Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts

Endpoint summary

Description of key information

Skin irritation: The test item was not corrosive or irritant in two in vitro tests (OECD TG 431, OECD TG 439). These results confirm the findings of an in vivo test performed using New Zealand White Rabbits (OECD TG 404; EU Method B.4) and a supporting investigation on test material containing 36 % active ingredient performed using New Zealand White Rabbits (OECD TG 404).

Eye irritation: In the key study, one animal exhibited distinct ocular effects that required premature termination (OECD TG 405; EU Method B.5) and a supporting study involving six animals and test material containing 36 % active ingredient provided evidence for irreversible effect on the eye in two animals (FHSA/CPSC Design, 16 CRD 1500).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th January - 9th January 1994

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)

Version / remarks:

Commission Directive 92/69/EEC

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: David Percival Ltd., Moston, Sandbach, Cheshire, UK
- Age at study initiation: 12-16 weeks old
- Weight at study initiation: 2.41-2.51 kg
- Housing:Individually housed in suspended metal cages.
- Diet: ad libitum - STANRAB SQC Rabbit Diet, Special Diets Services Ltd, Witham, Essex, UK
- Water: ad libitum
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-19
- Humidity (%): 44-49
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12/12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

not required

Amount / concentration applied:

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

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

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm
- % coverage: no data
- Type of wrap if used: 2.5 cm x 2.5 cm gauze patch, secured in position with a strip of surgical adhesive tape (BLENDERM; 2.5 cm x 4 cm) and then the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP).

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) one hour after removal of patches and 24, 48 and 72 hours later.

SCORING SYSTEM:
- Method of calculation: Draize scale

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

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

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Very slight erythema was noted at all treated skin sites one and 24 hours after patch removal and at one treated skin site at the 48-hour observation. Very slight oedema was noted at two treated skin sites one hour after patch removal.

Refer to Table 1 below for individual scores.

Other effects:

none

Table 1: Individual skin reactions

Skin Reaction	Observation Time (Hours)	Individual scores – Rabbit Number and Sex			Total
		109 Female (2.47)	126 (Male (2.51)	129 Female (2.41)
Erythema/Eschar Formation	1	1	1	1	3
	24	1	1	1	3
	48	1	0	0	1
	72	0	0	0	0
Oedema Formation	1	0	1	1	2
	24	0	0	0	0
	48	0	0	0	0
	72	0	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, the test item is not classified for skin irritation.

Executive summary:

In a study performed according to OECD TG 404 and EU Method B.4, the test item, moistened with water, was applied to the clipped backs of three New Zealand White rabbits under semi-occlusive conditions for 4 hours. After 4 hours, any remaining test item was removed by gently swabbing the skin with cotton wool soaked with distilled water. Approximately one hour after removal of patches and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the Draize scale. Very slight erythema was noted at all treated skin sites one and 24 hours after patch removal and at one treated skin site at the 48-hour observation. In all cases, no effects were noted after 72 hours. Very slight oedema was noted at two treated skin sites one hour after patch removal, but was not present by the 24 hour observation. On the basis of this study, the test item is not classified for skin irritation.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August - 02 September, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EPISKIN (SM) reconstructed epidermis with a functional stratum corneum

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Epidermis

Justification for test system used:

The EPISKIN (SM) model was considered suitable for the study because it has been validated for irritation testing in an international validation study and its use is recommended by the relevant guideline for irritation testing (OECD 439).

Vehicle:

unchanged (no vehicle)

Details on test system:

Human Skin
EPISKINTM (SM) (Manufacturer: SkinEthic, France, Batch No.:16-EKIN-035, Expiry Date: 05 September 2016) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Quality Control
EPISKINTM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM (SM) test kits used in the present study) and are documented in Appendix 2.

Kit Contents
Units: EPISKINTM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for
transport.
Plate: 12-well assay plate
Punch: EPISKINTM (SM) biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium”
(Batch No.: 16 MAIN3 060; Exp. Date: 07 September 2016)
A flask of sterile “Assay Medium”
(Batch No.: 16 ESSC 038; Exp. Date: 07 September 2016)

Number of Replicate Wells
In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay.

Kit Reception
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKINTM (SM) kit was kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

MTT solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 30 August 2016) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

Acidified Isopropanol
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed in the following paragraphs.

Check-method for possible direct MTT reduction with test item
Approximately 10 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: yellow
-Test items reacting with MTT: blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.

Check-method to detect the colouring potential of test-items
Based on the result of ”Alpha-Step PC-48 Dried: In Vitro Skin Corrosivity test in the EPISKINTM (SM) Model” study (CiToxLAB study code: 16/219-039B) where the Non-Specific Colour % was 1.0%, thus to detect colouring potential was not necessary in this study.

PERFORMANCE OF THE STUDY
Procedures were performed under aseptic conditions (in sterile hood using sterile equipments).

Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

Application and rinsing (Day 0)
Test Item
As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then approximately 20.6 mg* of the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

*Note: The treatment amount of the test item was determined based on the measurement of the remaining test item.

Negative and positive controls
50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).

Note: The negative and positive controls were also part of a concurrent study (CiToxLAB study code: 16/090-043B, 16/235-043B and 16/236-043B) performed in the same experimental period using the same batch of chemicals and same batch of skin units.

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (26.3-27.5°C).

After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

MTT test (Day 2)
After the 42 hours incubation, all EPISKINTM (SM) units were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere, protected from light.

Formazan extraction (Day 2)
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).

The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (Day 2)
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

CALCULATIONS OF VIABILITY PERCENTAGES - see attachment

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18.
The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability values should be ≤ 18.
The SD calculated from individual % tissue viability values of the three test item treated replicates should be <18.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

INTERPRETATION OF TEST RESULTS
- The irritation potential of test items can be classified according to the United Nations globally Harmonized System of Classification and Labelling of Chemicals and a similar system is used in CLP.
- In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item.
- The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
- If there is clear evidence that the test item is not corrosive, then it can be determined as No Category according to the UN GHS. It is plausible that some weaker corrosives could be classified as non-irritant in this in vitro assay.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

20.6 mg

Duration of treatment / exposure:

15 minutes (± 0.5 min) at room temperature (26.3 to 27.5 °C)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h) at 37 °C in an incubator with 5% CO2.

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

94.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

85.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

80.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value

Value:

87

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

VALIDITY OF THE TEST

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the three negative control tissues was in the recommended range (0.756). Standard deviation of the viability results for negative control samples was 4.5.

The positive control treated tissues showed 4.0% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.0.

The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 7.2.

The mean OD value of the blank samples (acidified isopropanol) was 0.046.

All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Note: Two individual opacity density data (0.029 and 0.023) and the mean optical density data of the positive control (0.030) in this study was slightly lower than the lower limit of the historical control range (0.032). This fact had no impact on the results or integrity of the study since the positive control material showed severe effect.

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPISKIN model test the results indicate that the test item is non-irritant to skin (No Category).

Executive summary:

An in vitro skin irritation test was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with the test item, the mean cell viability was 87.0% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Remarks:

36 % active ingredient

Adequacy of study:

supporting study

Study period:

26 July 1995 to 29 July 1995

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

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- Female New Zealand White rabbits aged approximately 8-10 weeks were used in the study.
- Rabbits were sourced from Kuiper Rabbitry, Gary, Indiana, USA.
- Rabbits weighed 2275 to 2710 g at the start of the study.
- Animals were housed in stainless steel cages in a temperature, humidity and light controlled room.
- Rabbits were nulliparous and non-pregnant.
- Animals were conditioned for at least three days prior to study initiation.
- Purina Rabbit Chow and water were available ad libitum.
- All animals were considered to be in good health at the start of the study.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

Six

Details on study design:

COMPOUND ADMINISTRATION
- Rabbits were individually identified by ear tags.
- The day before study initiation, electric clippers were used to remove hair from the abdomen and sides of six rabbits.
- An aliquot (0.5 mL) of the liquid test material was applied to an intact site approximately 6 cm2 on each rabbit.
- Test material was applied to the site and covered with a two-layer gauze patch held in place with non-irritating Kendall Curity Standard Porous Tape. The patch was then covered with a semi-occlusive plastic overwrap secured in place with Kendall Curity Standard Porous Tape for the duration of the exposure period.
- At the end of the four hour contact period, excess material was removed and the application site was observed and scored.
- The study was terminated after 72 hours.

CLINICAL OBSERVATIONS
- Dermal irritation readings for erythema and oedema were performed approximately 30 minutes after patches were removed and at 24, 48 and 72 hours after treatment.
- Grading and scoring of irritation were performed in accordance with the Draize scoring system (attached).

TERMINAL SACRIFICE
- After completion of the study, all animals were sacrificed by injection with Bauthanasia-D Special euthanasia solution and discarded.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Rabbit 46

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

Remarks on result:

other: Rabbit 47

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

Remarks on result:

other: Rabbit 48

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Rabbit 49

Irritation parameter:

erythema score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Rabbit 50

Irritation parameter:

erythema score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Rabbit 52

Irritation parameter:

edema score

Basis:

animal: all

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Rabbits 46, 47, 48, 49, 50, 52

Irritant / corrosive response data:

- Very slight erythema was noted at all treated skin sites 0.5 hours after patch removal and at five treated skin site at the 24-hour observation.
- Very slight oedema was reported for two skin sites at the 0.5 hour observation.
- No skin effects were found for any animal after 48 hours.
- On the basis of this study, the test item is not classified for skin irritation.

Interpretation of results:

GHS criteria not met

Conclusions:

Irritation was absent at the 72-hour observation and the test material was not considered to be a skin irritant.

Executive summary:

In a study performed according to OECD TG 404, test material containing 36 % active ingredient was applied to the clipped backs of six New Zealand White rabbits under semi-occlusive conditions for 4 hours. After 4 hours, any remaining test item was removed. Dermal irritation readings according to the Draize scale were performed approximately 30 minutes after patches were removed and at 24, 48 and 72 hours. Very slight erythema was noted at all treated skin sites 0.5 hours after patch removal and at five treated skin site at the 24-hour observation. Very slight oedema was reported for two skin sites at the 0.5 hour observation. No skin effects were found for any animal after 48 hours. On the basis of this study, the test item is not classified for skin irritation.

Reference 4

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-15 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Epidermis

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

Human Skin
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 16-EKIN-028, Expiry Date: 18 July 2016) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Quality Control
EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkinTM(SM) Test Kits used in the present study) and are documented in Appendix 2.

Kit Contents
Units: EPISKINTM(SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for
transport.
Plate: 12-well assay plate
Punch: EPISKINTM(SM) biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium”
(Batch No.: 16 MAIN3 045; Exp. Date: 20 July 2016)
A flask of sterile “Assay Medium”
(Batch No.: 16 ESSC 028; Exp. Date: 20 July 2016)

Number of Replicate Wells
In this assay, two replicates per test item were used. Two negative controls and two positive controls were also run in each assay. Furthermore, as the test item was coloured, two additional control tissue samples were used in the experiment for determination of the non-specific colour.

Kit Reception
In each case, the pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKINTM(SM) kits were kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

MTT solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 13 July 2016) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

Acidified isopropanol
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed in the following paragraphs.

Check-method for possible direct MTT reduction with test item
20 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours and then any colour change was observed:
-Test items which do not interact with MTT: yellow
-Test items interacting with MTT: blue or purple
After three hours incubation, yellow colour of the mixture was detected: Thus, the test item did not interact with MTT and therefore the use of additional controls was not necessary.

Check-method to detect the colouring potential of test item
Prior to treatment, the test item was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test item had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non-Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

PERFORMANCE OF THE STUDY
Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2 in a >95% humidified atmosphere.

Application (Day 0)
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of test item* was applied evenly to the epidermal surface of each of two test units and each additional control skin units and then 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
Chemicals might spread gently with the pipette tip in order to cover evenly all the
epidermal surface if necessary (without damaging the epidermis).
The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (25.2-26.4°C) covered with the plate lids.

Note: The negative and positive controls were also part of a concurrent study (CiToxLAB study code: 16/067-039B, 16/221-039B and 16/206-039B) performed in the same experimental period using the same batch of chemicals and same batch of skin units

*Note: The treatment amount of the test item was determined based on the measurement of the remaining test item.

Rinsing (Day 0)
After the incubation times, all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT test (Day 0)
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours, protected from light.

Formazan extraction (Day 0)
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

Cell viability measurements (Day 1)
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 02 September 2014, calibration is valid until September 2016) at the required wavelength on each day before use.

CALCULATIONS OF VIABILITY PERCENTAGES - see attachment

VALIDITY OF THE TEST
The mean OD value of the two negative control tissues should be ≥ 0.6 and ≤ 1.5 and negative control OD values should not be below historically established boundaries.
The acceptable mean viability % range for positive control is ≤ 20%.
The difference of viability between the two tissue replicates should not exceed 30%.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

INTERPRETATION OF TEST RESULTS
The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2015).
The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).

For 2 disks:
If both disks have mean viability of ≥35% = Non Corrosive
If both disks have mean viability of <35% = Corrosive (at the corresponding incubation period)
Otherwise:
If the mean value is ≥35% and the variability is less than 50% = Non Corrosive
If the mean value is <35% and the variability is less than 50% = Corrosive
Otherwise:
If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

20 mg

Duration of treatment / exposure:

4 hours

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

105.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

90.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

VALIDITY OF THE TEST

After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions in each case.

The mean OD value of the two negative control tissues was in the recommended range (0.834).

The two positive control treated tissues showed 1.9% viability demonstrating the proper performance of the assay.

The difference of viability between the two test item-treated tissue samples in the MTT assay was 14.7%.

The mean OD value of the blank samples (acidified isopropanol) was 0.046.

All these parameters were within acceptable limits and therefore the study was considered to be valid.

Historical control data are presented in Appendix 3.

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPISKIN™(SM) model test, the results indicate that the test item is non-corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKINTM(SM) (two units) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls (two units/control). For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test substance is considered to be corrosive to skin.

Following exposure with the test item, the mean cell viability was 98.0% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 - 15 January, 1994

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)

Version / remarks:

Commission Directive 92/69/EEC

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: David Percival Ltd, Moston, Sandbach, Cheshire, UK
- Age at study initiation: 12-16 weeks old
- Weight at study initiation: 2.96 kg
- Housing: Individually housed in a suspended metal cage.
- Diet: ad libitum - STANRAB SQC Rabbit Diet, Special Diet Services Ltd., Witham, Essex, UK
- Water: ad libitum - mains water
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-21
- Humidity (%): 51-58
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

One animal was used.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not washed
- Time after start of exposure:Not applicable

SCORING SYSTEM: Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment using the Draize system.

TOOL USED TO ASSESS SCORE: Light source from a standard ophthalmoscope

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

not reversible

Remarks:

based on 72 h score

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not reversible

Remarks:

based on 72h score

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not reversible

Remarks:

based on 72 h score

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.3

Max. score:

4

Reversibility:

not reversible

Remarks:

based on 72 h score

Irritant / corrosive response data:

Refer to Table 1 below for scores at each time point.

Other effects:

Vocalisation was noted approximately 15 seconds after dosing.
Residual test material was noted around the treated eye one hour after treatment.
A dulling of the normal lustre of the corneal surface was noted in the treated eye one hour after treatment. Areas of diffuse corneal opacity were noted in teh treated eye at the 24 and 48-hour observations with areas of translucent corneal opacity at the 72-hour observation.
Iridial inflammation was noted in the treated eye one hour after treatment and at subsequent observations.
Sever conjuctival irritation was noted in the treated eye one hour after treatment with moderate conjunctival irritation at the 24 and 48-hour observations and severe conjunctival irritation at the 72-hour observations. Haemorrhage of the nictitating membrane was noted in the treated eye at the 24, 48 and 72-hour observations. Pale appearance of the nictitating membrane and blood stained discharge were noted in the treated eye at the 72-hour observation.
The animal was killed for humane reasons immediately after the 72-hour observation. No further animals were treated.

Table 1: Scores at each time point

Rabbit Number and Sex (Bodyweight Kg)	IPR 4 Vo                                          29 Male (2.96)
Time after treatment	1 hour	24 hours	48 hours	72 hours^K
CORNEA
E = Degree of Opacity	d	1	1	2^+
F = Area of Opacity	4	4	4	4
Score (E x F) x 5	0	20	20	40
IRIS
D	1	1^+	1^+	1^+
Score (D x 5)	5	5	5	5
CONJUNCTIVAE
A = Redness	2	2^H	2^H	2^HBsP
B = Chemosis	3	2^+	2^+	3^+
C = Discharge	3^Re	3	3	3
Score (A + B + C) x 2	16	14	14	16
Total Score	21	39	39	61

Key:

IPR = initial pain reaction; D = dulling of the normal lustre of the corneal surface; Vo = vocalisation of approx. 15 seconds after dosing; P = pale appearance of the nictitating membrane; K = animal killed immediately after observation; H = Haemorrhage of the nictitating membrane; Re = residual test material around the treated eye; Bs = blood-stained discharge; + = positive criterion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Mean scores following grading at 24, 48 and 72 hours for corneal opacity (1.3), iritis (1), conjunctival redness (2) and chemosis (2.3) do not meet CLP criteria for irreversible effects on the eye (category 1). However, the animal exhibited distinct ocular effects and was killed for humane reasons immediately after the 72-hour observation. There are consequently grounds for concluding that the effects seen would not have been reversible and that classification for category 1 eye effects is warranted.

Executive summary:

In a study performed according to OECD TG 405 and EU Method B.5, a single application of the test material was made to the non-irrigated eye of one rabbit. Areas of translucent corneal opacity, iridial inflammation and severe conjunctival irritation were produced. Other ocular effects noted were a dulling of the normal lustre of the corneal surface, haemorrhage and pale appearance of the nictitating membrane and blood stained discharge. The animal was killed for humane reasons immediately after the 72-hour observation. No further animals were treated. Mean scores following grading at 24, 48 and 72 hours for corneal opacity (1.3), iritis (1), conjunctival redness (2) and chemosis (2.3) do not meet CLP criteria for irreversible effects on the eye (category 1). However, the animal exhibited distinct ocular effects that required premature termination. There are consequently grounds for concluding that the effects seen would not have been reversible and that classification for category 1 eye effects is warranted.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Remarks:

36 % active ingredient

Adequacy of study:

supporting study

Study period:

March 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: FHSA/CPSC Design, 16 CFR 1500

Deviations:

no

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Remarks:

albino

Details on test animals or tissues and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- New Zealand White rabbits aged approximately 8-10 weeks were used in the study.
- Rabbits were sourced from Scientific Small Animal Laboratory, Arlington Heights, Illinois, USA.
- Animals were housed in stainless steel cages in a temperature, humidity and light controlled room.
- Animals were acclimated for at least four days prior to study initiation.
- Purina Rabbit Chow and water were available ad libitum.
- All animals were considered to be in good health at the start of the study.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

14 days (the eye was not rinsed after instillation of test material containing 36 % active ingredient)

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

Six

Details on study design:

OBSERVATION AND GRADING
- Eyes were examined at 24, 48, 72, 168 and 336 hours after treatment.
- Sodium fluorescein (2 %) and ultraviolet light were employed to reveal possible corneal injury commencing at the 24-hour observation.

EVALUATION
- The Draize scale used for scoring ocular lesions is attached.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Rabbit 67

Remarks:

Corneal surface positive to sodium fluorescein 100 % at 72 h; 50 % at 14 d

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: Rabbit 68

Remarks:

Corneal surface positive to sodium fluorescein 75 % at 72 h; 0 % at 7 d

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: Rabbit 69

Remarks:

Corneal surface positive to sodium fluorescein 50 % at 72 h; 0 % at 7 d

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Rabbit 70

Remarks:

Corneal surface positive to sodium fluorescein 75 % at 72 h; 50 % at 14 d

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

14 d

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks:

based on worsening symptoms for at least part of cornea between 7d and 14 d

Remarks on result:

other: Rabbit 70

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Rabbit 71

Remarks:

Corneal surface positive to sodium fluorescein 100 % at 72 h; 50 % at 14 d

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

14 d

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks:

based on worsening symptoms for at least part of cornea between 7d and 14 d

Remarks on result:

other: Rabbit 71

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: Rabbit 72

Remarks:

Corneal surface positive to sodium fluorescein 75 % at 72 h; 0 % at 14 d

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: Rabbit 67

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: Rabbit 68

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: Rabbit 69

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: Rabbit 70

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not reversible

Remarks:

based on unchanged score 7d and 14 d

Remarks on result:

other: Rabbit 71

Irritation parameter:

iris score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: Rabbit 72

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Rabbit 67

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Rabbit 68

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: Rabbit 69

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #4

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Rabbit 70

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #5

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not reversible

Remarks:

based on unchanged score 7d and 14 d

Remarks on result:

other: Rabbit 71

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #6

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Rabbit 72

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: Rabbit 67

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: Rabbit 68

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: Rabbit 69

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Rabbit 70

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Rabbit 71

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: Rabbit 72

Irritant / corrosive response data:

- Full details of individual eye irritation scores and other findings are given in Table 2 (attached).
- Group mean eye irritation scores are given in Table 3 (attached).

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Mean scores following grading at 24, 48 and 72 hours were reported as corneal opacity (≥ 1 for 6/6 animals), iritis (≥ 1 for 5/6 animals), conjunctival redness (≥ 2 for 5/6 animals) and chemosis (≥ 2 for 5/6 animals). However, two animals exhibited corneal opacity at the 14-day observation that appeared to be worsening in parts of the cornea and, of those two animals, one also exhibited iritis and chemosis of unchanged severity throughout the 14-day study. There are consequently grounds for concluding that the effects seen would not have been reversible and that classification for category 1 eye effects is warranted.

Executive summary:

In a study performed according to FHSA/CPSC Design, 16 CFR 1500, a single application of the test material containing 36 % active ingredient was made to the non-irrigated eye of six rabbits. Mean scores following grading at 24, 48 and 72 hours for corneal opacity (≥ 1 for 6/6 animals), iritis (≥ 1 for 5/6 animals), conjunctival redness (≥ 2 for 5/6 animals) and chemosis (≥ 2 for 5/6 animals) do not meet CLP criteria for irreversible effects on the eye (Category 1) and would normally lead to classification of the test item for eye irritation (Category 2). Nevertheless, examination of Rabbits 70 and 71 showed that whilst the area of the cornea positive to sodium fluorescein reduced from 75 % to 50 % between day 7 and day 14, the score for corneal opacity increased from 1 to 2 for both animals. This pattern of effects suggests that symptoms worsened in parts of the two animal corneas during the second week of observation. In addition, based on unchanged scores for iritis (≥ 1) and chemosis (≥ 2) throughout the 14-day observation period, data suggest that these effects would not have reversed in Rabbit 71 over a longer period of time. It is therefore appropriate to conclude that irreversible effects on the eye were observed in two animals even at the reduced active ingredient concentration of 36 %.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

An in vitro skin corrosivity test was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKINTM(SM) (two units) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls (two units/control). For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test substance is considered to be corrosive to skin.

Following exposure with the test item, the mean cell viability was 98.0% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

An in vitro skin irritation test was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with the test item, the mean cell viability was 87.0% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In the key in vivo study performed according to OECD TG 404 and EU Method B.4, the test item, moistened with water, was applied to the clipped backs of three New Zealand White rabbits under semi-occlusive conditions for 4 hours. After 4 hours, any remaining test item was removed by gently swabbing the skin with cotton wool soaked with distilled water. Approximately one hour after removal of patches and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the Draize scale. Very slight erythema was noted at all treated skin sites one and 24 hours after patch removal and at one treated skin site at the 48-hour observation. In all cases, no effects were noted after 72 hours. Very slight oedema was noted at two treated skin sites one hour after patch removal, but was not present by the 24 hour observation. On the basis of this study, the test item is not classified for skin irritation.

In a supporting study performed according to OECD TG 404, test material containing 36 % active ingredient was applied to the clipped backs of six New Zealand White rabbits under semi-occlusive conditions for 4 hours. After 4 hours, any remaining test item was removed. Dermal irritation readings according to the Draize scale were performed approximately 30 minutes after patches were removed and at 24, 48 and 72 hours. Very slight erythema was noted at all treated skin sites 0.5 hours after patch removal and at five treated skin site at the 24-hour observation. Very slight oedema was reported for two skin sites at the 0.5 hour observation. No skin effects were found for any animal after 48 hours. On the basis of this study, the test item is not classified for skin irritation.

Eye irritation:

In the key study performed according to OECD TG 405 and EU Method B.5, a single application of the test material was made to the non-irrigated eye of one rabbit. Areas of translucent corneal opacity, iridial inflammation and severe conjunctival irritation were produced. Other ocular effects noted were a dulling of the normal lustre of the corneal surface, haemorrhage and pale appearance of the nictitating membrane and blood stained discharge. The animal was killed for humane reasons immediately after the 72-hour observation. No further animals were treated. Mean scores following grading at 24, 48 and 72 hours for corneal opacity (1.3), iritis (1), conjunctival redness (2) and chemosis (2.3) do not meet CLP criteria for irreversible effects on the eye (category 1). However, the animal exhibited distinct ocular effects that required premature termination. There are consequently grounds for concluding that the effects seen would not have been reversible and that classification for category 1 eye effects is warranted.

In a supporting study performed according to FHSA/CPSC Design, 16 CFR 1500, a single application of the test material containing 36 % active ingredient was made to the non-irrigated eye of six rabbits. Mean scores following grading at 24, 48 and 72 hours for corneal opacity (≥ 1 for 6/6 animals), iritis (≥ 1 for 5/6 animals), conjunctival redness (≥ 2 for 5/6 animals) and chemosis (≥ 2 for 5/6 animals) do not meet CLP criteria for irreversible effects on the eye (Category 1) and would normally lead to classification of the test item for eye irritation (Category 2). Nevertheless, examination of Rabbits 70 and 71 showed that whilst the area of the cornea positive to sodium fluorescein reduced from 75 % to 50 % between day 7 and day 14, the score for corneal opacity increased from 1 to 2 for both animals. This pattern of effects suggests that symptoms worsened in parts of the two animal corneas during the second week of observation. In addition, based on unchanged scores for iritis (≥ 1) and chemosis (≥ 2) throughout the 14-day observation period, data suggest that these effects would not have reversed in Rabbit 71 over a longer period of time. It is therefore appropriate to conclude that irreversible effects on the eye were observed in two animals even at the reduced active ingredient concentration of 36 %.

Justification for classification or non-classification

Skin irritation:

The substance was found to be non-corrosive in an in vitro skin corrosivity test performed in a reconstructed human epidermis model (OECD TG 431) and non-irritant in an in vitro skin irritation test performed in a reconstructed human epidermis model (OECD TG 439).

In the key in vivo study, mean scores in three animals following grading at 24, 48 and 72 hours were all > 1 for erythema (fully reversible by 72 h in all animals) and for oedema were zero. On the basis of these results the substance is not classified as a skin irritant according to CLP. These data are supported by a study involving test material containing 36 % active ingredient in which erythema scores of ≥ 1 at 24 hours were fully reversible by 48 hours and oedema scores were zero at 24, 48 and 72 hours.

Eye irritation: In the key study, mean scores following grading at 24, 48 and 72 hours for corneal opacity (1.3), iritis (1), conjunctival redness (2) and chemosis (2.3) do not meet CLP criteria for irreversible effects on the eye (Category 1). However, the animal exhibited distinct ocular effects that required premature termination and gave grounds for concluding that the effects seen would not have been reversible. These data are supported by a study in which grading at 24, 48 and 72 hours for corneal opacity (≥ 1 for 6/6 animals), iritis (≥ 1 for 5/6 animals), conjunctival redness (≥ 2 for 5/6 animals) and chemosis (≥ 2 for 5/6 animals) would normally lead to classification of the test item for eye irritation (Category 2). Nevertheless, data suggests that damage to sections of the cornea became more severe in two animals during the second week of observation. In addition, iritis and chemosis were not expected to reverse in one of the animals. Classification for irreversible effects on the eye (Category 1) under the terms of CLP is therefore warranted.