2,6-dimethyloct-7-en-2-yl formate

Endpoint summary

Description of key information

Skin irritation: Key study: OECD TG 439. GLP study. Tissue viability after exposure and post-treatment incubation was 27.6%, i.e. less that 50%. Therefore, the test item is irritating to the skin (Category 2).

Key study: OECD TG 431. GLP study. The test item is not corrosive to the skin (viability = 92.7 and 105.3% after 3 and 60 min exposure).

Eye irritation: Key study: OECD TG 437. GLP study. The IVIS score for the corneas treated with test item was found to be 0.87 (No category according to OECD 437). Thus, the test item was determined to be not irritating to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2018 - 07 May 2018

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This study addresses the human health endpoint skin irritation. It makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RhE) is also recommended by the OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies. The results of the study are believed to be of value in predicting the potential of inducing skin irritation by the test item in humans.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model
- Tissue batch number(s): N°18-RHE-029

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 times in a constant soft stream of 1 mL DPBS from 5-8 cm distance from the insert.
- Observable damage in the tissue due to washing: No.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 180 minutes
- Wavelength: 570 nm

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 is considered to be irritant to skin, if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
The test item is considered as non-irritant to skin, if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

16 ± 2 mg of test item/0.5 cm2

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 (mean of 3 replicates)

Value:

27.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: Test item did not produce direct MTT reduction when compared to concurrent negative control (Maintenance meduim).
- Colour interference with MTT: Difference in absorbance due to color interference was not observed between negative control (isopropanol) and dihydromyrcenyl formate. The test item did not form color in isopropanol, therefore results shows no interference in OD due to test item.

DEMONSTRATION OF TECHNICAL PROFICIENCY: JRF Study Number: 618-1-06-9641

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The data met the acceptance criteria since the mean OD value of the 3 tissues was ≥ 1.2 at 570 ± 30 nm according to the historical database. The Standard Deviation value is considered as valid if it is ≤ 18%. The OD values (Corrected ODs) of negative controls in all tissues were between 1.221 to 1.273

- Acceptance criteria met for positive control:
The data met the acceptance criteria since the mean viability, expressed as % of the NC, was < 40 % and the Standard Deviation value is ≤ 18 %. Mean % viability of the positive control was 1.5%

- Acceptance criteria met for variability between replicate measurements:
Standard deviation of each intra-batch mean (3 Replicates/Tissue and 3 Tissue/Run) was < 18%.

- Range of historical values if different from the ones specified in the test guideline:
Optical Density at 570±30 nm
Exposure time: 42-minute exposure time
Negative Control (Dulbecco's phosphate-buffered saline):
Mean: 2.081
Standard deviation: 0.208
Minimum: 1.964
Maximum:2.776
Positive Control (Sodium dodecyl sulfate, 5% aqueous)
Mean: 0.028
Standard deviation: 0.007
Minimum: 0.021
Maximum: 0.053

Data summary of percent viability:

Treatment	Tissue Replicate	O.D. at 570 nm	Blank Corrected O.D.	Mean of Corrected O.D.	Mean O.D. of Three Tissues	% Viability/ Tissue	Mean % Viability	S.D. of % Viability	C.V. of % Viability	Corrosivity Class
Negative Control (Dulbecco’s Phosphate Buffered Saline (DPBS))	1	1.274	1.231	1.238	1.238	100	100	0.01	0.81	NA
		1.282	1.239
		1.287	1.244
	2	1.280	1.237	1.231
		1.264	1.221
		1.277	1.234
	3	1.270	1.227	1.244
		1.276	1.233
		1.316	1.273
Dihydromyrcenyl Formate	1	0.361	0.319	0.336	0.342	27.1	27.6	0.61	2.21	Category 2
		0.384	0.342
		0.388	0.346
	2	0.381	0.339	0.34		27.5
		0.384	0.342
		0.382	0.340
	3	0.395	0.353	0.35		28.3
		0.388	0.346
		0.393	0.351
Positive control (Sodium dodecyl sulphate (5% aq.))	1	0.061	0.018	0.018	0.018	1.5	1.5	0	0.00	Category 2
		0.061	0.018
		0.060	0.017
	2	0.062	0.019	0.019		1.5
		0.062	0.019
		0.061	0.018
	3	0.061	0.018	0.018		1.5
		0.061	0.018
		0.061	0.018

Keys: O.D. = Optical Density, S.D. = Standard Deviation, C.V. = Coefficient of Variation, NA = Not Applicable

Note: For Negative control, SD and CV of % viability was calculated using corrected OD at 570 nm and for the test item and positive control SD and CV of % viability was calculated using % viability/tissue."

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The substance was determined to be irritating to the skin.

Executive summary:

An in-vitro Skin Irritation test with Reconstructed Human Epidermis (RHE) Tissues was performed according to the OECD Guideline 439 (GLP study). Tissues were exposed to the negative control (Dulbecco’s Phosphate Buffered Saline (DPBS)), positive control (sodium dodecyl sulfate, 5% aqueous (SDS)) and test item in triplicate for 42 minutes at room temperature. The mean cell viability in tissues treated with the test item was 27.6% after 42 minutes exposure. A significant reduction in percent cell viability was observed in treated tissues when compared with the concurrent negative control. The Optical density (OD) values for the negative control replicates were between 1.221 to 1.273, against the guideline requirement of ≥ 0.8 and ≤ 3.0 (≥ 1.2 as per SkinEthic SOP). The OD of the blank was between 0.041 to 0.045 which met the guideline requirement of OD < 0.1. The positive control showed a 1.5% cell viability, against the acceptance criteria of <40% for the SkinEthic RHE model, compared to concurrent negative control. Variation between tissue replicates (i.e. CV% value) was 4.54% for the test item group, 0.00% for positive control and 0.81% for negative control against the guideline requirement of ≤ 18%, which demonstrate the efficiency of the test system, SkinEthicTM RHE model. All criteria for a valid study were met. Based on these results, the test item was determined to be irritating to the skin (category 2).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 July 2018 - 14 July 2018

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:

This study addresses the human health endpoint skin corrosion. It makes use of reconstructed human epidermis (RHE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RhE) is also recommended by the OECD and other regulatory authorities. SkinEthic™ RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies. The results of the study are believed to be of value in predicting the potential of inducing skin corrosivity by the test item in humans.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE model
- Tissue batch number(s): N°18-RHE-077

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ± 1 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 20 times in a constant soft stream of 1 mL DPBS from 5-8 cm distance from the insert.
- Observable damage in the tissue due to washing: No.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 180 minutes
- Wavelength: 570 nm

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 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:

40 μL of test item/0.5 cm2

Duration of treatment / exposure:

3 minutes / 1 hour

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure (mean of 3 replicates)

Value:

92.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure (mean of 3 replicates)

Value:

105.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

0.24%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: Test item did not produce direct MTT reduction when compared to concurrent negative control (Maintenance meduim).
- Colour interference with MTT: Difference in absorbance due to color interference was not observed between negative control (isopropanol) and dihydromyrcenyl formate. The test item did not form color in isopropanol, therefore results shows no interference in OD due to test item.

DEMONSTRATION OF TECHNICAL PROFICIENCY: JRF Study Number: 616-1-06-9542

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The data met the acceptance criteria since the mean OD value of the 3 tissues was ≥ 0.8 and ≤ 3.0 for each exposure time.
- Acceptance criteria met for positive control: Yes. The data met the acceptance criteria since the mean viability of the PC, expressed as % of the NC, was < 15 %.
- Acceptance criteria met for variability between replicate measurements: Standard deviation of each intra-batch mean (3 Replicates/Tissue and 3 Tissue/Run) was < 30%.

- Range of historical values if different from the ones specified in the test guideline (OD at 560 nm):
Negative Control (Distilled water), 3-min exposure: mean = 2.375, SD = 0.264, min = 1.610, max = 2.594
Negative Control (Distilled water), 3-min exposure: mean = 2.470, SD = 0.224, min = 1.806, max = 2.693
Positive Control (8N KOH), 60-min exposure: mean = 0.061, SD = 0.072, min = 0.004, max = 0.284

Data summary of percent viability:

Treatment	Exposure Time	Tissue Replicate	O.D.	Corrected O.D.	Mean of Corrected O.D.	Mean O.D. of Replicate tissues	% Viability/ Tissue	Mean % Viability	S.D. of % Viability	% C.V. of % Viability	Corrosivity Class
Test item	3 Minutes	1	1.009	0.966	0.970	0.969	92.80	92.70	0.115	0.12	Non-corrosive
			1.015	0.972
			1.016	0.973
		2	1.008	0.965	0.968		92.60
			1.013	0.970
			1.013	0.970
		3	1.017	0.974	0.970		92.80
			1.012	0.969
			1.009	0.966
	60 Minutes	1	1.076	1.033	1.030	1.028	105.50	105.30	0.208	0.20
			1.070	1.027
			1.072	1.029
		2	1.070	1.027	1.029		105.40
			1.076	1.033
			1.069	1.026
		3	1.072	1.029	1.026		105.10
			1.063	1.020
			1.073	1.030

Treatment	Exposure Time	Tissue Replicate	O.D.	Corrected O.D.	Mean of Corrected O.D.	Mean O.D. of Replicate tissues	% Viability/ Tissue	Mean % Viability	S.D. of % Viability	% C.V. of % Viability	Corrosivity Class
Negative Control (Sterile Distilled water)	3 Minutes	1	1.081	1.038	1.043	1.045	100	100	0.002	0.19	NA
			1.066	1.023
			1.112	1.069
		2	1.085	1.042	1.045
			1.093	1.050
			1.085	1.042
		3	1.086	1.043	1.046
			1.089	1.046
			1.092	1.049
	60 Minutes	1	1.010	0.967	0.973	0.976	100	100	0.004	0.41
			1.026	0.983
			1.011	0.968
		2	1.017	0.974	0.981
			1.034	0.991
			1.022	0.979
		3	1.035	0.992	0.975
			1.008	0.965
			1.012	0.969
Positive Control (8N KOH)	60 Minutes	1	0.045	0.002	0.002	0.002	0.20	0.24	0.064	26.67	Corrosive
			0.045	0.002
			0.045	0.002
		2	0.047	0.004	0.003		0.31
			0.046	0.003
			0.046	0.003
		3	0.045	0.002	0.002		0.20
			0.045	0.002
			0.045	0.002

Key: O.D. = Optical Density, S.D. = Standard Deviation, C.V. = Coefficient of Variation, NA = Not Applicable

Note: For Negative control, SD and CV of % viability was calculated using corrected OD at 570 nm and for the test item and positive control SD and CV of % viability was calculated using % viability/tissue."

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The substance was determined to be non-corrosive to the skin.

Executive summary:

An in-vitro Skin Corrosion test with Reconstructed Human Epidermis (RHE) Tissues was performed according to the OECD Guideline 431 (GLP study). SkinEthic™ RHE tissues were exposed to the test item for 3 minutes at room temperature and 60 minutes at 37ºC in an incubator with 5% CO2. Negative (distilled water) and positive (8N KOH, 60 min) controls were run in parallel. Three replicates units per group and time point were used. Exposure of the test item was terminated by rinsing with 20 x 1 mL of DPBS. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals with isopropanol overnight and measuring the concentration of formazan by determining the OD at 570 nm. All criteria for a valid study were met. Under test conditions, the mean corrected percent viability of the treated tissues was 92.7 and 105.3 % after 3 and 60 minutes exposure, respectively, versus 0.24% in the positive control. From results of this study, it is concluded that the test item is non-corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

02 January 2018 - 20 January 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Cow

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra
- Characteristics of donor animals (e.g. age, sex, weight): Between 1 to 5 years
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported (in a sealed plastic container) under cold condition in Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL]
- Time interval prior to initiating testing: The eyes were used within 24 hours from slaughter
- indication of any existing defects or lesions in ocular tissue samples: Corneas from eyes free of visible defects were used.

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): 750 µL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes were examined prior to use. Corneas from eyes free of visible defects were used. Corneas that have opacity lesser than seven opacity units or equivalent for the opacitometer were used in the study.

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : Yes (nomral saline)

POSITIVE CONTROL USED : Yes (dimethylformamide)

APPLICATION DOSE AND EXPOSURE TIME : 750 µL of test item; corneas were exposed for approx. 10 min.

TREATMENT METHOD: Open chamber
A volume of 750 μL was introduced into the anterior chamber through the dosing holes on the top surface of the chamber. The anterior compartment was then plugged. Post application of the test item the holders were turned to a horizontal position and slightly rotated to ensure uniform covering of the test item over the cornea.

POST-INCUBATION PERIOD: yes (2 h)
Once the medium was free of test item, the corneas were given a final rinse with EMEM (without phenol red). Media in the anterior and the posterior chamber was removed and fresh EMEM (without phenol red) was filled. The compartments were plugged and post treatment opacity of each cornea was recorded. Once the medium was free of test item, the corneas were given a final rinse with EMEM (without phenol red). Anterior chamber was then refilled with fresh EMEM without phenol red. After rinsing, the corneas were incubated for an additional period of approximately 2 hours ± 10 minutes at 32 ± 1ºC. At the end of post-exposure incubation period the media in the anterior and the posterior chamber were removed and fresh EMEM (without phenol red) was filled. The compartments were plugged and post treatment opacity of each cornea recorded.

After opacity measurement, the medium was removed from the anterior chamber and was filled with 1 mL of 4 mg/mL fluorescein solution whilst the posterior chamber was filled with fresh EMEM (without phenol red). The holders were then incubated in a horizontal position for approximately 90 ± 5 min at 32 ± 1 ºC. After the incubation the medium in the posterior chamber was transferred into labelled tubes. An aliquot of the medium from the posterior chamber was transferred to a 96-well plate. The plate reader was set to read at OD490. A reading of EMEM (without phenol red) was also recorded which served as blank.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Once, until no visual evidence of test item.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: passage of sodium fluorescein dye (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

0.34

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

1.31

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

0.96

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean IVIS score for the corneas treated with test item was found to be 0.87.

OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: JRF Study nº 530-01-01-10123 (2015)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (mean IVIS = -0.49)
- Acceptance criteria met for positive control: Yes (mean IVIS = 160.97)
- Range of historical values if different from the ones specified in the test guideline:
Dimethylformide (positive control): Mean ± SD = 120.37 ± 30.51 (min 58.94, max 165.18)

Corneal Opacity

The mean final opacity values for dihydromyrcenyl formate treated eyes (0.68) did not show any observable increase in comparison to the control group (-0.78). An observable marked increase in final mean opacity was observed in the corneas treated with N,N-dimethylformamide (124.96).

Corneal Permeability

The mean final corneal permeability values for dihydromyrcenyl formate treated eyes (0.012) did not show any observable increase in comparison to the control group (0.019). An observable marked increase in mean final corneal permeability was observed in the corneas treated with N,N- dimethylformamide (2.400).

In vitro Irritancy Score (IVIS)

The mean In-Vitro Irritancy Score (IVIS) of normal saline (control) and N,N-dimethylformamide (positive control) treated corneas were found to be -0.49 and 160.97 which confirmed the reliability of the test procedure.

The mean IVIS score for the corneas treated with dihydromyrcenyl formate was found to be 0.87.

Dihydromyrcenyl Formate (0.75 mL)
Cornea holder N°	Io  (LUX)	I (Initial) (LUX)	Initial Opacity Value	I (Post Treatment) (LUX)	Post Treatment Opacity Value	Corr. Opacity Value	Final Opacity Value	OD490 Value	Corr. OD490Value	Final OD490 Value	IVIS
20	1083	1019	2.92	1029	2.51	-0.41	0.37	0.065	0.017	-0.002	0.34
21	1103	1011	4.05	1011	4.05	0.00	0.78	0.102	0.054	0.035	1.31
22	1112	1061	2.34	1058	2.46	0.12	0.90	0.071	0.023	0.004	0.96
Mean							0.68	-	0.031	0.012	0.87
SD							0.28	-	0.020	0.020	0.49

Normal Saline (0.75 mL)
Cornea Holder N°	Io  (LUX)	I (Initial) (LUX)	Initial Opacity Value	I (Post Treatment) (LUX)	Post Treatment Opacity Value	Corr. Opacity Value	OD490 Value	Corr. OD490 Value	IVIS
13	1112	1054	2.61	1053	2.65	0.04	0.060	0.012	0.22
14	1067	961	4.82	1021	2.22	-2.60	0.091	0.043	-1.96
15	1052	990	2.92	985	3.13	0.21	0.051	0.003	0.26
Mean						-0.78	-	0.019	-0.49
SD						1.58	-	0.021	1.27

N,N-Dimethylformamide (0.75 mL)
Cornea holder N°	Io  (LUX)	I (Initial) (LUX)	Initial Opacity Value	I (Post Treatment) (LUX)	Post Treatment Opacity Value	Corr. Opacity Value	Final Opacity Value	OD490 Value	Corr. OD490 Value	Final OD490 Value	IVIS
17	1088	1016	3.25	233	146.62	143.37	144.15	1.908	1.860	1.841	171.77
18	1094	969	5.56	287	112.45	106.89	107.67	3.385	3.337	3.318	157.44
19	1103	1024	3.50	266	125.79	122.29	123.07	2.108	2.060	2.041	153.69
Mean							124.96	-	2.419	2.400	160.97
SD							18.31	-	0.801	0.801	9.54

Keys: IVIS = In Vitro Irritation Score, Io =Baseline Reading (With medium but without cornea), I = LUX Reading with Medium and Cornea, OD₄₉₀= Optical Density at 490 Wave Length, - = Not Applicable, Corr. = Corrected. Blank OD₄₉₀value = 0.048.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was determined to be not irritating to the eye.

Executive summary:

An in-vitro eye irritation bovine corneal opacity and permeability test was performed according to OECD Guideline 437 (GLP study). Three sets, each consisting of three corneas were tested. The first set served as control and was treated with 750 μL normal saline. The second set served as positive control and was treated with 750 μL dimethylformamide with the third set treated with 750 μL test item. Post application the corneas were incubated for approximately 10 minutes after which the test item was washed-off the cornea and the corneas kept in incubation for approximately 2 h at 32 ± 1 ºC. At the end of the incubation period opacity readings were taken. Post opacity permeability reading was measured by applying 1 mL of fluorescein sodium solution (4 mg/mL) on to the anterior surface of the cornea and was incubated for approximately 90 min at 32 ºC. At the end of the incubation period the Optical Density (OD) was measured at 490 nm for the fluid collected from the posterior chamber. The mean In-Vitro Irritancy Score (IVIS) of normal saline (control) and dimethylformamide (positive control) treated corneas were found to be -0.49 and 160.97 which confirmed the reliability of the test procedure. The IVIS score for the corneas treated with test item was found to be 0.87 (No category according to OECD 437). Thus, the test item was determined to be not irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation: Based on available data, the substance is classified as Skin Irritant, Category 2 (H315) according to the CLP Regulation (EC) No. 1272/2008.

Eye irritation: Based on available data, the substance is not classified for Eye Damage according to the CLP Regulation (EC) No. 1272/2008.