2-methyldecan-1-al

Endpoint summary

Description of key information

Skin Corrosion: non-corrosive; OECD 431; Anon, 2017

Skin Irritation: skin irritant (GHS Category 2); OECD 439; Anon, 2017

Eye Irritation: non-irritant; OECD 437; Anon, 2016

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:

06 Jan 2017 - 02 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014

Deviations:

yes

Remarks:

For the MTT Assay: the formazan salt was extracted in the refrigerator instead of at room temperature. No impact on study outcome. 50µL test item used in MTT reduction/colour intereference AND test substance exposure, rather than 30µL

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Epi-200 kits and MTT-100 assays purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes, cultured to form a multilayered, highly differentiated model of the human epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: At least 1 hour before dosing, EpiDerm™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. A 24-well plate was prepared as holding plate containing 300 µL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.
- Quality control for skin discs: Robustness of the test system (or rather of the test kit) was demonstrated following treatment with 1% Triton X-100: 4.77 hours ≤ ET50 ≤ 8.72 hours

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Rinsed 20 times
- Observable damage in the tissue due to washing: N/A
- Modifications to validated SOP: N/A

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)
- Wavelength: 570 nm
- Filter: no reference filter
- Filter bandwidth: N/A

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Two

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin as follows: < 50% mean tissue viability after 3 minutes exposure and < 15% after 60 minutes exposure results in a prediciton of corrosivity.
- The test substance is considered to be non-corrosive to skin as follows: ≥ 50% mean tissue viability after 3 minutes exposure and ≥ 15% after 60 minutes exposure results in a predition of non-corrosivity.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430: N/A

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): N/A

VEHICLE - N/A


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): N/A

Duration of treatment / exposure:

(Test item, negative and positive controls): 3 ± 0.5 minutes, 60 ± 5 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Two

Controls:

yes, concurrent positive control

yes, concurrent negative control

Details on study design:

TEST SITE
- Area of exposure: The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diameter).
- % coverage: Not specified
- Type of wrap if used: N/A

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a multipipette containing PBS to remove any residual test material (20 times).
- Time after start of exposure: ('At the end of exposure', no time specified)

OBSERVATION TIME POINTS
3 ± 0.5 minutes, 60 ± 5 minutes

SCORING SYSTEM:
- Method of calculation:
The mean OD of the duplicate negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [(mean OD test item/positive control)/ (mean OD negative )]x 100

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

109.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

107.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: N/A
- Direct-MTT reduction: The colourless test item reduced MTT
Test for Direct MTT Reduction and Colour Interference:
Step 1
50 µL of the test item was added to 0.3 ml of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue and an additional test on viable tissues (without MTT addition) should be performed (step 2). Since the test item did not dye water when mixed with it, step 2 did not have to be performed.
Step 3
All test items (including those already evaluated in step 1 and step 2) should be further evaluated for their potential to interfere with MTT. To test if an item directly reduces MTT, 50 µL of the test item was added to 1 ml of a MTT/DMEM solution (1 mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. Since the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test item proved to reduce MTT and an additional test on freeze-killed tissues (step 4) was performed.
Step 4:
The procedure employed freeze-killed tissues that possess no metabolic activity but absorb and bind the test item similar to viable tissues.
The test item was applied to two freeze-killed tissues. In addition, two freeze killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues.

Data correction procedure:
True viability = Viability of treated tissue – Interference from test chemical =
ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)

- Colour interference with MTT: The colourless test item did not change colour when mixed with deionised water.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time
- Acceptance criteria met for positive control: the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control
- Acceptance criteria met for variability between replicate measurements: Yes
- Mean of historical values if different from the ones specified in the test guideline:
3 minutes, positive control:
Mean viability (%)= 21.60
CV (%): 9.52
Range of viabilities (%): 4.60-39.83

3 minutes, negative control:
Mean OD: 1.68
CV (OD): 4.87
Range of ODs: 1.34-1.93

60 minutes, positive control:
Mean viability (%)= 7.02
CV (%): 14.72
Range of viabilities (%): 2.80-14.77

60 minutes, negative control:
Mean OD: 1.65
CV (OD): 3.19
Range of ODs: 1.32-1.85

Table 1 details the results from all tests performed. Table 2 details the corrosivity classification based on relative mean viability.

Table 1. Results after treatment with test item and controls

DOSE GROUP	EXPOSURE INTERVAL [MIN]		AB-SORBANCE (OD) 570 NM WELL 1	AB-SORBANCE (OD) 570 NM WELL 2	AB-SORBANCE (OD) 570 NM WELL 3	MEAN AB-SORBANCE (OD) OF 3 WELLS MINUS BLANK	MEAN AB-SORBANCE (OD) OF 2 TISSUES	REL. ABSORBANCE [% OF NEGATIVE CONTROL]*	MEAN REL. ABSORBANCE [% OF NEGATIVE CONTROL]*	CV [%]	CORRECTED ODTEST ITEM**	CORRECTED REL. ABSORBANCE [% OF NEGATIVE CONTROL]***
Blank		0.046		0.041	0.035	0.000
Negative Control Tissue 1	3	1.734		1.538	1.694	1.615	1.606	100.5	100	0.8
Negative Control Tissue 2		1.659		1.624	1.631	1.597		99.5
Positive Control Tissue 1		0.497		0.479	0.472	0.442	0.473	27.5	29.4	9.2
Positive Control Tissue 2		0.540		0.547	0.546	0.503		31.3
Test Item Tissue 1		1.675		1.646	1.653	1.617	1.723	100.7	107.3	8.7	1.723- (0.164-0.199) = 1.758	109.5
Test Item Tissue 2		1.864		1.877	1.868	1.829		113.9
Negative ControlFreeze Killed TissueTissue 1		0.232		0.246	0.245	0.200	0.199
Negative ControlFreeze Killed TissueTissue 2		0.239		0.238	0.235	0.197
Test ItemFreeze Killed TissueTissue 1		0.201		0.198	0.199	0.159	0.164
Test ItemFreeze Killed TissueTissue 2		0.211		0.210	0.210	0.170
		0.035		0.035	0.034	0.000
Negative Control Tissue 1	60	1.698		1.625	1.679	1.633	1.622	100.7	100	1.0
Negative Control Tissue 2		1.668		1.648	1.617	1.610		99.3
Positive Control Tissue 1		0.158		0.163	0.161	0.126	0.140	7.8	8.7	14.5
Positive Control Tissue 2		0.195		0.192	0.182	0.155		9.5
Test Item Tissue 1		1.753		1.752	1.714	1.705	1.756	105.2	108.3	4.1	1.756- (0.182-0.166) = 1.740	107.3
Test Item Tissue 2		1.840		1.850	1.833	1.807		111.4
Negative ControlFreeze Killed TissueTissue 1		0.234		0.233	0.227	0.191	0.166
Negative ControlFreeze Killed TissueTissue 2		0.180		0.181	0.186	0.142
Test ItemFreeze Killed TissueTissue 1		0.219		0.222	0.214	0.178	0.182
Test ItemFreeze Killed TissueTissue 2		0.228		0.227	0.226	0.187

* Relative absorbance (rounded values): ( [100 x (absorbance_{test item/positive control})]/ (absorbance _{negative control} ) ) x 100

** OD_{test item} – (OD_{test item freeze-killed} – OD_{negative control freeze-killed})

*** Corrected relative viability (%) = [mean OD _{test item} - (OD _{test item freeze killed} - OD _{negative control freeze killed})/mean OD _{negative control}]x 100

Table 2. Classification of corrosivity potential based on relative viability

VIABILITY MEASURED AFTER EXPOSURE TIME POINTS	PREDICTION TO BE CONSIDERED
< 50% after 3 minutes exposure	Corrosive
≥ 50% after 3 minutes exposure AND < 15% after 60 minutes exposure	Corrosive
≥ 50% after 3 minutes exposureAND ≥ 15% after 60 minutes exposure	Non-corrosive
TEST ITEM IDENTIFIED AS CORROSIVE
< 25% after 3 minutes exposure	Optional Sub-category 1A*
≥ 25% after 3 minutes exposure	A combination of optional Sub-categories 1B and 1C

*According to the data generated in view of assessing the usefulness of the RhE test methods for supporting sub-categorisation, it was shown that around 29%, 31% and 33% of the Sub-category 1A results of the EpiDERM^TM test method may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications)

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is considered to be non-corrosive to skin since the viability after 3 minutes exposure is greater than 50% and the viability after 1 hour exposure is greater than 15%.

Executive summary:

Skin corrosion potential was measured using the Human Skin Model Test with EpiDerm™tissues models, in accordance with OECD Guideline for Testing of Chemicals 431: In vitro Skin Corrosion: Human Skin Model Test (Updated Guideline adopted July 29, 2016) and the MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014.

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Due to the test item’s MTT-reducing property, an additional test with freeze-killed tissues to determine a correction factor for true viability calculation was performed.

Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and subsequently 1 hour.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 20.5 hours in the refrigerator.

The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance compared to the negative control, both for the 3 minutes exposure period (29.4%) and for the 1 hour exposure period (8.7%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item, the corrected relative absorbance values were not reduced (109.4% after 3 minutes exposure, 107.3% after 1 hour exposure). The threshold for corrosivity defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure was not affected. Therefore, the test item is not considered to be corrosive under experimental conditions, in accordance with EU CLP and UN GHS.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Oct - 25 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

SOURCE
- Source and Supplier: EpiSkin™ kits purchased from SkinEthic Laboratories (69007 Lyon, France).
- Kit information: EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
- EpiSkin™ Kit Lot No.: 16-EKIN-047
- Transportation: shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate
- Date received: 22 November 2016

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts. Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for 23.5 hours.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ±1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ±1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: The tissues were gently rinsed with PBS to remove any residual test material. After a 3 hour incubation period, the MTT solution was aspirated from the wells and the wells were rinsed three times with PBS.
- Observable damage in the tissue due to washing: N/A
- Modifications to validated SOP: The tissues were exposed to the different test groups at room temperature.

DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:] MTT Formazan salt was dissolved in DMEM or assay medium to reach a final concentration of 0.3 mg/mL.
- Spectrophotometer: Microplate reader - Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1
- Wavelength:
- Filter: 570 nm
- Filter bandwidth: N/A

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3
An irritation potential of a test item leading to H315 classification of EU (according to directive 67/548/EEC and according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (3rd) revision 2009) is recommended if the mean relative tissue viability of three individual tissues is reduced by < 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): 10 µL of the liquid test item were applied to each tissue, spread to match the tissue size.
- Concentration (if solution): N/A
VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): N/A

Duration of treatment / exposure:

The tissues were treated with the test substance for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

Approx. 42.5 hours.

Number of replicates:

3 (triplicates)

Details on test animals or test system and environmental conditions:

N/A

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): 10 µL of the liquid test item were applied to each tissue
- Concentration (if solution): N/A

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of the liquid test item were applied to each tissue
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of the liquid test item were applied to each tissue
- Concentration (if solution): N/A

Duration of treatment / exposure:

15 minutes

Details on study design:

TEST SITE
- Area of exposure: EpiSkin™ tissues surface 0.38 cm²
- % coverage: Liquid test item was applied to each tissue, spread to match the tissue size
- Type of wrap if used:

REMOVAL OF TEST SUBSTANCE
- Washing: The test item and the positive and negative controls were washed off the skin tissues
- Time after start of exposure: 15 minutes

OBSERVATION TIME POINTS
Test duration was 15 minutes - observation at 15 minutes

SCORING SYSTEM:
- Method of calculation: The mean OD (optical density) of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) =
(mean ODtest item/positive control)/(mean ODnegative control)*100

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

7.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction: The test item did not reduce MTT.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

DEMONSTRATION OF TECHNICAL PROFICIENCY: N/A

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Positive control: Range of Variabilities - 1.70%-35.40%. Negative Control: Range of ODs: 0.61-1.52

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 8.1%. The test item relative absorbance (corresponding to cell viability) percentage values were as follows for the triplicates: 7.1, 8.0, 9.0.

Table 1 demonstrates the results following treatment with the test substance.

Table 1. Tissue absorbance results following treatment

Dose Group	Treatment Interval	Absorbance 570 nm Tissue 1	Absorbance 570 nm Tissue 2	Absorbance 570 nm Tissue 3	Mean Absorbance of 3 Tissues*	Relative Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Rel. Absorbance [% of Negative Control]***
Blank					0.039
Negative Control	15 min	0.651	0.651	0.656	0.614	99.8 99.7 100.5	0.5	100.0
Positive Control	15 min	0.062	0.065	0.068	0.026	3.7 4.3 4.8	12.5	4.3
Test Item	15 min	0.083	0.089	0.094	0.050	7.1 8.0 9.0	11.7	8.1

*Mean of two replicate wells after blank correction

**Relative absorbance pre tissue [rounded values]:

100 x(absorbance_tissue ) / (mean absorbance_{negative control})

***Relative absorbance per treatment group [rounded values]:

(mean absorbance_{test item}) / (mean absorbance_{negative control})

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 8.1% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

Executive summary:

The substance was tested for skin irritation as per OECD Guideline 439, 28 July 2015 (In vitro skin irritation: reconstructed human epidermis test method).

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes (26.3 µL/cm2). 10 µL of the liquid test item were applied to each tissue, spread to match the tissue size. The test item relative absorbance (corresponding to cell viability) percentage values were as follows for the triplicates: 7.1, 8.0, 9.0.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 8.1% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to 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:

27 Feb 2017 - 27 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (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:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Age at study initiation: corneae from at least 9 month old donors

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

VEHICLE -N/A

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

x3 replicates per test group
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin). The corneae were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

QUALITY CHECK OF THE ISOLATED CORNEAS: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

NUMBER OF REPLICATES: 3 per group

NEGATIVE CONTROL USED: Yes - Saline (0.9% NaCl in deionised water)

SOLVENT CONTROL USED (if applicable) N/A

POSITIVE CONTROL USED: Yes - 2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes incubation

TREATMENT METHOD: [closed chamber / open chamber] Not specified

POST-INCUBATION PERIOD: Yes - two hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 1 step
- POST-EXPOSURE INCUBATION: After exposure of the corneae to the test groups, corneae were rinsed and incubated for a further two hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify) The permeability endpoint was measured, as an indication of the integrity of the epithelial cell sheets.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Irritation parameter:

cornea opacity score

Value:

>= 0.67 - <= 1.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

>= 0 - <=1

Positive controls validity:

valid

Remarks:

>=74.67 - <=95.67

Remarks on result:

no indication of irritation

Remarks:

IVIS score: <= 3

Irritation parameter:

in vitro irritation score

Value:

1.02

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

1.44

Positive controls validity:

valid

Remarks:

98.08

Table 1 demonstrates the results of the test groups after 10 minutes incubation.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.44). The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 98.08) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The test item was tested undiluted. Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.02 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorized.

Table 1. Results after 10 minutes incubation time

TEST GROUP	OPACITY VALUE = DIFFERENCE (t130-t0) OF OPACITY		PERMEABILITY AT 490 nm (OD490)		IVIS	MEAN IVIS	PROPOSED IN VITRO IRRITANCY SCORE
		MEAN		MEAN
Negative control	1	0.33	0.069	0.074	2.04	1.44	Not categorised
	0		0.081		1.22
	0		0.072		1.08
Positive control	95.67*		0.643*		105.31	98.08	Category 1
	84.67*		0.890*		98.02
	74.67*		1.063*		90.91
Test item	0.67*		-0.009*		0.53	1.02	Not categorised
	1.67*		0.011*		1.83
	0.67*		0.002*		0.70

*corrected values

Interpretation of results:

GHS criteria not met

Conclusions:

The calculated mean in vitro irritancy score was 1.02. Therefore, the test item is not categorized (GHS).

Executive summary:

The test item was assessed for eye irritation as per OECD Guideline 437, 26 July 2013.

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

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

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.02 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not classified for eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion:

Skin corrosion potential was measured using the Human Skin Model Test with EpiDerm™tissues models, in accordance with OECD Guideline for Testing of Chemicals 431: In vitro Skin Corrosion: Human Skin Model Test (Updated Guideline adopted July 29, 2016) and the MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014.

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Due to the test item’s MTT-reducing property, an additional test with freeze-killed tissues to determine a correction factor for true viability calculation was performed.

Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and subsequently 1 hour.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 20.5 hours in the refrigerator.

The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance compared to the negative control, both for the 3 minutes exposure period (29.4%) and for the 1 hour exposure period (8.7%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item, the corrected relative absorbance values were not reduced (109.4% after 3 minutes exposure, 107.3% after 1 hour exposure). The threshold for corrosivity defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure was not affected. Therefore, the test item is not considered to be corrosive under experimental conditions, in accordance with EU CLP and UN GHS.

Skin Irritation:

The substance was tested for skin irritation as per OECD Guideline 439, 28 July 2015 (In vitro skin irritation: reconstructed human epidermis test method).

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes (26.3 µL/cm2). 10 µL of the liquid test item were applied to each tissue, spread to match the tissue size. The test item relative absorbance (corresponding to cell viability) percentage values were as follows for the triplicates: 7.1, 8.0, 9.0.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 8.1% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

Eye Irritation:

The test item was assessed for eye irritation as per OECD Guideline 437, 26 July 2013.

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

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

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.02 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not classified for eye irritation.

Justification for classification or non-classification

The substance meets the criteria for classification as Skin Irritant Category 2 (Skin Irrit. 2) in accordance with Regulation (EC) No 1272/2008 (CLP).