Decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane

Endpoint summary

Description of key information

H2925 (CAS No. 68130-53-0) is considered to be non-corrosive to skin (OECD Guideline 431) and not irritating to skin (OECD Guideline 439).

H2925 is not considered to be irritating to eyes (OECD Guideline 437).

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:

03 April 2014 to 02 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: EPA 40 CFR 158.340

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

EpiDerm™ tissues, Lot 19981 Kit D, were received from MatTek Corporation (Ashland, MA) on 08 Apr 2014 and refrigerated at approximately 4°C. Before use, tissues were incubated (37°C ± 1°C, 5% ± 1% CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium before dosing.

Justification for test system used:

In accordance with test guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

Test Article Reduction of MIT: 100 µI of the test article were mixed with 1 ml of MTT solution (1 mg/ml Methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium (DMEM)), A Negative Control, 100 µI of tissue culture water (TCH2O), was tested concurrently, The solutions were incubated at room temperature in the dark for 60 minutes, After incubation, the solutions were visually inspected for purple coloration, which indicates that the test article reduced MTT Since tissue viability is based on MTT reduction, direct reduction by a test article can exaggerate viability, making a test article seem less irritating than it really is. The test article did not reduce MTT and the assay continued as per the protocol.

Mesh Compatibility: Pre-cut nylon meshes supplied with the tissues were placed on a slide and 30 µI of the test article or TCH2O Negative Control were applied. After 60 minutes exposure, the mesh was checked microscopically, No interaction between the test article or TCH2O and the mesh was observed so the test article was dosed using the mesh as a spreading aid,

Dosing:
30 µI of the test article or control articles were applied to the EpiDerm ™ tissue. The nylon mesh was then placed on top to facilitate even distribution of the test material. A Negative Control (phosphate buffered saline (PBS)) and a Positive Control (5% sodium dodecyl sulfate (SDS) solution, MatTek) were tested concurrently, with a nylon mesh placed on top to facilitate even distribution of the material. Each treatment with test article or control was conducted in triplicate.
The exposure period for the test article and controls was 60 minutes. The dosed tissues were placed in an incubator at 37° ± 1°C, 5% ± 1% CO2 for 35 ± 1 minutes, then returned to the sterile hood for the remainder of the 60-minute exposure period.
After dosing and incubation, the tissues were rinsed with PBS, blotted to remove the test substance and dry the tissue, and transferred to fresh medium. The rinsed EpiDerm™ tissues were returned to the incubator for 24 ± 2 hours. Medium was changed at 24 ± 2 hours. Tissues were returned to the incubator for an additional 18 ± 2 hours.

Tissue Viability (MTT Reduction): At the end of the incubation period, each EpiDerm™ tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well plate containing 300 µI of MTT solution (1 mg/ml MTT in DMEM). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm TM tissue was rinsed with PBS and then treated with 2.0 ml of extractant solution (isopropanol) per well for at least two hours, with shaking, at room temperature. Two aliquots of the extracted MTT formazan were measured at 540 nm using a plate reader (µQuant Plate Reader, BioTek Instruments, Winooski, VT).

Analysis of Data: The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability =100 X (OD sample/OD Negative Control)

Quality Controls: The assay meets the acceptance criterion if the mean OD540 of the Negative Control tissues is ≥ 1.0 and ≤ 2.5, and the mean viability of Positive Control tissues, expressed as percentage of the Negative Control tissues, is ≤ 20%. In addition, the standard deviation (SD) calculated from individual percent tissue viabilities of the three identically-treated replicates is < 18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µI of the test article were applied to the top of each EpiDerm™ tissue.

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

Not specified

Number of replicates:

Tests were repeated in triplicate.

Irritation / corrosion parameter:

% tissue viability

Value:

96.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 minutes. Max. score: 100.0. Remarks: Viability score are reported as %. The lower the score the more harmful a substance is expected to be. . (migrated information)

Interpretation of results:

GHS criteria not met

Conclusions:

H2925 (CAS No. 68130-53-0), Lot# 2013090401 is considered to be a non-irritant and therefore it is not classified in accordance with the CLP Regulation.

Executive summary:

OBJECTIVE: To predict dermal irritation potential of test articles in the context of identification and classification of skin irritation hazard according to the European Union (EU) classification, United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS) classification system (Category 2 and non-irritants), and OECD Guideline for the Testing of Chemicals No. 439 - In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. This study is designed based on MatTek protocol in vitro EpiDerm™ Skin Irritation Test.

METHOD SYNOPSIS: MatTek EpiDerm™ tissue samples were treated in triplicate with the test article, Negative Control and Positive Control for 60 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using Methylthiazole tetrazolium (MTT) uptake and reduction. The absorbance of each sample was measured at 540 nm. The viability was then expressed as a percent of control values. If the mean tissue viability was ≤ 50%, the test material was classified as an irritant; if the mean tissue Viability was >50%, the test material was classified as a non-irritant.

SUMMARY/CONCLUSION:

Test and Control Article Identity	Mean Tissue Viability	Irritancy Classification
H2925 (CAS No. 68130-53-0), Lot# 2013090401	96.4%	Non-Irritant
Phosphate Buffered Saline (Negative Control)	100.0%	Non-Irritant
5% Sodium Dodecyl Sulfate (Positive Control)	2.4%	Irritant

 

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with restrictions. Limited documentation but relevant data given.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

Adopted 24 April 2002

Deviations:

yes

Remarks:

limited documentation, limited information on test substance given, 2 h exposure only

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: mean: 3.7 kg

Type of coverage:

occlusive

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL
- Concentration: 100%

Duration of treatment / exposure:

2 h

Observation period:

72 h
Reading time points: 1, 24, 48 and 72 h

Number of animals:

5 males

Details on study design:

TEST SITE
- Area of exposure: 9 cm²
- Type of wrap if used: the treated skin was covered with occlusive tape.

REMOVAL OF TEST SUBSTANCE
- Washing: Residual test material was removed with water
- Time after start of exposure: 2 h

SCORING SYSTEM: Draize scoring system

Irritation parameter:

erythema score

Basis:

mean

Remarks:

out of all 5 animals

Time point:

24/48/72 h

Score:

0.24

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

out of all 5 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

At tape removal and after 1 h, no effects on the skin were observed. After 24 h, 3 animals showed erythema, which persisted in 1 animal for 48 h. 72 h after exposure, all animals were free of erythema.
Edema were not observed at all.

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Nov - 06 Nov 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study. For read-across, maximum reliability score is 2.

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

September 1984

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Broekman Institute, Someren, The Netherlands
- Age at study initiation: 20 - 21 weeks
- Weight at study initiation: 3.1 kg ± 202.53 g
- Housing: individually in plastic cages with perforated floors
- Diet: 100 g / day, LK-01, Hope Farms, Woerden, The Netherlands
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 55 -65
- Air changes: air conditioned room
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

semiocclusive

Preparation of test site:

other: clipping

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated site of the same animal served as control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL
- Concentration: 100 %

Duration of treatment / exposure:

4 h

Observation period:

72 h
Reading time points: 50 min, 24, 48, 72 h

Number of animals:

3 females

Details on study design:

TEST SITE
- Area of exposure: 6 cm²
- Type of wrap: gauze patch attached with a drop of petrolatum to aluminium foil and mounted on permeable tape, held in place with flexible bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: remaining test substance was removed, using a dry tissue and subsequently a tissue moistened with tap-water.
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize scoring system

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritant / corrosive response data:

Very slight erythema and edema were observed in all animals 24 h after treatment which were fully reversible latest within 72 h.

Other effects:

No signs of systemic toxicity were observed in any of the animals.

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Nov - 16 Nov 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. For read-across, maximum reliability score is 2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted in 1981

Deviations:

yes

Remarks:

Lack of details on test substance

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Cunicole du Val de Selle, France
- Weight at study initiation: 2.3 kg
- Diet: ad libitum; Rabbit certified pellet diet (Rabbit sustenance ref. 112 C), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days
-other: the animals received a preventive coccidiosis treatment during acclimation (Mucoxid, 137.5 mg/kg bw/day), via drinking water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: not required, untreated sites of the same animal served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL

Duration of treatment / exposure:

4 h

Observation period:

72 h
Reading time points: 1, 24, 48 and 72 h

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 6 cm², flanks were clipped a day before treatment
- Type of wrap if used: adhesive hypoallergic aerated semi-occlusive dressing (Laboratoires de Pansements et d´Hygiène, France)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No

SCORING SYSTEM: According to Draize

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of all three animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

of all three animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No cutaneous reactions were observed in all the animals.

Erythema score

Animal Number	1 h	24 h	48 h	72 h
1	0	0	0	0
2	0	0	0	0
3	0	0	0	0

 

Edema Score

Animal Number	1 h	24 h	48 h	72 h
1	0	0	0	0
2	0	0	0	0
3	0	0	0	0

 

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Reference 5

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 2014 to 02 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: EPA 40 CFR 158.340

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

EpiDerm™ tissues, Lot 19981 Kit D, were received from MatTek Corporation (Ashland, MA) on 08 Apr 2014 and refrigerated at approximately 4'C. Before use, tissues were incubated (37°C ± 1°C, 5% ± 1% CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium before dosing.

Justification for test system used:

In accordance with the test guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

Test Article Reduction of MTT: 100 µI of the test article were mixed with 1 ml of MTT solution (1 mg/ml Methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium (DMEM)). A Negative Control, 100 µI of tissue culture water, was tested concurrently. The solutions were incubated at room temperature in the dark for 60 minutes. After incubation, the solutions were visually inspected for purple coloration, which indicates that the test article reduced MTT. Since tissue Viability is based on MTT reduction, direct reduction by a test article can exaggerate viability, making a test article seem less irritating than it really is. The test article did not reduce MTT and the assay continued as per the protocol.

Mesh Compatibility: Pre-cut nylon meshes supplied with the tissues were placed on a slide and 30 µI of the test article or tissue culture water were applied. After 60 minutes exposure, the mesh was checked microscopically. No interaction between the test article or tissue culture water and the mesh was observed so the test article was dosed using the mesh as a spreading aid.

Dosing: 50 µI of the test article were applied to the top of each EpiDerm™ tissue. A nylon mesh was then placed on top to facilitate even distribution of the test article. The test article remained in contact with the EpiDerm™ tissue for 3 and 60 minutes. A Negative Control (TCH2O) and a Positive Control (KOH) were tested at 3 and 60 minutes. A nylon mesh was placed on top of each liquid control to facilitate even distribution of the material. Each treatment with test article or control was conducted in duplicate.
All tissues were dosed at ambient temperature. Tissues treated with the test article and controls for 3 minutes were maintained at ambient temperature until analyzed for Tissue Viability (MTT Reduction). Tissues to be treated for 60 minutes were dosed and then returned to the Incubator (37°C ± 1°C, 5% ± 1% CO2) for the remainder of the 60-minute exposure period

Tissue Viability (MTT Reduction): At the end of the exposure period, each EpiDerm™ tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well plate containing 300 µI of MTT solution (1 mg/ml MTT in DMEM). The tissues were then returned to the Incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well. The absorbency of an aliquot of the extracted MTT formazan was measured In triplicate at 540 nm using a microplate reader (µQuant Plate Reader, BioTek Instruments, Winooski, VT).

Analysis of Data: The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate tissues and expressed as percent viability for each sample using the following formula:
% viability = 100 X (OD sample/OD Negative Control)

Quality Controls: The Negative Controls meets the acceptance criterion if the mean OD of the two tissues at each time point is greater than or equal to 0.8 and is less than or equal to 2.8 (0.8 ≤ OD ≤ 2.8).
The Positive Control meets the acceptance criterion If the mean relative tissue viability at the 60 minute time point is less than 15% < 15%).
Inter-tissue viability meets the acceptance criterion if the difference between two identically treated tissues is no greater than 30%. Re-testing of the chemical is recommended if the resulting viability is near to a classification cut-off. Note: If necessary, the percent difference will be calculated between the mean of the two tissues and one of the tissues. If this difference is greater than 15%, then rejection should be considered.

Interpretation of Results: Skin Corrosion is defined as the production of irreversible tissue damage in skin following the application of test material. The percent viability calculated was used to determine corrosivity potential in the following manner:
Mean Viability Mean Viability
(3 min.) (60 min.) Predicted Corrosivity
<50% Not Applicable Corrosive
≥50% <15% Corrosive
≥50% ≥15% Non-Corrosive

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µI of the test article were applied to the top of each EpiDerm™ tissue.

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

Not specified

Number of replicates:

Tests were repeated in triplicate.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

85.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

EXPERIMENTAL DATA

Test Article:		H2925 (CAS No. 68130-53-0), Lot# 2013090401
Dose:		50µl
Conc:		Neat
TIME (min)	OD 1	OD 2	OD 3	MEAN (OD)	SD	VIABILITY %	ERROR %
3.0	1.824	1.833	1.837	1.831	0.007	89.1	0.3
	1.670	1.679	1.671	1.673	0.005	81.4	0.2
Neg control				2.056		100.0
60.0	1.725	1.721	1.717	1.721	0.004	89.4	0.2
	2.112	2.153	2.135	2.133	0.021	110.9	1.1
Neg control				1.924		100.0
Mean % Viability at 3 min:						85.2
Mean % Viability at 60 min:						100.1
Predicted Corrosivity:			Non-corrosive

 

Negative Control:		Tissue Culture Water
Dose:		50µl
Conc:		Neat
TIME (min)	OD 1	OD 2	OD 3	MEAN (OD)	SD	VIABILITY %	ERROR %
3.0	2.026	2.078	2.066	2.057	0.027	100.0	1.3
3.0	2.00	2.073	2.094	2.056	0.049	100.0	2.4
Mean				2.056		100.0
60.0	1.959	1.936	1.961	1.952	0.014	101.4	0.7
60.0	1.859	1.932	1.899	1.897	0.037	98.6	1.9
Mean				1.924		100.0
Predicted Corrosivity:			Non-corrosive

 

Positive Control:		Potassium Hydroxide, 8.0 N
Dose:		50µl
Conc:		Neat
TIME (min)	OD 1	OD 2	OD 3	MEAN (OD)	SD	VIABILITY %	ERROR %
3.0	0.587	0.602	0.602	0.597	0.009	29.0	0.4
3.0	0.705	0.708	0.708	0.707	0.002	34.4	0.1
Neg control				2.056		100.0
60.0	0.038	0.039	0.040	0.039	0.001	2.0	0.1
60.0	0.043	0.043	0.043	0.043	0.000	2.2	0.0
Neg control				1.924		100.0
Mean % Viability at 3 min:						31.7
Mean % Viability at 60 min:						2.1
Predicted Corrosivity:			Corrosive

 

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

H2925 (CAS No. 68130-53-0), Lot# 2013090401 is predicted to be non-corrosive.

Executive summary:

OBJECTIVE: To predict and classify the skin corrosivity potential of a chemical by using a three-dimensional human epidermis model. This study is designed based on MatTek protocol "In Vitro EpiDerm™ Skin Corrosion Test" and follows the OECD Guideline for the Testing of Chemicals No. 431 In Vitro Skin Corrosion: Human Skin Model Test.

METHOD SYNOPSIS: MatTek EpiDerm^TMtissues were treated in duplicate with the test article. Negative Control and Positive Control for 3 minutes and 60 minutes. Following treatment, the viability of thetissues was determined using Methyl thiazole tetrazolium (MTT) uptake and conversion, and theabsorbance of each tissue was measured at 540 nm. The mean viability was then expressed as apercent of control values. The percent viability was used to determine corrosivity potential.

 

SUMMARY/CONCLUSION:

Test and Control Article Identity	Mean Viability (3 min.)	Mean Viability (60 min.)	Predicted Corrosivity
H2925 (CAS No. 68130-53-0), Lot# 2013090401	85.2%	100.1%	Non-Corrosive
Tissue culture water (Negative Control)	100.0%	100.0%	Non-Corrosive
Potassium Hydroxide, 8.0 N (Positive Control)	31.7%	2.1%	Corrosive

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

key study

Study period:

03 April 2014 to 02 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The bovine eyes were received from Spear Products on 10 Apr 2014 and transported to MB Research in Hank's Balanced Salt Solution with Penstrep in a refrigerated container.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

Using the closed chamber method, a volume of 0.75 ml of the ethanol, MEM or liquid test article was applied to the epithelium of each of the three positive controls, three negative controls, or three test article-treated corneas in a manner, which ensured the entire cornea was covered.

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

2 hours

Number of animals or in vitro replicates:

9 cornea's used in total (3 positive control, 3 negative controls and 3 test article treated)

Details on study design:

Pretest Procedures
Fresh assay solutions were prepared prior to use. MEM solution was prepared by stirring together one jar of MEM powder (sufficient to make one liter of solution), 2.2 g Sodium Bicarbonate, 0.292 g L-Glutamine, 10 ml of Fetal Bovine Serum (FBS) and brought to a final volume of 1000 ml with distilled water. The MEM solution was kept in a 32°C (± 1°C) incubator for the duration of testing. Hanks Balanced Salt Solution (HBSS) was prepared by stirring together one jar of HBSS powder (sufficient to make one liter), 0.35 g Sodium Bicarbonate and brought to a final volume of 1000 ml with distilled water. HBSS was maintained at room temperature.
In addition, MEM solution with Phenol Red was prepared by stirring together 9.3 g MEM with Phenol Red (sufficient to make one liter), 2.2 g Sodium Bicarbonate, 0.292 g L-Glutamine, 10 mL Fetal bovine Serum and brought to a final volume of 1000 mL with distilled water. The MEM solution with Phenol Red was kept in a 32°C (± 1°C) incubator for the duration of testing.
The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded.
Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The Corneas were then placed in a container of fresh HBSS.
The dissected Corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each Cornea was mounted allowing the epithelium of the Cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each Cornea was visually inspected again to ensure there were no defects.
The entire holder was incubated at 32°C (± 1°C) and allowed to equilibrate for at least one hour but not longer than two hours.
Following the equilibration period, a pre-exposure (baseline) determination of opacity was made for each cornea by measuring each against the blank supplied with the opacitometer. Any cornea with a value greater than 7 opacity units was discarded.

Study Procedure
Following the pretest observations, the MEM solution was removed from the anterior chamber. Using the closed chamber method, a volume of 0.75 ml of the ethanol, MEM or liquid test article was applied to the epithelium of each of the three positive controls, three negative controls, or three test article-treated corneas in a manner, which ensured the entire cornea was covered.
All holders and corneas were placed in a horizontal position (anterior side up) in the 32°C (± 1°C) incubator. After 10 (± 1) minutes, the test article, ethanol or MEM solution in the controls were removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. Following the 1O-minute (±1 minute) exposure, the corneal epithelium was thoroughly rinsed with MEM solution containing phenol red until the test article, ethanol, or MEM solution (in the negative controls) were washed off. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.
All corneas were incubated at 32°C (± 1°G) for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OPKIT. This reading was used in the final IVIS calculations.
The corneas were visually inspected for abnormalities immediately following the 10-minute exposure period, and again at 2 hours post-exposure. There were no abnormalities noted.
Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium. fluorescein solution in Dulbecco's Phosphate Buffered Saline (PBS). Each holder was returned to the 32°C (±1°C) incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea.
After 90 (± 5) minutes, the fluid from the posterior chamber was removed and the amount of dye, which passed through the cornea (permeability), was measured as the optical density at 490 nm by a spectrophotometer. A 1: 1000 dilution of the fluorescein was prepared and measured in the spectrophotometer as a measure of consistency.

Analysis of Data
Individual corrected opacity scores were calculated by subtracting the pretest score from the ten minute and two-hour scores. Corrected mean opacity scores were calculated by averaging the individual two-hour corrected opacity scores for a given dose group and subtracting the mean opacity score for the negative control group. A corrected mean opacity score was not calculated for the negative control rather only the mean of the individual two-hour corrected opacity scores were calculated (with no subtraction of mean opacity score for negative control).
Individual corrected optical densities were calculated by subtracting the mean optical density for the negative control group from the individual optical density values. Individual corrected optical densities were not calculated for the negative control group. Corrected mean optical densities were calculated by averaging the individual corrected optical density values for a given dose group. A corrected mean optical density was not calculated for the negative control, rather only the mean of the individual optical densities.
The In Vitro Irritancy Score (IVIS) for the test article and positive control were calculated by adding the corrected mean opacity score to fifteen times the corrected mean optical density as shown by the equation below. The calculations to obtain an IVIS for the positive control was performed in the same manner as the test article.

In Vitro Irritancy Score = Corrected Mean + 15 (Corrected Mean Optical Density Score)
(IVIS) Opacity Score

OECD Guideline #437 defies a substance, which produces an IVIS of >55.1 as Category 1, a substance that causes “Serious eye damage”.

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

Irritation parameter:

in vitro irritation score

Value:

-2.42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

RESULTS

TEST ARTICLE – H2925 (CAS No. 68130-53-0), Lot# 2013090401

 

INDIVIDUAL TEST VALUES

CORNEA#	Pretest Opacity Scores	10-Minute Scores	2-Hour Scores	O.D. ay 490 nm (Permeability)
7	2	2	2	0.028
8	2	2	3	0.020
9	1	1	0	0.007

 

INDIVIDUAL AND MEAN CALCULATED VALUES

CORNEA#	Corrected Opacity Scores		Individual Corrected O.D.^2
	Individual 10-Minute Corrected Opacity Score^1	Individual 2-Hour Corrected Opacity Score^1
7	0	0	0.004
8	0	1	-0.004
9	0	-1	-0.017
Corrected Mean Opacity Density^3=			-0.006
2-Hour Corrected Mean Opacity Score^4=			-2.33

¹Individual Corrected Opacity Score = 10-minute or 2-hour opacity score minus the pretest opacity score

²Individual Corrected Opacity Density = Individual test article OD minus the mean OD for negative control group. No correction was made for the negative control group.

³Corrected Mean Optical Density = Mean of the individual corrected optical density values for a given dose group.

⁴2-Hour Corrected Mean Opacity Score = Mean of the individual 2-hour corrected opacity scores for a given dose group minus the mean opacity score for the negative control group.

 

NEGATIVE CONTROL – MEM

INDIVIDUAL AND MEAN CALCULATED VALUES

Cornea#	Pretest Opacity Score	10-Minute Scores	10-Minute Corrected Opacity Score^1	2-Hour Scores	2-Hour Corrected Opacity Score^1	O.D. at 490 nm (Permeability)
C1 (neg)	2	3	1	4	2	0.027
C2 (neg)	2	3	1	3	1	0.027
C3 (neg)	5	5	0	9	4	0.018
Mean of Individual Optical Density =						0.024
2-Hour Corrected Mean Opacity Score =						2.33

 

POSITIVE CONTROL – ETHANOL

INDIVIDUAL AND MEAN CALCULATED VALUES

Cornea#	Individual Pretest Opacity Score	Individual 10-Minute Scores	Individual 10-Minute Corrected Opacity Score^1	Individual 2-Hour Scores	Individual 2-Hour Corrected Opacity Score^1	O.D. at 490 nm (Permeability)	Individual Corrected O.D.^2
C4 (pos)	3	9	6	3	0	0.192	0.168
C5 (pos)	3	34	31	34	31	0.266	0.242
C6 (pos)	1	32	31	30	29	0.175	0.151
Corrected Mean Optical Density^3=							0.187
2-Hour Corrected Mean Opacity Score^4=							17.67

¹Individual Corrected Opacity Score = 10-Minute or 2-Hour opacity score minus pretest opacity score.

²Individual Corrected Optical Density = Individual positive control OD minus the mean OD for negative control group.

³Corrected Mean Optical Density = Mean of the individual corrected optical density values for a given dose group.

⁴2-Hour Corrected Mean Opacity Score = Mean of the individual 2-hour corrected opacity scores for a given dose group minus the mean opacity score for the negative control group.

 

CALCULATED IN VITRO IRRITATION SCORES

Test Article (H2925 (CAS No. 68130-53-0), Lot# 2013090401) -2.33 + 15 (-0.006) -2.33 + (-0.09)      IVIS:                 -2.42

Negative Control (MEM) 2.33 + 15 (0.024) 2.33 + 0.36      IVIS:                 2.69

Positive Control (Ethanol) 17.67 + 15 (0.187) 17.67 + 2.805      IVIS:                  20.48

 

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the In Vitro Irritation Score, H2925 (CAS No. 68130-53-0), Lot# 2013090401, is not classfied as an eye irritant in accordance with the CLP Regulation.

Executive summary:

Objective: To determine the potential for ocular irritation using an alternative to the Draize methodology.

This protocol is based on the methodology described in the current OECD Guideline for the Testing of Chemicals #437.

Method Synopsis: Three bovine corneas per group were dosed with 0.75 ml of H2925 (CAS No. 6813053-0), Lot# 2013090401, Minimal Essential Media (MEM) (negative control), or 100% Ethanol (positive control). Following a ten-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined.

Summary/Conclusion:

Test Article: The corrected mean opacity score was -2.33. The corrected mean optical density (permeability) score was -0.006. The in vitro irritancy score (IVIS) was calculated as -2.42.

Negative Control: The corrected mean opacity score was 2.33. The mean optical density (permeability) score was 0.024. The IVIS was calculated as 2.69.

Positive Control: The corrected mean opacity score was 17.67. The corrected mean optical density (permeability) score was 0.187. The IVIS was calculated as 20.48.

All controls were within normal limits.

Based on the In Vitro Irritation Score, H2925 (CAS No. 68130-53-0), Lot# 2013090401, is not classfied as an eye irritant in accordance with the CLP Regulation.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions. Limited documentation but relevant data given.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted 24 April 2002

Deviations:

yes

Remarks:

limited documentation, limited information on test substance given

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL
- Concentration: 100 %

Duration of treatment / exposure:

Group 1: 10 s
Group 2: 72 h (continuous treatment)

Observation period (in vivo):

72 h
Reading time points: 1, 24, 48 and 72 h

Number of animals or in vitro replicates:

Group 1: 4 males
Group 2: 4 males

Details on study design:

REMOVAL OF TEST SUBSTANCE
Group 1
- Washing: yes
- Time after start of exposure: 10 s
Group 2
- Washing: no

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

out of all 4 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Irritation parameter:

iris score

Basis:

mean

Remarks:

out of all 4 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility not applicable

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

out of all 4 animals

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

other: reversibility not applicable

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

out of all 4 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Irritant / corrosive response data:

No effects on cornea and iris were observed. Slight redness of the conjunctivae was observed both, after 10 s and after continuous treatment. The effects were obvious after 1 and 6 h. After 10 s treatment, the effects were reversible within 48 h. After continuous treatment, redness reversed within 24 h. In addition, exsudation was observed in both treatment groups, being reversible within 24 h (continuous treatment) and 48 h (10 s treatment), respectively. Chemosis was not observed.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted: 24 April 2002

Deviations:

yes

Remarks:

limited information on test substance

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 3408 - 3935 g

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL
- Concentration: 100 %

Duration of treatment / exposure:

5 days (single application without washing)

Observation period (in vivo):

5 days
Reading time points: 2, 24, 48 and 72 h

Number of animals or in vitro replicates:

3 males

Details on study design:

REMOVAL OF TEST SUBSTANCE: no

SCORING SYSTEM: Draize scoring system

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Irritation parameter:

iris score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility not applicable

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: reversibility not applicable

Irritant / corrosive response data:

No effects on the cornea and the iris were observed. Conjunctival effects included slight to moderate redness and slight to severe discharge. No chemosis was seen. Additional signs of irritation included erythema and dried secretion on the upper and lower eyelids and convoluted eyelids. All effects were completely reversible within 48 h after application.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Reference 4

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

other: 16 CFR 1500.42

Deviations:

yes

Remarks:

limited documentation

GLP compliance:

no

Species:

rabbit

Strain:

other: albino rabbit, not further specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Acclimation period was carried out, not further specified.

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye served as control

Amount / concentration applied:

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

Duration of treatment / exposure:

single application without washing

Observation period (in vivo):

72 h
Reading time points: 24, 48 and 72 h

Number of animals or in vitro replicates:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: no washing was done

SCORING SYSTEM: Draize scoring system

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

out of all 6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Irritation parameter:

iris score

Basis:

mean

Remarks:

out of all 6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility: not applicable

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

out of all 6 animals

Time point:

24/48/72 h

Score:

0.4

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

out of all 6 animals

Time point:

24/48/72 h

Score:

0.1

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritant / corrosive response data:

No effects on the cornea or iris were observed in test animals. Slight redness of the conjunctivae was observed after 24 h in 3 animals and after 48 h in 4 animals which were fully reversible in all animals within 72 h.
Slight chemosis of the conjunctiva was detected in 1 animal after 24 h which was fully reversible within 48 h.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Reference 5

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 Nov - 23 Nov 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study; only few details on study substance). For read-across, maximum reliability score is 2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted in 1987

Deviations:

yes

Remarks:

lack of details on test substance

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Elevage Cunicole du Val de Selle, France
- Weight at study initiation: 2.6 kg
- Housing: individually
- Diet: Certified pellet (Rabbits sustenance ref. 112 C; ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days
-other: the animals received a preventive coccidiosis treatment during acclimation (Mucoxid, 137.5 mg/kg bw/day, via drinking water)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye served as control

Amount / concentration applied:

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

Duration of treatment / exposure:

single dose, no rinsing

Observation period (in vivo):

72 h
Reading time points: 1, 24, 48 and 72 h

Number of animals or in vitro replicates:

3 males

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No

SCORING SYSTEM: according to 84/499/EEC appendix V B5, i.e., according to the Draize scoring system

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: only slight exsudation at 1h (2 cases) and 24 h (1 case) was seen

Irritation parameter:

iris score

Basis:

mean

Remarks:

of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: none of the animals showed effects in the iris

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Remarks on result:

other: none of the aniamals showed corneal opacity

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Remarks on result:

other: none of the animals showed chemosis

Irritant / corrosive response data:

In 2 animals slight exsudation (grade 1) was obsevered at 1 h. At 24 h slight exsudation (grade 1) was observed in 1 animals. These effects were completely reversible at 48 h.

Animal	Hours after application
	1					24					48					72
	A	B	C	D	E	A	B	C	D	E	A	B	C	D	E	A	B	C	D	E
1	0	0	0	0	1	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0
2	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

A = Cornea Score

B = Iris Score

C = Erythema Score

D = Chemosis Score

E = Exsudation Score

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A GLP study was conducted in accordance with OECD Guideline 431; based on the results of this study, H2925 (CAS No. 68130-53-0), Lot# 2013090401 is considered to be non-corrosive.

A GLP study H2925 was conducted in accordance with OECD Guideline 439; based on the results of this study, H2925 (CAS No. 68130-53-0), Lot# 2013090401 is considered to be a non-irritant and therefore it is not classified in accordance with the CLP Regulation.

Three in vivo skin irritation studies are also available for read-across substances:

The first study was conducted with CAS 11138-60-6 in accordance with OECD Guideline 404. No cutaneous reactions occurred in the 3 New Zealand White rabbits tested. Therefore, it was concluded that the test substance is not irritating to the skin.

The second study was conducted with CAS 91050-89-4 in accordance with OECD Guideline 404. After 24 h, 3 animals (New Zealand White rabbits) showed erythema, which persisted in 1 animal for 48 h. 72 h after exposure, all animals were free of erythema. Therefore, it was concluded that the test substance is not irritating to the skin.

The third study was conducted with CAS 78-16-0 in accordance with EU Method B.4. Very slight erythema and edema were observed in all animals (3 New Zealand White rabbits) 24 h after treatment which were fully reversible latest within 72 h. Therefore, it was concluded that the test substance is not irritating to the skin.

A GLP study was conducted in accordance with OECD Guideline 437; based on the results of this study, H2925 (CAS No. 68130-53-0), Lot# 2013090401, is not classfied as an eye irritant in accordance with the CLP Regulation.

Four in vivo eye irritation studies are available for read-across substances:

The first study was conducted with CAS 11138-60-6 in accordance with OECD Guideline 405. In 2 animals (New Zealand White rabbits) slight exudation (grade 1) was observed at 1 h. At 24 h slight exudation (grade 1) was observed in 1 animals. These effects were completely reversible at 48 h. Therefore, it was concluded that the test substance is not irritating to the eyes.

The second study was conducted with CAS 78-16-0 in accordance with guideline 16 CFR 1500.42. No effects on the cornea or iris were observed in test animals. Slight redness of the conjunctivae was observed after 24 h in 3 animals (albino rabbits) and after 48 h in 4 animals which were fully reversible in all animals within 72 h. Slight chemosis of the conjunctiva was detected in 1 animal after 24 h which was fully reversible within 48 h.Therefore, it was concluded that the test substance is not irritating to the eyes.

The third study was conducted with CAS 91050-89-4 in accordance with OECD Guideline 405. No effects on cornea and iris were observed. Slight redness of the conjunctivae was observed both, after 10 s and after continuous treatment. The effects were obvious after 1 and 6 h. After 10 s treatment, the effects were reversible within 48 h. After continuous treatment, redness reversed within 24 h. In addition, exudation was observed in both treatment groups, being reversible within 24 h (continuous treatment) and 48 h (10 s treatment), respectively. Chemosis was not observed. Therefore, it was concluded that the test substance is not irritating to the eyes.

The fourth study was conducted with CAS 78-16-0 in accordance with OECD Guideline 405 in New Zealand White rabbits. No effects on the cornea and the iris were observed. Conjunctival effects included slight to moderate redness and slight to severe discharge. No chemosis was seen. Additional signs of irritation included erythema and dried secretion on the upper and lower eyelids and convoluted eyelids. All effects were completely reversible within 48 h after application. Therefore, it was concluded that the test substance is not irritating to the eyes.

In conclusion, the substance is not considered to be irritating to either the skin or the eyes.

Justification for selection of skin irritation / corrosion endpoint:

Klimisch 1 study.

Justification for selection of eye irritation endpoint:

Klimisch 1 study.

Justification for classification or non-classification

Based on the above mentioned results, classification according to the CLP Regulation (EC)1272/2008 is not required.