Manganese dioxide

Endpoint summary

Description of key information

SKIN
Not irritating, OECD 404, EU Method B.4, Pooles (2010)
EYE
Not irritating, OECD 405, EU Method B.5, Pooles (2010)

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:

supporting study

Study period:

2009-07-07 to 2009-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU method B.46 (in vitro skin irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EPISKIN™ Reconstituted Human Epidermis model

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

Not applicable

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Positive and negative controls were included in study

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg

VEHICLE
Test material was used as supplied

Duration of treatment / exposure:

15 minutes

Observation period:

42 hours

Number of animals:

The test was performed in triplicate

Details on study design:

APPLICATION OF TEST MATERIAL
- Area of exposure: The test material was applied topically to the reconstituted epidermis ensuring uniform coverage. The epidermis surface had been moistened with 5 µL of sterile distilled water to improve contact between the solid test material and the epidermis.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period each tissue was rinsed with a Phosphate Buffered Saline solution containing Ca2+ and Mg2+ before incubating for approximately 42 hours at 37 °C in 5% CO2 air
- Time after start of exposure: 15 minutes

SCORING SYSTEM:
At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Irritation / corrosion parameter:

% tissue viability

Value:

>= 100.1 - <= 109.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean viability of the test material treated tissues was 106.1% after a 15-minute exposure.

QUANTITATIVE MTT ASSESSMENT (percentage tissue viability):
For the test material, the relative mean tissue viabilities were compared to the mean of the negative control treated tissues (n = 3). The relative mean viabilities were calculated in the following way:

% Relative mean viability = (mean OD540 of test material/mean OD540 of negative control) x 100

The test material was found not to directly reduce MTT

Table 1: Mean OD₅₄₀ values and % viabilities^b for the negative control material, positive control material and test material

Material	OD540of tissues	Mean OD540of triplicate tissues	± SD of OD540	Relative individual tissue viability %	Relative mean % viability	± SD of % viability
Negative control material	0.826	0.756	0.061	109.3	100^a	8.15
	0.710			93.9
	0.733			97.0
Positive control material	0.069	0.074	0.012	9.1	9.7	1.55
	0.065			8.6
	0.087			11.5
Test material	0.757	0.802	0.039	100.1	106.1	5.18
	0.822			108.7
	0.827			109.4

^aThe mean viability of the negative control tissues is set at 100%;

^bData are presented in the form of % viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Table 2: Qualitative evaluation of tissue viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative control Material	-	-	-
Positive Control Material	++	++	++
Test Material	-	-	-

MTT visual scoring scheme:

-         - = blue tissue (viable)

-         + = blue/white tissue (semi-viable)

-         ++ = tissue is completely white (dead)

Quality criteria

The quality criteria required for acceptance of results in the test were satisfied, i.e. positive control and negative control acceptance criteria.

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

The test material was considered to be a non-irritant to the reconstituted human epidermis model EPISKIN™.

Executive summary:

The skin irritation potential of the test material was determined in vitro in a study which was conducted under GLP conditions and in accordance with the standardised guideline EU Method B.46.

Under the conditions of the study the test material was considered to be a non-irritant to the reconstituted human epidermis model EPISKIN™.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-13 to 2009-10-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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)

Species:

other: Reconstituted Human Epidermal Model

Strain:

other: SkinEthic

Details on test animals or test system and environmental conditions:

- Supplier: SkinEthic Laboratories, Nice, France
- Date received : 13 October 2009
- Pre-incubation: Tissues were aseptically transferred into 6-well plates containing 1 mL maintenance medium at room temperature (Each tissue was inspected for any air bubble between the agarose gel and the tissue culture prior to transfer). The 6-well plated containing the tissues were placed into an incubator overnight at 37 °C, 5 % CO2 in air.
- Medium: Produced in serum-free SkinEthic maintenance medium

Type of coverage:

other: Topical treatment

Preparation of test site:

other: Tissues on polycarbonate inserts.

Vehicle:

unchanged (no vehicle)

Controls:

other: Positive and negative controls

Amount / concentration applied:

TEST MATERIAL
- Amount applied : 20 mg
- Preparation of test material : Used as received

CONTROL
- Negative control: 40 µL of sterile distilled water
- Positive control : 40 µL of 8.0 N potassium hydroxide (used as supplied)

Duration of treatment / exposure:

3 or 60 minutes

Observation period:

3 hours

Number of animals:

All tests were performed in duplicate

Details on study design:

TEST SITE
- Area of exposure: Tissue surface area
- Wetting : 20 µL of sterile distilled water was used for wetting the test material to ensure adequate contact with the tissue surface.

REMOVAL OF TEST SUBSTANCE
- Washing : At the end of each exposure period, each tissue was removed from the well of the treatment plate using forceps and rinsed using a wash bottle containing DPBS (Dulbecco's Phosphate Buffered Saline). Rinsing was achieved by filling and emptying each tissue insert with a constant soft stream of DPBS for approximately 40 seconds to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the tissue culture insert with absorbent paper.
- Time after start of exposure: 3 or 60 minutes.


SCORING SYSTEM: Corrosivity was determined by measuring the absorbency at 540 nm (OD 540) after treatment with MTT. The scoring system used is detailed in table 1.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of two tissues. Time point: 3 minutes.

Value:

95.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of two tissues. Time point: 60 minutes.

Value:

83

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The OD540 for the 3 and 60 minute exposure of the SkinEthic model to MnO2 were 1.189 and 1.146, respectively.

The relative mean viability of cell cultures compared to negative control tissues were calculated as follows:

Relative mean viability (%) = ((mean OD540 of test material)/(mean OD540 of negative control)) x 100

The test material was found not to directly reduce MTT.

Table 2: Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

 

Material	Exposure Time	Mean OD540^1	Relative Mean Viability (%)
Negative control	3 Minute	1.243	100*
	60 Minute	1.381	100*
Positive control	3 Minute	0.058	4.7
	60 Minute	0.048	3.5
Test Material	3 Minute	1.189	95.7
	60 Minute	1.146	83.0
^1Mean of SkinEthic tissues tested in duplicate *Mean percentage viability of the negative control tissue is set at 100 %.

 

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

The test material was not corrosive in vitro using human skin.

Executive summary:

The skin corrosion potential of the test material was investigated in vitro in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 431.

Under the conditions of the study the test material was considered not to have the potential to be corrosive.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 October 2009 to 13 November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Hillcrest, Belton, Loughborough, UK
- Weight at study initiation: 2.33 to 2.67 kg
- Housing: The animals were individually housed in suspended cages.
- Diet: Free access to food (2030 Teklad Global Rabbit diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study
- Water: Free access to mains drinking water was allowed throughout the study
- Acclimation period: an acclimatisation period of at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 30 to 70% respectively
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

other: tets material moistened with 0.5 mL distilled water to achieve a paste

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of test material
- Concentration: At each test site a quantity of 0.5 g of the test material, moistened sufficiently with 0.5 mL of distilled water to achieve a paste

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3 animals

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm cotton gauze
- Type of wrap if used: a strip of surgical adhesive tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: four hours

SCORING SYSTEM:
Draize scoring system

Irritation parameter:

erythema score

Remarks:

68623 Male

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

erythema score

Remarks:

68624 Male

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

erythema score

Remarks:

68663 Male

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Remarks:

68623 Male

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Remarks:

68624 Male

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Remarks:

68663 Male

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritant / corrosive response data:

The individual scores for erythema/eschar and oedema are given in Table 1 (all tables are in the any other information on results section). Black staining of the fur was noted at all treated skin sites. No evidence of skin irritation was noted during the study.

Other effects:

Individual bodyweights and bodyweight changes are given in Table 2. All animals showed expected gain in bodyweight during the study.

Table 1: Individual Skin Reactions

Skin Reaction	Observation (following patch removal)	Individual Scores – Rabbit Number and Sex			Total
		68623Male	68624Male	68663Male
Erythema/Eschar Formation	Immediately	0STA	0STA	0STA	(0 )
	1 Hour	0STA	0STA	0STA	( 0 )
	24 Hours	0STA	0STA	0STA	0
	48 Hours	0STA	0STA	0	( 0 )
	72 Hours	0STA	0STA	0	0
Oedema Formation	Immediately	0	0	0	( 0 )
	1 Hour	0	0	0	( 0 )
	24 Hours	0	0	0	0
	48 Hours	0	0	0	( 0 )
	72 Hours	0	0	0	0
Sum of 24 and 72-hour Readings (S)       :          0
Primary Irritation Index (S/6)                   :          0/6 = 0.0
Classification                                        :          NON-IRRITANT

(   ) = Total values not used for calculation of primary irritation index

STA = Black staining of the fur

Table 2: Individual Bodyweights and Bodyweight Changes

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 3
68623Male	2.33	2.43	0.10
68624Male	2.33	2.45	0.12
68663Male	2.67	2.84	0.17

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

The test material was not considered to be an irritant under the conditions of the study.

Executive summary:

The skin irritation potential of the test material was investigated in vivo in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 404 and EU Method B.4.

During the study no signs of skin irritation were noted at any of the observation points. The test material was therefore concluded to be a non-irritant.

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:

25 August 2009 to 27 August 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed according to GLP and follows an appropriate protocol for in vitro eye irritation potential without any deviations from the study design. As the method is not yet included in a standardised guideline the data have been assigned a reliability score of 2.

Qualifier:

no guideline available

Principles of method if other than guideline:

The aim of the study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

GLP compliance:

yes (incl. QA statement)

Species:

other: Reconstituted Corneal Epithelium model

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST CULTURE
- Source: SkinEthic Laboratories, Nice, France
- Date Received : 25/08/09
- Culture details: Day 6 cultures
- Storage of culture: Cultures were stored at room temperature on arrival and then transferred into 24-well plates containing 300 µL of maintenance medium. No air bubbles were present under the tissue inserts. Tissues were incubated overnight at 37 °C, 5% CO2 in air.
Tissue preparation: Using sterile techniques, 1 mL of maintenance medium at room temperature dispensed into the required number of wells in a 6-well plate. Each well was labelled with the details of treatment and the appropriate exposure time. Separate treatment plates were used for the test material and controls (negative and positive) to avoid cross contamination. Before treatment, 7 day old cultures were transferred into the treatment plates containing maintenance medium.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied : Tissues were treated with 30 mg of the test material.

VEHICLE
Test material was used as supplied

CONTROLS:
- Amount(s) applied: 30 µL of Solution A was applied as a negative control and 30 µL of SDS 1.0% (w/v) as a positive control. Solution A was comprised of Na2HPO4 0.142 g/L, Glucose 1.802 g/L, HEPES 7.149 g/L, KCl 0.224 g/L and NaCl 7.597 g/L.
Sodium Dodecyl Sulphate (SDS) was prepared as a 1% w/v solution in sterile distilled water.

Duration of treatment / exposure:

Cultures were exposed for 10 minutes to the test material.

Observation period (in vivo):

Skin cultures were examined after three hours.

Number of animals or in vitro replicates:

All test material were tested in triplicate (including controls)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Cultures were rinsed by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated "holding plate" containing 300 µL of maintenance medium (at room temperature) until all tissues were rinsed.
- Time after start of exposure: 10 minutes

STAINING PROCEDURE:
Staining: After rinsing, the tissues (two per group) were transferred into a pre-labelled 24-well plate containing 300 µL of a 0.5 mg/mL MMT solution prepared in maintenance medium. The MMT loading plate was placed into an incubator for approximately three hours at 37 °C, 5 % CO2 in air.


SCORING SYSTEM:
- Tissue viability (OD): After incubation with MMT, the tissues were visually examined and the degree of MMT staining was evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MMT and transferred to a pre-labelled 24-well plate containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.

- Histology: If deemed necessary, the histopathology of the remaining insert was examined. At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded for each tissue triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of Isopropanol alone was added to three wells designated as blanks. The optical density was measured (quantitative measurement of tissue viability) at 540 nm (OD540) using Anthos 2001 microplate reader. One tissue for each treatment group was retained for assessment of tissue histopathology. Tissues were cut out of the polycarbonate inserts with a sharp scalpel. The tissues were cut in half. Both halves were placed into a pre-labelled 1.5 mL Eppendorf tube containing 1 mL of 10% Formalin and stored at room temperature.
To determine histopathological changes the tissues were observed for any changes in thickness or organisation of the cells. The negative control tissues should have a constant thickness devoid of terminally differentiated cell, and feature a regular and compact shape. Cells must maintain attachment via multiple desmosomes. Positive control tissues should have a disintegration of most of the upper cell layers of the epithelial tissue. The remaining basal cells should be loosely attached to the polycarbonate substratum.

- Interpretation of data: Quantitative MMT Assessment (percentage of viable tissue)
The relative mean tissue viabilities were compared to the mean of the untreated negative control tissues (n = 2). The relative mean viabilities were calculated using the following: (mean OD540 of test material/mean OD540 of negative control) x 100

TOOL USED TO ASSESS SCORE: Anthos 2001 microplate reader.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

mean

Value:

97.2

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:

The relative mean viability of the test material treated tissues after a 10 minute exposure was 97.2%.

-Interpretation of data: Quantitative MMT Assessment (percentage of viable tissue)
The relative mean tissue viabilities were compared to the mean of the untreated negative control tissues (n = 2). The relative mean viabilities were calculated using the following: (mean OD540 of test material/mean OD540 of negative control) x 100

The test material was found to not directly reduce MMT. It was deemed unnecessary to examine tissue histopathology.

Please refer to Table 1 for all results.

Table 1: Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material	Mean Tissue Viability	Mean OD540	Viability (%)
Negative control	1.042	1.010	100*
	0.977
Positive control	0.282	0.218	21.6
	0.154
Test material	1.021	0.982	97.2
	0.942
* The mean viability of the negative control tissues is set at 100 %

 

Table 2: Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Score
	Tissue 1	Tissue 2
Negative control	-	-
Positive control	+	+
Test material	-	-
- = Blue tissue (viable) + = Blue/White tissue (semi viable) ++ = Tissue completely white (dead)

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

According to the protocol followed, the test material was found to be a Non-Irritant (NI) to the reconstituted human corneal epithelial model, SkinEthic.

Executive summary:

The aim of the study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

According to the protocol followed, the test material was found to be a Non-Irritant (NI) to the reconstituted human corneal epithelial model, SkinEthic.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 November 2009 to 19 November 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Limited, Hillcrest, Belton, Loughborough, UK
- Weight at study initiation: 2.31 to 2.82 kg
- Housing: The animals were individually housed in suspended cages
- Diet: Free access to mains drinking water and food (2030 Teklad Global Rabbit diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study.
- Water:Free access to mains drinking water was allowed throughout the study.
- Acclimation period: of at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 30 to 70% respectively
- Air changes (per hr): The rate of air exchange was at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A volume of 0.1 mL of the test material, which was found to weigh approximately 32 mg
- Concentration (if solution): A volume of 0.1 mL of the test material, which was found to weigh approximately 32 mg

VEHICLE
Not applicable

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3

Details on study design:

APPLICATION OF TEST SUBSTANCE:
Initially a single rabbit was treated with 0.1 mL (32 mg) of test material. The test material was administered into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment to prevent loss of test material and then released. The left eye served as a control and remained untreated. Immediately after administration of the test material an assessment of the initial pain reaction was made. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

SCORING SYSTEM:
Assessment of ocular damage/irritation was made 1, 24, 48 and 72 hours post dosing using the scoring system from Draize JH (1977) “ Dermal and Eye Toxicity Tests” In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p. 48 to 49. Any other ocular effects were also noted at this point along with any clinical signs of toxicity if present. Individual bodyweights were recorded on day 0 and 3. The numerical values corresponding to each animal, tissue and observation time were recorded. These scores were then assessed using a modified version of the system described by Kay JH and Calandra JC (1962), J. Soc. Cosmet. Chem. 13, 281-289. If evidence of irreversible ocular damage is noted, the test material will be classified as corrosive to the eye.

TOOL USED TO ASSESS SCORE:
Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

animal: 68633 Male

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal: 68650 Male

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal: 68672 Male

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal: 68633 Male

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal: 68650 Male

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal: 68672 Male

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: 68633 Male

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: 68650 Male

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: 68672 Male

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal: 68633 Male

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal: 68650 Male

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal: 68672 Male

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Table 1 and Table 2 (all tables are in the any other information on results section).
Black staining of the fur was noted around all treated eyes throughout the study.
No corneal or iridial effects were noted during the study.
Moderate conjunctival irritation was noted in all treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24-hour observation. All treated eyes appeared normal at the 48-hour observation.

Other effects:

Individual bodyweights and bodyweight changes are given in Table 3. All animals showed expected gain in bodyweight during the study.

Table 1: Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	68633 Male				68650 Male				68672 Male
	IPR= 2				IPR= 2				IPR= 2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	1	0	0	2	1	0	0	2	1	0	0
B = Chemosis	2	1	0	0	2	1	0	0	2	1	0	0
C = Discharge	1Sf	0Sf	0Sf	0Sf	1Sf	0Sf	0Sf	0Sf	1Sf	0Sf	0Sf	0Sf
Score (A + B + C) x 2	10	4	0	0	10	4	0	0	10	4	0	0
Total Score	10	4	0	0	10	4	0	0	10	4	0	0

IPR= Initial pain reaction

Sf = Black staining of the fur around treated eye

Table 2: Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
68633 Male	10	4	0	0
68650 Male	10	4	0	0
68672 Male	10	4	0	0
Group Total	30	12	0	0
Group Mean Score	10.0	4.0	0.0	0.0

Table 3: Individual Bodyweights and Bodyweight Changes

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 3
68633Male	2.31	2.37	0.06
68650Male	2.82	2.85	0.03
68672Male	2.55	2.57	0.02

 

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of the study the test material was determined to be not irritating to eyes.

Executive summary:

The eye irritation potential of the test material was investigated in vivo in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 405 and EU Method B.5.

No corneal or iridial effects was noted during the study. Moderate conjunctival irritation was noted in all treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24-hour observation. All treated eyes appeared normal at the 48-hour observation.

Therefore, under the conditions of the study the test material was determined to be not irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

In vitro testing concluded that the test material was unlikely to be either corrosive to skin, or irritating to skin, following exposure. An in vivo test was subsequently performed to confirm findings of the in vitro studies.

The skin irritation potential of the test material was therefore investigated in vivo in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 404 and EU Method B.4.

During the study no signs of skin irritation were noted at any of the observation points. The test material was therefore concluded to be a non-irritant.

 

Eye

In vitro testing concluded that the test material was unlikely to be an eye irritant. An in vivo test was subsequently performed to confirm findings of the in vitro study.

The eye irritation potential of the test material was therefore investigated in vivo in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 405 and EU Method B.5.

No corneal or iridial effects was noted during the study. Moderate conjunctival irritation was noted in all treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24-hour observation. All treated eyes appeared normal at the 48-hour observation. Therefore, under the conditions of the study the test material was determined to be not irritating to eyes.

 

Justification for selection of skin irritation / corrosion endpoint:
The key study was selected as such because it represents the only available in vivo study.

Justification for selection of eye irritation endpoint:
The key study was selected as such because it represents the only available in vivo study.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to either skin or eye irritation.