Manganese ores, reduced

Endpoint summary

Description of key information

Skin and eye irritation / corrosion were assessed in vitro and in vivo according to the following methods/guidelines:

- In vitro skin corrosion: OECD Guideline 436

- In vitro skin irritation EU method B.46

- In vivo skin irritation: OECD Guideline 404

- In vitro eye irritation: SkinEthic Reconstituted Human Corneal model

- In vivo eye irritation: OECD Guideline 405

Manganese ores, reduced is considered to be irritating to the eyes but not irritating to skin based on the results of the in vivo studies in rabbits. The substance is not predicted to be a respiratory irritant.

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:

2009-07-07 to 2009-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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: EPISKIIN™ 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:

other: other: Relative tissue viability %

Value:

>= 92.39 - <= 101.3

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Please refer to table 1 for tabulated results including control.

Other effects / acceptance of results:

The relative mean viability of the test material treated tissues was 97.3% 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	0.744	0.736	0.035	98.4	97.3	4.59
	0.698			92.3
	0.766			101.3

^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 in accordance with 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 assessed in vitro using a method equivalent to EU Method B.46 and in compliance with GLP using an EPISKIIN™ Reconstituted Human Epidermis model.

The relative mean viability of the test material treated tissues was 97.3 % after a 15-minute exposure and was therefore considered to be a non-irritant.

Reference 2

Endpoint:

skin irritation: 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:

guideline study

Qualifier:

according to guideline

Guideline:

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

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 polaced 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 :2 0 mg
- Preparation of test material : The test material was used as supplied. The test material was a combination of powder and large particles. The powder form was selected for dosing.

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:

other: other: % viability of tissues

Value:

90.1

Remarks on result:

other:

Remarks:

Basis: mean of two tissues. Time point: 3 minutes. Max. score: 100.0. Reversibility: other: Not applicable.

Irritation / corrosion parameter:

other: other: % viability of tissues

Value:

90.5

Remarks on result:

other:

Remarks:

Basis: mean of two tissues. Time point: 60 minutes. Max. score: 100.0. Reversibility: other: Not applicable.

Other effects / acceptance of results:

The OD540 for the 3 and 60 minute exposure of the SkinEthic model to sinter ore were 1.120 and 1.250 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))x100

The test material was found not to directly reduce MTT.

Table 1: 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	1.120	90.1
	60 Minute	1.250	90.5
^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 in accordance with EU criteria.

Conclusions:

The test material was considered not to have the potential to be corrosive in vivo.

Executive summary:

The skin corrosion potential of the test material was assessed in vitro according to ECD Test Guideline 431 and in compliance with GLP using a SkinEthic Reconstituted Human Epidermal Model.

After 3 and 60 minutes exposure there was 90.1 % and 90.5 % viability of the tissues respectively and is therefore considered not to have the potential to be corrosive in vivo.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-03 to 2009-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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
- Age at study initiation: 12 to 20 weeks old
- Weight at study initiation: 2.17 to 2.69 kg
- Housing: Individually housed in suspended cages.
- Diet: 2030 Teklad Global Rabbit diet (Harlan Teklad, Blackthorn, Bicester, Oxon, UK) available ad libitum.
- Water : Mains drinking water available ad libitum.
- Acclimation period: Minimum of 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): A minimum of 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hour cycle

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 g moistened sufficiently with 0.5 mL of distilled water to achieve a paste.

Duration of treatment / exposure:

Animal were exposed for 4 hours.

Observation period:

Animals were observed for 72 hours for skin reactions

Number of animals:

3 animals in total (1 animal used in the initial test, with a further 2 animals in the main test.)

Details on study design:

TEST SITE
- Area of exposure: The test material was introduced a 2.5 cm x 2.5 cm cotton gauze patch at three sites on the first rabbit, and 1 patch on both of the remaining two animals.
- Type of wrap if used: The trunk of each rabbit was wrapped in an elasticated corset.


REMOVAL OF TEST SUBSTANCE
- Washing : Any residual test material was removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 3 minutes, then 1 and 4 hours post administration of test material for the first rabbit, and 1 and 4 hours post administration on the remaining 2 rabbits.


SCORING SYSTEM: Draize JH (1959) “Dermal Toxicity” In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Associations of Food and Drug Officials of the United States, Austin, Texas p.46-59

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Remarks:

of three animals

Time point:

other: 24, and 72 hours post dosing

Score:

0

Max. score:

8

Reversibility:

other: Not applicable, no effects were observed

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritant / corrosive response data:

The scores for erythema and oedema at the 24 and 72-hour readings were totalled for the three test rabbits (12 values) and this total was divided by six to give the primary irritation index of the test material.

The test material produced a primary irritation index of 0.0.

Other effects:

Not reported

Measurement of pH

The pH of the test material was determined prior to commencement of the study and found to be as follows:

 

Preparation	pH Measurement
	immediately	after 10 minutes
10 % w/w aqueous preparation of the test material	11.4	11.5

Table 1: Individual Skin Reactions Following 3-Minute and 1-Hour Exposures

 

Skin Reaction	Observation Time (following patch removal)	Individual Scores – Rabbit Number and Sex
		68636 Male
		3-Minute Exposure	1-Hour Exposure
Erythema/Eschar Formation	Immediately	0	0
	1 Hour	0	0
	24 Hours	0	0
	48 Hours	0	0
	72 Hours	0	0
Oedema Formation	Immediately	0	0
	1 Hour	0	0
	24 Hours	0	0
	48 Hours	0	0
	72 Hours	0	0

 

Table 2: Individual Skin Reactions Following 4-Hour Exposure

Skin Reaction	Observation Time (following patch removal)	Individual Scores – Rabbit Number and Sex			Total
		68636 Male	68658 Male	68659 Male
Erythema/Eschar 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
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 Reading (S) : 0
Primary Irritation Index (S/6) : 0/6 = 0.0
Classification : Non-Irritant

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

 

Table 3:Individual Bodyweights and Bodyweight Changes

 

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 3
68636 Male	2.69	2.72	0.03
68658 Male	2.17	2.22	0.05
68659 Male	2.33	2.41	0.08

Interpretation of results:

other: Not classified in accordance with EU criteria.

Conclusions:

The test material produced a primary irritation index of 0.0 and was classified as a non-irritant to rabbit skin. No corrosive effects were noted.

Executive summary:

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

Under the conditions of the study no signs of erythema or oedema were noted at the 24, 48 and 72 hours readings and therefore the test material was classified as a non-irritant to rabbit skin.

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to GLP. Study follows an appropriate protocol for in vitro eye irritation potential without any deviations from the study design.

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

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 substances 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 groups 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 are 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.

TOOL USED TO ASSESS SCORE: Anthos 2001 microplate reader.

Irritation parameter:

other: Mean tissue viability

Run / experiment:

Mean

Value:

> 0.92 - < 1

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Other effects / acceptance of results:

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

The relative mean viability of the test material treated tissues after a 10 minute exposure was 95.2%.

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

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	0.996	0.962	95.2
	0.927
* 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 in accordance with 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 eye irritation potential of the test material was assessed in vitro using a SkinEthic Reconstituted Human Corneal model for a treatment period of 10 minutes and in compliance with GLP. 

Under the conditions of the study, the relative mean viability of the test material treated tissues after a 10 minute exposure was 95.2 % and was considered non-irritant.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-16 to 2009-12-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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
- Age at study initiation: 12 to 20 weeks old
- Weight at study initiation: 2.36 to 2.75 kg
- Housing: Animals were housed individually in suspended cages.
- Diet : Free access to 2030 Teklad Global Rabbit diet (Harlan Teklad, Blackthorn, Bicester, Oxon, UK).
- Water : Free access to mains drinking water
- Acclimation period: A minimum of 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): At least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour cycle.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied : 0.1 mL (weighing approximately 17 mg)

Duration of treatment / exposure:

Up to 72 hours

Observation period (in vivo):

Animals were assessed up to 72 hours after exposure (assessments were made at 1, 24, 48 and 72 hours post treatment).

Number of animals or in vitro replicates:

1 animal was initially treated, with a further two animals

Details on study design:

APPLICATION OF TEST SUBSTANCE:
Initially a single rabbit was treated with a volume of 0.1 mL of test material, weighing approximately 17 mg in an adapted syringe. 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 and 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: standard ophthalmoscope

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Remarks:

of three animals

Time point:

other: 24 Hours

Score:

27

Max. score:

110

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.66

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible

Irritant / corrosive response data:

The test material produced a maximum group mean score of 27.0

To calculate the maximum group mean score, encompassing all reactions, the following equations were used:
Cornea = (E x F) x 5
Where:
E = Degree of opacity
F = Area of Cornea Involved

Iris = (D x 5)

Conjunctivae = (A + B + C) x 2
Where:
A = Redness
B = Chemosis
C = Discharge

Other effects:

Attaining of the fur around the treated eye was noted in all animals.

Measurement of pH

The pH of the test material was determined prior to commencement of the study and found to be as follows:

Preparation	pH Measurement
	immediately	after 10 minutes
10 % w/w aqueous preparation of the test material	11.4	11.5

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

 

Rabbit Number and Sex	68670 Male					68685 Male					68686 Male
	IPR = 0 +					IPR = 0+					IPR = 0+
	1 Hr	24 Hrs	48 Hrs	72 Hrs	7 Dys	1 Hr	24 Hrs	48 Hrs	72 Hrs	7 Dys	1 Hr	24 Hrs	48 Hrs	72 Hrs	7 Dys
Cornea
E = Degree of opacity	0	1	0	0	0	0	1	1	1	0	0	1	1	1	0
F = Area of Cornea Involved	0	2	0	0	0	0	2	2	3	0	0	2	2	2	0
Score (E x F) x 5	0	10	0	0	0	0	10	10	15	0	0	10	10	10	0
Iris
D	1	1	0	0	0	1	1	1	1	0	1	1	1	1	0
Score (D x 5)	5	5	0	0	0	5	5	5	5	0	5	5	5	5	0
Conjunctivae
A = Redness	2	2	2	2	0	2	2	2	2	0	2	2	2	2	0
B = Chemosis	2	2	2	1	0	2	2	2	2	0	2	2	2	2	0
C = Discharge	2Sf	2Sf	1	0	0	2Sf	2Sf	2Sf	2Sf	0	2Sf	2Sf	2Sf	2Sf	0
Score (A + B + C) x 2	12	12	10	6	0	12	12	12	12	0	12	12	12	12	0
Total Score	17	27	10	6	0	17	27	27	32	0	17	27	27	27	0

IPR = Initial pain reaction

Sf = Staining of the fur around the treated eye

+ = One drop of local anaesthetic instilled into both eyes 1 to 2 minutes before treatment

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	7 Days
68670 Male	17	27	10	6	0
68685 Male	17	27	27	32	0
68686 Male	17	27	27	27	0
Group Total	51	81	64	65	0
Group Mean Score	17.0	27.0	21.3	21.7	0.0

 

Table 3:Individual Bodyweights and Bodyweight Changes

 

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 7
68670 Male	2.75	3.00	0.25
68685 Male	2.73	2.94	0.21
68686 Male	2.36	2.58	0.22

 

Interpretation of results:

other: Classified Category 2 in accordance with EU criteria.

Conclusions:

Moderate conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24 and 48-hour observations. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation noted in one treated eye at the 72-hour observation. All treated eyes appeared normal at the 7-day observation. The substance is therefore considered to be an eye irritant in accordance with EU criteria.

Executive summary:

The eye irritation potential of the test material was assessed in vivo according to OECD Test Guideline 405 and EU Method B.5 and in compliance with GLP using New Zealand White rabbits. 

Moderate conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24 and 48-hour observations. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation noted in one treated eye at the 72-hour observation. All treated eyes appeared normal at the 7-day observation. The substance is therefore considered to be an eye irritant in accordance with EU criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation / Corrosion

The skin corrosion potential of the test material was assessed in vitro according to ECD Test Guideline 431 and in compliance with GLP using a SkinEthic Reconstituted Human Epidermal Model. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

After 3 and 60 minutes exposure there was 90.1 % and 90.5 % viability of the tissues respectively and is therefore considered not to have the potential to be corrosive in vivo.

The skin irritation potential of the test material was assessed in vitro using a method equivalent to EU Method B.46 and in compliance with GLP using an EPISKIIN™ Reconstituted Human Epidermis model. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The relative mean viability of the test material treated tissues was 97.3 % after a 15-minute exposure and was therefore considered to be a non-irritant to the reconstituted human epidermis model EPISKIN™.

The skin irritation potential of the test material was assessed in vivo according to OECD Test Guideline 404 and EU Method B.4 and in compliance with GLP using three New Zealand White rabbits. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

No erythema or oedema were noted for the 24, 48 and 72 hours readings and therefore the test material was classified as a non-irritant to rabbit skin.

 

Eye Irritation

The eye irritation potential of the test material was assessed in vitro using a SkinEthic Reconstituted Human Corneal model for a treatment period of 10 minutes and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Under the conditions of the study, the relative mean viability of the test material treated tissues after a 10 minute exposure was 95.2 % and was considered non-irritant.

The eye irritation potential of the test material was assessed in vivo according to OECD Test Guideline 405 and EU Method B.5 and in compliance with GLP using New Zealand White rabbits. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Moderate conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24 and 48-hour observations. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation noted in one treated eye at the 72-hour observation. All treated eyes appeared normal at the 7-day observation. The substance is therefore considered to be an eye irritant in accordance with EU criteria.

Respiratory Irritation

A test for respiratory irritation is not considered appropriate considering the low level of inhalable particles in the substance (based on particle size distribution test)

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 skin, but is classified Category 2 for eye irritation.