean cOD Positive Control = mean ODPC - mean ODblank
DECISION CRITERIA
Mean relative viability is = or < 50%: category 2 (H315)
Mean relative viability is > 50% : no category but the concentration of the inflammatory mediator interleukine 1 alpha (IL-1a) is evaluated from the culture medium retained following the 42 hours recovery period. This quantification, based on an ELISA assay is performed in order to confirm a non-irritant result or to override the non-irritant result (see table 7.3.1/1 in Any Other Information on Materials and Methods).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: 100%

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5% w/v in water

Duration of treatment / exposure:

Exposure period of 15 minutes, following rinsing.

Duration of post-treatment incubation (if applicable):

MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells

Number of replicates:

Triplicate tissues for each tested substance (test item, negative control and positive control).

Species:

other: in vitro test

Irritation / corrosion parameter:

% tissue viability

Remarks:

15 min

Run / experiment:

Mean

Value:

97

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: IL1a release

Run / experiment:

Mean

Value:

20.2

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no abnormality noted
- Direct-MTT reduction:The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties.
- Colour interference with MTT:as the water solution containing the test item did not change colour, the test item was found not to have a colouring potential.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean cOD of the three negative controls should be equal or above 0.6 and the Standard Deviation (SD) value of the % viability should be equal or below 18%,
- Acceptance criteria met for positive control: the relative mean viability of the positive control should be equal or below 40% of the relative mean viability of the negative control and the SD value of the % viability should be equal or below 18%.

7.3.1/2 Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

Group	Tissue n°	OD measurements		Mean OD blank	cOD			Mean cOD	Viability (%)
		1st	2nd		1st		2nd
Negative control	1	1.012	0.997		0.972	0.957		0.964	99
	2	1.001	1.012	0.041	0.961	0.972		0.966	99
	3	1.048	1.018		1.008	0.978		0.993	102
Positive control	1	0.087	0.087		0.047	0.047		0.047	5
	2	0.132	0.135	0.041	0.092	0.095		0.093	10
	3	0.090	0.089		0.050	0.049		0.049	5
Test item	1	1.019	1.019		0.979	0.979		0.979	100
	2	0.901	0.900	0.040	0.861	0.860		0.861	88
	3	1.019	1.045		0.979	1.005		0.992	102

OD= Optical Density

cOD= blank Corrected Optical Density

7.3.1/3 Mean tissue viability and standard deviation for the test item, the negative and positive controls

Group	cOD		Viability (%)
	Mean	SD	Mean	SD
Negative control	0.974	0.016	100	2
Positive control	0.063	0.026	6	3
Test item	0.944	0.072	97	7

 

SD= Standard Deviation

cOD= blank Corrected Optical Density

7.3.1/4 Determination of IL-1a concentration (pg/mL) in Episkin samples

Sample identification	IL-1a measured concentration (pg/mL)	Mean IL-1a measured concentration (pg/mL) n=3 tissues	Inter tissue %CV, n =3 tissues
Negative control 1	BLQ	NC	NC
Negative control 2	5.81
Negative control 3	10.8
Test sample 1	14.4	20.2	26.1
Test sample 2	24.7
Test sample 3	21.4

% CV : Coefficient of Variation

BLQ : Below Limit of Quantification (<5.00 pg/mL)

NC: Not Calculated

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is considered to be non-irritant to skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test substance using the EPISKIN^TM reconstructed human epidermis model. The study was performed according to OECD guideline 439 in compliance with Good Laboratory Practice.

Preliminary tests were performed to detect the ability of the test substance to directly reduce MTT as well as its colouring potential. following these preliminary tests, triplicate tissues (Episkin epidermis surface area of 0.38 cm²) were treated with 10 µL of the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed to remove residual test substance before incubating for 42 hours at 37°C in a humidified atmosphere of 5% CO2 in air. At the end of the post-exposure incubation period each tissue was taken for MTT-loading (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm with acidified isopropanol solution as a blank.

In addition, the concentration of the inflammatory mediator IL-1a was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

Negative ( Dulbecco's Phosphate Buffered Saline (DPBS)) and positive controls (5% sodium dodecyl sulphate (SDS) in distilled water) were used in the study. The mean absorbance of the triplicate negative control values was 0.974 which was above the minimum acceptance value of 0.6. The standard deviation (SD) of the % viability was 2 which was below the maximum acceptance value of 18. The mean % viability of the positive control was 6 +/- 3 of the negative control. This was below the maximum acceptance values of 40% viability and SD of 18. Therefore the study was considered as valid.

The relative mean viability of the test item treated tissues was 97 +/- 7 % after the 15-minute exposure period. As the mean viability was above 50% after MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The mean IL-1a concentration for treated tissues was 20.2 pg/mL. Due to this value being below 60 pg/mL, the results met the criteria for a non-irritant response.

 

 

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:

06 June 2017 - 13 September 2017

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 July 2013

Deviations:

yes

Remarks:

see table 7.3.2/1 in Any Other Information on Materials and Methods

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine cornea

Details on test animals or tissues and environmental conditions:

Species: bovine cattle

Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
Age: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).

Reason for choice: bovine corneas are recommended by Regulatory Authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.

Transport from Supplier to laboratory: the eyes were transported to the laboratory at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas. Upon arrival at the laboratory, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were rinced in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature.
The corneas were used immediately.

(Pre)Incubation T°C: 32°C

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: undiluted

VEHICLE
- none

Duration of treatment / exposure:

Exposure period of 10 minutes (± 30 seconds) at +32°C, followed by rinsing.

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes) at +32°C

Number of animals or in vitro replicates:

The test item and the negative control were tested on three corneas each.
The positive control was tested on two corneas, instead of three.

Details on study design:

REMOVAL OF THE TEST SUBSTANCE
On completion of the treatment period, the test item was removed from the front opening of the anterior chamber and the epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- residual test item adhering to the walls of the anterior chamber was removed with a cotton bud,
- the corneas were rinsed four times with pre-warmed cMEM containing phenol red (i.e. until the test item had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.
Some difficulties were encountered since residual amounts of test item were observed on the walls of the anterior chamber, but they were removed at the end of the rinsing step.
- Time after start of exposure: 10 minutes

SCORING SYSTEM / TOOL USED TO ASSESS SCORE:
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values of positive control and test item treated corneas.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values of positive control and test item treated corneas.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

53

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

0.3

Negative controls validity:

valid

Remarks:

0.0

Positive controls validity:

valid

Remarks:

18.0

Irritation parameter:

fluorescein leakage

Run / experiment:

mean

Value:

0.062

Negative controls validity:

valid

Remarks:

0.023

Positive controls validity:

valid

Remarks:

2.315

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
No notable opaque spots or irregularities were observed on all negative control-treated corneas.
Fluorescein fixation was observed on the three corneas treated with the test item.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (Negative control: opacity = 0.0 +/- 0.0; permeability = 0.023 +/- 0.005, Upper limit of the historical mean for opacity and permeability 3 and 0.036 respectively)
- Acceptance criteria met for positive control: yes ( IVIS = 53 +/-1.7 The positive control IVIS was within the range of historical mean +/- 2 SD: 27 - 63).

Table 7.3.2/3: Individual and Mean Corneal Opacity and Permeability Measurements

 

Treatment	Cornea number	OPACITY				PERMEABILITY		In Vitro Irritancy Score (IVIS)
Negative control		Pre-Treatment	Post-Treatment	Change Post- Pre-Treatment	Corrected Value	OD490 nm	Corrected Value
	71	2	1	-1	0	0.024	0.024
	77	2	1	-1	0	0.018	0.018
	74	2	1	-1	0	0.027	0.027
	Mean				0.0		0.023
	SD				0.0		0.005
Test item	Cornea number	Pre-Treatment	Post-Treatment	Change Post- Pre-Treatment	Corrected Value	OD490 nm	Corrected Value	In Vitro Irritancy Score (IVIS)
	85	2	3	1	1	0.010	0.010	1
	82	3	2	-1	0	0.155	0.132	2
	67	3	3	0	0	0.078	0.055	1
	Mean				0.3		0.062	1
	SD				0.6		0.066	0.6
Positive control	Cornea number	Pre-Treatment	Post-Treatment	Change Post- Pre-Treatment	Corrected Value	OD490 nm	Corrected Value	In Vitro Irritancy Score (IVIS)
	59	2	23	21	21	2.060	2.037	52
	62	2	17	15	15	2.616	2.593	54
	Mean				18.0		2.315	53
	SD				4.2		0.393	1.7

OD= Optical Density

SD: Standard Deviation

Negative control: 0.9% NaCl

Positive control: 100% Ethanol

 

Interpretation of results:

GHS criteria not met

Conclusions:

the test substance is considered as not requiring classification for eye irritation or serious eye damage.

Executive summary:

A study was performed to assess the ocular irritancy potential of the test substance to the isolated bovine cornea. The study was conducted according to the OECD guideline No. 437 and in compliance with the principles of Good Laboratory Practice .Negative (Sodium chloride 0.9%) and positive (Ethanol 100%) control items were tested concurrently. 

A single experiment was performed using three corneas for test substance and negative control; and two corneas for positive control.

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test substance, applied undiluted, the negative and the positive controls were evaluated in a single experiment using a treatment time of 10 minutes and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours at +32 °C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated for 90 minutes at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined to generate an In Vitro Irritancy Score (IVIS). 

To consider the study as valid, the positive control should elicit an In Vitro Score that falls within two standard deviations of the historical mean for the laboratory, which was the case; IVIS of ethanol was found to be 53 +/- 1.7. Furthermore, the negative control mean opacity and mean OD490nm should be less than the established upper limit of historical means which was the case as opacity of sodium chloride 0.9% was 0.0 +/- 0.0 and historical value was 3.00 while the permeability of sodium chloride 0.9% was 0.023 +/-0.005 and historical value was 0.036.

 IVIS of the test substance was determined to be 1 +/- 0.6, which was below the cut-off value of 3 and thus indicating that the test substance was not requiring classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Several in vitro tests were available to assess the skin and eye irritation potential of the registered substance.

In the key study, the skin irritation potential of the registered substance was assessed using the EPISKIN^TMreconstructed human epidermis model. The test procedure was equivalent to the one described in the OECD guideline 439 and the study was performed in compliance with the GLP. Triplicate tissues (Episkin epidermis surface area of 0.38 cm²) were treated with 10 µL of the test substance for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed to remove residual test substance before incubating for 42 hours at 37°C in a humidified atmosphere of 5% CO2 in air. At the end of the post-exposure incubation period each tissue was tested for viability using a MTT test. Negative (sterile Dulbecco's Phosphate Buffered Saline (DPBS)) and positive controls (5% sodium dodecyl sulphate (SDS) in distilled water) were used in the study and gave appropriate results. Therefore the study was considered as valid.

The relative mean viability of the test item treated tissues was 97 +/- 7 % after the 15-minute exposure period. The test material was therefore considered to be non-irritant which was confirmed by the IL-1a concentration measurement in the culture media samples. The mean IL-1a concentration analyzed by ELISA was 20.2 pg/mL. This value below 60 pg/mL confirmed that the substance was not a skin irritant.

The non-ocular irritant potential of the registered substance was assessed using a BCOP (Bovine Corneal opacity and permeability) assay. The study was conducted according to the OECD guideline No. 437 and in compliance with GLP. Negative (Sodium chloride 0.9%) and positive (ethanol 100%) control items were tested concurrently and gave appropriate response. Therefore the study was considered as valid. 

The test substance was applied undiluted on each cornea (3 corneas/treatment condition). The corneas were incubated for 4 hours at 32°C. Following incubation, the test substance, positive and negative controls were removed and the epithelial surface of the cornea was washed.

The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined to generate an In Vitro Irritancy Score (IVIS). IVIS of the test substance was determined to be 1 +/- 0.6 which was below the cut-off value of 3 and thus indicating that the test substance was not requiring classification for eye irritation or serious eye damage.

Justification for classification or non-classification

Based on the results from in vitro studies, the substance is not classified as a skin or eye irritant according to regulation EC No. 1272/2008and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.