Phosphoric acid, mono and bis-linear butyl esters, potassium salts

Endpoint summary

Description of key information

Skin irritation in vivo: Following grading at 24, 48 and 72 hours in a GLP study, the mean scores for erythema were ≥ 2.3 in 1/6 rabbits and the mean scores for edema were < 2.3 in 6/6 animals. Study duration did not permit demonstration of full reversibility in all animals (equivalent or similar to OECD 404). In a pre-GLP study, no skin reactions were observed in 5/6 surviving animals at 24, 72 or 144 hours after treatment (equivalent or similar to OECD 404).

Skin irritation in vitro: Following exposure to test material, the mean cell viability was 66.4 % compared to the negative control. This value is above the threshold of 50 % and the test item was considered to be non-irritant to skin (OECD 439).

Eye irritation in vivo: Reversible effects on the cornea, iris and conjunctivae were seen in one washed and one unwashed rabbit eye and the test item was considered to be a severe eye irritant under the conditions of the study. However, the reversibility of the effects was not confirmed by exposing a third animal to test material for 7 days without rinsing of the treated eye, and these data cannot be used to classify for eye irritation under the terms of Regulation (EC) No 1272/2008 in the absence of supporting information (equivalent or similar to OECD 405).

Eye irritation in vitro: In an in vitro eye irritation test in the isolated chicken eyes test, the test item could not be classified as a severe irritant and not classified as non-irritant.

The weight of evidence from these data would suggest that skin or eye irritation may result after topical exposure (skin or eye) to the test substance.

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:

27 October 1987 to 30 October 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

exposure to test material took place over 24 hours and reversibility of skin reactions was not investigated

GLP compliance:

yes

Specific details on test material used for the study:

- Physical form: Colourless to light amber liquid
- Purity: 50 % active ingredient
- Lot: 369

Species:

rabbit

Strain:

New Zealand White

Remarks:

male

Details on test animals or test system and environmental conditions:

ANIMAL HUSBANDRY
- Young adult male and female New Zealand White rabbits were received from Hare Marland, Hewitt, New Jersey.
- Rabbits were housed singly in suspended, stainless steel, wire-mesh cages.
- Each rabbit was assigned a unique identification number which was recorded on a card affixed to the cage.
- Purina Certified Rabbit Chow #5322 and water were available ad libitum except during the 24-hour period when exposure to test material took place.
- Rabbits were weighed and observed for general health for approximately two weeks.
- Animal rooms were maintained on a timer-controlled 12-hour light and 12-hour dark cycle.
- Target environmental conditions of the animal rooms were 20 ± 2 °C with relative humidity of 50 ± 10 %. Any excursions outside these ranges were of small magnitude and/or brief duration and did not adversely affect the validity of the study.

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

24 hours

Observation period:

72 hours

Number of animals:

Six

Details on study design:

PROTOCOL
- On the day prior to study initiation, the hair of six male rabbits was closely clipped to expose the skin from the scapular to the lumbar region of the back.
- Rabbits weighed 2625 to 3130 g on the day of treatment.
- Each rabbit was placed into a stock which had been fitted with a piece of rubber sheeting approximately 8" x 18 ".
- Rabbits remained in the stocks throughout the exposure period and did not have access to food or water during that time.
- An aliquot (0.5 mL) of test material was applied directly onto the test site beneath a 1" gauze square that was held in place with tape.
- The rubber sheeting was then wrapped around the animal and secured with clips to retard evaporation and to keep the test material in contact with the skin without undue pressure.
- Three other test materials were applied to three other sites on the same animal.
- Approximately 24 hours after application of the test material, the rubber sheeting was loosened and the skin at the corners of the gauze squares was marked with a waterproof pen. Wrappings were then removed.
- The test sites were gently washed with warm water to remove excess test material.
- The skin was gently patted dry and the animals were returned to their cages.
- Approximately 24, 48 and 72 hours after application of the test material, the test sites were evaluated for erythema, edema and other evidence of dermal effects.
- The test sites were scored according to the Draize scale (see Table 1, attached). Adjacent areas of skin were used for comparison.

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: Animal 21955

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: Animal 21956

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

mean score

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: Animal 21957

Irritation parameter:

erythema score

Basis:

animal #4

Remarks:

mean score

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: Animal 21958

Irritation parameter:

erythema score

Basis:

animal #5

Remarks:

mean score

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: Animal 21959

Irritation parameter:

erythema score

Basis:

animal #6

Remarks:

mean score

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: Animal 21960

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: Animal 21955

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Animal 21956

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

mean score

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Animal 21957

Irritation parameter:

edema score

Basis:

animal #4

Remarks:

mean score

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Animal 21959

Irritation parameter:

edema score

Basis:

animal #6

Remarks:

mean score

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Animal 21960

Irritant / corrosive response data:

- By 24 hours following treatment, the test item produced severe erythema with moderate edema in one rabbit and moderate erythema with slight edema in five rabbits.
- By 48 hours following treatment, one rabbit exhibited moderate erythema with slight edema and five rabbits exhibited slight or mild erythema with no edema.
- The skin irritation had subsided by 72 hours with slight erythema being observed in five rabbits.
- A summary of skin responses to test material is attached.
- Individual skin responses following exposure to test material are shown in Table II (attached).

Interpretation of results:

study cannot be used for classification

Conclusions:

Following exposure to test material for 24 hours and grading at 24, 48 and 72 hours, the mean scores for erythema were ≥ 2.3 in 1/6 rabbits and the mean scores for edema were < 2.3 in 6/6 animals. Study duration did not permit demonstration of full reversibility of skin irritation in all animals.

Executive summary:

In a study similar to OECD 404, the test material was evaluated for acute skin irritation in six male rabbits after 24 hours exposure to the substance under occlusive conditions. Severe erythema with moderate edema was seen in one rabbit and moderate erythema with slight edema in five rabbits at 24 hours after treatment. By 48 hours, one rabbit exhibited moderate erythema with slight edema and five rabbits exhibited slight or mild erythema with no edema. Following grading at 24, 48 and 72 hours, the mean scores for erythema were ≥ 2.3 in 1/6 rabbits and the mean scores for edema were < 2.3 in 6/6 animals. The results do not provide evidence of skin corrosivity but study duration did not permit demonstration that skin irritation would be fully reversible in all animals.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1973

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

animals were exposed to test material for 24 h

GLP compliance:

no

Remarks:

pre-GLP

Specific details on test material used for the study:

- Heskell No: 7774
- Chemical composition: 50 % active ingredient in water

Species:

rabbit

Strain:

other: albino

Remarks:

male

Details on test animals or test system and environmental conditions:

- Not reported

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

24 hours

Observation period:

Six days

Number of animals:

Six

Details on study design:

PROCEDURE
- Six male albino rabbits were clipped free of hair on the trunk and lateral areas and placed in FDA-type stocks.
- Doses of 0.1 mL of undiluted test material were applied to intact skin under double thickness 1" x 1" gauze squares.
- Rubber sheeting was secured around the trunk and held in place with adhesive tape.
- After 24 hours, the rabbits were removed from the stocks, the wrappings were removed and skin reactions were observed.
- Observations were also made at 72 hours and 144 hours (three and six days).

Irritation parameter:

other:

Remarks:

erythema and edema

Basis:

other: all animals

Time point:

other: 24 h, 72 h and 144 h

Score:

0

Max. score:

4

Reversibility:

other:

Remarks:

not applicable

Remarks on result:

other:

Remarks:

no skin reactions were observed

Irritant / corrosive response data:

- One rabbit was found dead in the stocks from strangulation (not compound related).
- No skin reactions were observed in any of the remaining five rabbits at any examination.

Interpretation of results:

study cannot be used for classification

Conclusions:

Following exposure to test material for 24 hours and observation at 24, 72 and 144 hours, no skin reactions were observed in 5/6 surviving animals. One animal died in the stocks due to strangulation (not compound related) and was not evaluated for skin reactions.

Executive summary:

In a pre-GLP study similar to OECD 404, the test material was evaluated for acute skin irritation in 5/6 male rabbits after 24 hours exposure to the substance under occlusive conditions because one animal died in the stocks due to strangulation (not compound related). No skin reactions were observed in the 5/6 surviving animals at 24, 72 or 144 hours after treatment.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 June 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Lot/batch No.of test material: 7675229
- Appearance: White/grey solid
- Purity: 100%
- Manufacture date: August 2015
- Expiry date: August 2018
- Storage conditions: Controlled room temperature (15-20 °C) and humidity (below 70 % RH)

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
- EPISKIN (SM) was supplied by SkinEthic, France (batch 16-EKIN-024; expiry date 20 June 2016).
- Adult human-derived epidermal keratinocytes were seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- A highly differentiated and stratified epidermis model was obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers together with a functional stratum corneum (Tinois et al, 1994).
- The EPISKIN (SM) kit was manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium were tested for the presence of viruses, bacteria and mycoplasma.
- The quality of the final product was assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS) (see Appendix 2, attached).

KIT CONTENTS
- Units: EPISKIN (SM) plate containing up to 12 reconstructed epidermis units (area 0.38 cm2). Each reconstructed epidermis was 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: EPISKIN (SM) biopsy punch for sampling of epidermis.
- Medium: Flask of sterile maintenance medium (batch 16 MAIN3 039; expiry date 22 June 2016) and flask of sterile assay medium (batch 16 ESSC 024; expiry date 22 June 2016).

NUMBER OF REPLICATE WELLS
- Three replicates were used for the test item.
- Three negative controls and three positive controls were also run in the assay.
- Since the test item was coloured, two additional test item-treated tissues were used for the non-specific OD (optical density or absorbance) evaluation.

KIT RECEPTION
- The pH of the agar medium used for transport was checked by observing 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 had not been exposed to a temperature above 40 °C (white colour = good; grey or black colour = not acceptable; the colour change is irreversible irrespective of the length of the period above 40 °C).
- The kits were found to be in good order when received.

STORAGE
- The EPISKIN (SM) kit was kept in the original packaging at 37 °C.
- The maintenance medium and assay medium supplied with the kit were stored at 2 to 8 °C until the initiation of the test.

MTT SOLUTION
- MTT was diluted in phosphate buffered saline to give a stock solution of 3 mg/mL.
- The stock solution prepared on 14 June 2016 was stored in a refrigerator (2 to 8 °C) protected from light.
- The MTT stock solution was diluted with pre-warmed (37 °C) assay medium immediately before use to give a working solution of 0.3 mg/mL.

ACIDIFIED ISOPROPANOL
- Isopropanol was acidified with HCl to achieve a final concentration of 0.04 N HCl.
- HCl (1.8 mL of 12 N acid) was diluted with 500 mL isopropanol or a 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 below.

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, the mixture was a yellow colour 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 ITEM
- Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
- As the test item had an intrinsic colour, further evaluation to detect colouring potential was unnecessary.
- Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test items 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. This tissue 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 reading was conducted following the same conditions as for the other tissues.

PERFORMANCE OF THE STUDY
- Procedures were performed under aspectic conditions in a sterile hood using sterile equipment.

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).
- 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 20 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.

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: 16/185-043B and 16/206-043B) 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 (26.4 to 27.3 °C).
- After the 15 minutes incubation time, the EPISKIN (SM) units were removed and rinsed thoroughly with PBS to remove as far as possible any remaining material from the epidermal surface. Additional rinsing was used to remove test item stuck on the surface of the epidermis. 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.

MTT TEST (Day 2)
- After the 42 hours incubation, all EPISKIN (SM) units (except the two living colour control units) were transferred into the MTT working solution-filled wells (2 mL of 0.3 mg/mL MTT per well).
- All transferred EPISKIN (SM) units were then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 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 between material and 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 samples from each tube were placed into the wells of a 96-well plate (labelled appropriately).
- The OD 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 valid until September 2016) at the required wavelength on each day before use.

CALCULATIONS OF VIABILITY PERCENTAGES

BLANK
- The mean of the six blank OD values was calculated.

NEGATIVE CONTROL
– Individual negative control OD values (NCraw) were corrected with the mean blank OD using the equation OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The mean corrected OD of the 3 negative control samples were calculated: this value corresponds to 100% viability.

POSITIVE CONTROL
– Individual positive control OD values (PCraw) were corrected with the mean blank OD using the equation OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The mean corrected OD of the 3 positive control samples were calculated
– The % viability for each positive control replicate was calculated relative to the mean negative control: % Positive Control 1 = (ODPC1 / mean ODNC) ×100; % Positive Control 2 = (ODPC2 / mean ODNC) ×100; % Positive Control 3 = (ODPC3 / mean ODNC) ×100
– The mean value of the 3 individual relative viability % for positive control was calculated using the equation Mean PC % = (%PC1 +%PC2 +%PC3) / 3

TEST ITEM
– Individual test item OD values (TTraw) were corrected with the mean blank OD using the equation OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The mean corrected OD of the 3 test item samples were calculated
– The % viability for each test item replicate was calculated relative to the mean negative control: % Treated Tissue 1 = (ODTT1 / mean ODNC) ×100; % Treated Tissue 2 = (ODTT2 / mean ODNC) ×100; % Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
– The mean value of the 3 individual relative viability % for test item was calculated using the equation Mean TT % = (%TT1 +%TT2 +%TT3) / 3

DATA CALCULATION FOR TEST ITEMS HAVING MTT-INTERACTING POTENTIAL
- Test items that interfere with MTT can produce non specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability% as detailed below:
- Non specific MTT reduction calculation (NSMTT): NSMTT (%) = [(ODKT- ODKNC) / ODNC] × 100 where ODKNC = negative control killed tissues OD; ODKT = test item treated killed tissues OD; ODNC = negative control OD
- If NSMTT is ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken using the equation TODTT = [ODTT – (ODKT – ODKNC)] where ODTT = test item treated viable tissues
- The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control: % RV 1 = [TODTT1 / mean ODNC] × 100; % RV 2 = [TODTT2 / mean ODNC] × 100; % RV 3 = [TODTT3 / mean ODNC] × 100
- The mean value of the 3 individual relative viability % for test item is calculated using the equation Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3
- If NSMTT is > 30% relative to the negative control, then additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

DATA CALCULATION FOR TEST ITEMS HAVING COLOURING POTENTIAL
- For test items detected as able to stain the tissues the non specific OD due to the residual chemical colour (unrelated to mitochondrial activity) is evaluated and subtracted before calculation of the “true” viability % as detailed below.
- Non Specific Colour % with viable tissues (NSCliving %) is calculated using the equation NSCliving % = (mean ODCTV / mean ODNC)×100 where ODCTV = test substance treated viable tissues (not incubated with MTT); ODNC = negative control OD (incubated with MTT).
- If NSCliving % is ≤ 5 % then the normal calculation mode is used.
- If NSC % i s > 5 % a nd ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken using the equation TODTT = [ODTT – mean ODCTV] where ODTT = test substance treated viable tissue (incubated with MTT); ODCTV = test substance treated viable tissue (not incubated with MTT).
- The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control: % RV 1 = [TODTT1 / mean ODNC] × 100; % RV 2 = [TODTT2 / mean ODNC] × 100; % RV 3 = [TODTT3 / mean ODNC] × 100
- The mean value of the 3 individual relative viability % for test item is calculated using the equation Mean Relative Viability % = (% RV 1 +% RV 2 +% RV 3) / 3
- If NSCliving % is > 30 % relative to the negative control, additional steps must be undertaken if possible, or the test substance must be considered as incompatible with the test.

DATA CALCULATION FOR SUBSTANCE HAVIBG BOTH MTT INTERACTING POTENTIAL AND COLOURING POTENTIAL
- Test substances detected as able to stain the tissues and interfere with MTT will also require a third set of controls before calculation of the “true” viability %.
- Non Specific Colour % with killed tissues (NSCkilled%) is calculated using the equation NSCkilled % = (mean ODCTK / mean ODNC)×100 where ODCTK = test substance treated killed tissues (not incubated with MTT); ODNC = negative control OD (incubated with MTT)
- The % relative viability (% RV) for each test substance replicate will be calculated relative to the mean negative control: % RV 1 = [TODTT1 / mean ODNC] × 100; % RV 2 = [TODTT2 / mean ODNC] × 100; % RV 3 = [TODTT3 / mean ODNC] × 100 where TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV + mean ODCTK] and ODTT = test substance treated viable tissues (incubated with MTT); ODKT = test substance treated killed tissues OD; ODKNC = negative control killed tissues OD; ODCTV = test substance treated viable tissues (not incubated with MTT); ODCTK = test substance treated killed tissues (not incubated with MTT)
- The mean value of the 3 individual relative viability % for test substance will be calculated using the equation Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3

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 mg

Duration of treatment / exposure:

15 minutes (± 0.5 min) at room temperature (26.4 to 27.3 °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:

63.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

65.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

70.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value

Value:

66.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ADDITIONAL CONTROLS
- As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.
- The optical density (measured at 570 nm) of tissues were 0.020
- Non Specific Colour % was calculated as 2.5% (see Table 1, attached).
- The NCS value was below 5 % and additional data calculation was not necessary.

VIABILITY RESULTS
- The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2 (attached). The OD values for the test item treated skin samples showed 66.4% relative viability.

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 the proper condition.
- The mean OD value of the three negative control tissues was in the recommended range (0.819). Standard deviation of the viability results for negative control samples was 5.2.
- The three positive control treated tissues showed 7.1% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 4.8.
- The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 3.6.
- The mean OD value of the blank samples (acidified isopropanol) was 0.048.
- All these parameters met the acceptability criteria, therefore the study was considered to be valid.
- Historical control data are presented in Appendix 3 (attached).

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure to test material, the mean cell viability was 66.4 % compared to the negative control. This value is above the threshold of 50 % and the test item was considered to be non-irritant to skin.

Executive summary:

An in vitro skin irritation test on the test item was performed in a reconstructed human epidermis model (EPISKIN (SM) designed to predict and classify the irritation potential of chemicals by measuring cytotoxic effect as reflected in the MTT (3 -(4,5 -dimethylthiazol-2 -yl)-2,5 -diphenyltetrazolium bromide assay). The irritation potential of the test item was evaluated according to OECD 439. Disks of EPISKIN (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 and protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS solution treated epidermis was used as the negative control and 5 % w/v sodium dodecyl sulphate (SDS) solution treated epidermis was used as the positive control (three units/control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a percentage relative to the negative control. The test item was considered to be irritant to skin if the mean viability after 15 minutes exposure and 42 hours post-exposure incubation was ≤ 50 % of the negative control. Following exposure to test material, the mean cell viability was 66.4 % compared to the negative control. This value is above the threshold of 50 % and the test item was considered to be non-irritant to skin. The experiment met the validity criteria.

Reference 4

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May - 19 September 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Lot/batch No.of test material: 7675229
- Appearance: White/grey solid
- Purity: 100%
- Manufacture date: August 2015
- Expiry date: August 2018
- Storage conditions: Controlled room temperature (15-20 °C) and humidity (below 70 % RH)

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.

Details on test system:

HUMAN SKIN
- EPISKIN (SM) was supplied by SkinEthic, France (batch 16-EKIN-021; expiry date 30 May 2016).
- Adult human-derived epidermal keratinocytes were seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- A highly differentiated and stratified epidermis model was obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers together with a functional stratum corneum (Tinois et al, 1994).
- The EPISKIN (SM) kit was manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium were tested for the presence of viruses, bacteria and mycoplasma.
- The quality of the final product was assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS) (see Appendix 2, attached).

KIT CONTENTS
- Units: EPISKIN (SM) plate containing up to 12 reconstructed epidermis units (area 0.38 cm2). Each reconstructed epidermis was 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: EPISKIN (SM) biopsy punch for sampling of epidermis.
- Medium: Flask of sterile maintenance medium (batch 16 MAIN3 035; expiry date 01 June 2016) and flask of sterile assay medium (batch 16 ESSC 021; expiry date 01 June 2016).

NUMBER OF REPLICATE WELLS
- Two replicates were used for the test item.
- Two negative controls and two positive controls were also run in the assay.
- Since the test item was coloured, two additional test item-treated tissues were used for the non-specific OD (optical density or absorbance) evaluation.

KIT RECEPTION
- The pH of the agar medium used for transport was checked by observing 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 had not been exposed to a temperature above 40 °C (white colour = good; grey or black colour = not acceptable; the colour change is irreversible irrespective of the length of the period above 40 °C).
- The kits were found to be in good order when received.

STORAGE
- The EPISKIN (SM) kit was kept in the original packaging at 37 °C.
- The maintenance medium and assay medium supplied with the kit were stored at 2 to 8 °C until the initiation of the test.

MTT SOLUTION
- MTT was diluted in phosphate buffered saline to give a stock solution of 3 mg/mL.
- The stock solution prepared on 14 June 2016 was stored in a refrigerator (2 to 8 °C) protected from light.
- The MTT stock solution was diluted with pre-warmed (37 °C) assay medium immediately before use to give a working solution of 0.3 mg/mL.

ACIDIFIED ISOPROPANOL
- Isopropanol was acidified with HCl to achieve a final concentration of 0.04 N HCl.
- HCl (1.8 mL of 12 N acid) was diluted with 500 mL isopropanol or a 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 below.

CHECK METHOD FOR POSSIBLE DIRECT MTT REDUCTION WITH TEST ITEM
- Approximately 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 recorded.
- Test items which do not react with MTT: yellow.
- Test items reacting with MTT: blue or purple.
- After three hours incubation, the mixture was a yellow colour 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 ITEM
- Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
- As the test item had an intrinsic colour, further evaluation to detect colouring potential was unnecessary.
- Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test items 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. This tissue 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 reading was conducted following the same conditions as for the other tissues.

PERFORMANCE OF THE STUDY
- Procedures were performed under aseptic conditions in a sterile hood using sterile equipment.

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).
- 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 as 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.
- 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.
- The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (24.2-26.1°C) covered with the plate lids.
- The negative and positive controls were also part of a concurrent study (CiToxLAB study code: 16/149-039B and 16/159-039B) performed in the same experimental period using the same batch of chemicals and same batch of skin units

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 living 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 samples from each tube were placed into the wells of a 96-well plate (labelled appropriately).
- The OD 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 valid until September 2016) at the required wavelength on each day before use.

CALCULATIONS OF VIABILITY PERCENTAGES

BLANK
- The mean of the six blank OD values was calculated.

NEGATIVE CONTROL
– Individual negative control OD values (NCraw) were corrected with the mean blank OD using the equation OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The corrected mean OD of the 2 negative control samples were calculated: this value corresponds to 100% viability.

POSITIVE CONTROL
– Individual positive control OD values (PCraw) were corrected with the mean blank OD using the equation OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The corrected mean OD of the 3 positive control samples were calculated
– The % viability for each positive control replicate was calculated relative to the mean negative control: % Positive Control 1 = (ODPC1 / mean ODNC) ×100; % Positive Control 2 = (ODPC2 / mean ODNC) ×100;
– The mean value of the 2 individual viability % for positive control was calculated using the equation Mean PC % = (%PC1 +%PC2) / 2

TEST ITEM
– Individual test item OD values (TTraw) were corrected with the mean blank OD using the equation OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 2 test item samples were calculated
– The % viability for each test item replicate was calculated relative to the mean negative control: % Treated Tissue 1 = (ODTT1 / mean ODNC) ×100; % Treated Tissue 2 = (ODTT2 / mean ODNC) ×100;
– The mean value of the 2 individual relative viability % for test item was calculated using the equation Mean TT % = (%TT1 +%TT2) / 2
- The variability for 2 disks is calculated as: (Disk1-Disk2)/((Disk1+Disk2)/2) x 100%

DATA CALCULATION FOR TEST ITEMS HAVING COLOURING POTENTIAL
- For test items detected as able to stain the tissues the non specific OD due to the residual chemical colour (unrelated to mitochondrial activity) is evaluated and subtracted before calculation of the “true” viability % as detailed below.
- Non Specific Colour % with viable tissues (NSCliving %) is calculated using the equation NSCliving % = (mean ODCTV / mean ODNC)×100 where ODCTV = test substance treated viable tissues (not incubated with MTT); ODNC = negative control OD (incubated with MTT).
- If NSCliving % is ≤ 5 % then the normal calculation mode is used.
- If NSC % i s > 5 % a nd ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken using the equation TODTT = [ODTT – mean ODCTV] where ODTT = test substance treated viable tissue (incubated with MTT); ODCTV = test substance treated viable tissue (not incubated with MTT).
- The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control: % RV 1 = [TODTT1 / mean ODNC] × 100; % RV 2 = [TODTT2 / mean ODNC] × 100; % RV 3 = [TODTT3 / mean ODNC] × 100
- The mean value of the 2 individual relative viability % for test item is calculated using the equation Mean Relative Viability % = (% RV 1 +% RV 2) / 2
- If NSCliving % is > 30 % relative to the negative control, additional steps must be undertaken if possible, or the test substance must be considered as incompatible with the test.

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:

Mean value

Value:

146

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: No
- Colour interference with MTT: As the test item was coloured, two additional test item-treated living tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.027, Non Specific Colour % (NSCliving%) was calculated as 4.0%. This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: See Appendix 3

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Phosphoric acid, butyl ester, potassium salt, the mean cell viability was 146.0% compared to the negative control. 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.

Executive summary:

An in vitro skin corrosivity test of Phosphoric acid, butyl ester, potassium salt test item 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 Phosphoric acid, butyl ester, potassium salt 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, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. 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 item is considered to be corrosive to skin. Following exposure with Phosphoric acid, butyl ester, potassium salt, the mean cell viability was 146.0% compared to the negative control. 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:

adverse effect observed (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:

02 November 1987 to 09 November 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

only one animal exposed to test material without washing of the eye

GLP compliance:

yes

Specific details on test material used for the study:

- Physical form: Colourless to light amber liquid
- Purity: 50 % active ingredient
- Lot: 369

Species:

rabbit

Strain:

New Zealand White

Remarks:

female

Details on test animals or tissues and environmental conditions:

ANIMAL HUSBANDRY
- Young adult female New Zealand White rabbits were received from Hare Marland, Hewitt, New Jersey.
- Rabbits were housed singly in suspended, stainless steel, wire-mesh cages.
- Each rabbit was assigned a unique identification number which was recorded on a card affixed to the cage.
- Purina Certified Rabbit Chow #5322 and water were available ad libitum except during the 24-hour period when exposure to test material took place.
- Rabbits were quarantined, weighed and observed for general health for approximately two weeks.
- Animal rooms were maintained on a timer-controlled 12-hour light and 12-hour dark cycle.
- Target environmental conditions of the animal rooms were 20 ± 2 °C with relative humidity of 50 ± 10 %. Any excursions outside these ranges were of small magnitude and/or brief duration and did not adversely affect the validity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated left eye served as the control in both animals

Amount / concentration applied:

0.01 mL

Duration of treatment / exposure:

20 seconds in one animal (test item removed from eye by rinsing) and 7 days in the second animal

Duration of post- treatment incubation (in vitro):

20 seconds in one animal (test item removed from eye by rinsing) and 7 days in the second animal

Number of animals or in vitro replicates:

Two

Details on study design:

PROTOCOL
- On the day of the study initiation, the eyes of two female New Zealand White rabbits were examined using fluorescein dye.
- Animals showing pre-existing corneal or conjunctival injury or irritation were not used in the study.
- Rabbits weighed 2208 g and 2362 g on the day of treatment.
- An aliquot (0.01 mL) of test material was introduced into the lower conjunctival sac of the right eye in both rabbits. The left eyes served as controls.
- The treated and control eyes of one animal remained unwashed.
- Approximately 20 seconds after the test item was administered, both eyes of the second animal were washed with lukewarm tap water for one minute.
- Rabbits were examined for evidence of eye irritation after approximately 1 hour and 4 hours then 1, 2 and 3 days after treatment.
- The rabbit that had been exposed to test material without rinsing of the eyes was also examined 7 days after treatment.
- At each observation the treated eyes were examined using illumination and magnification. The treated eyes were scored for ocular reactions at each examination using the Draize scale (see Table I, attached). The untreated eye of each animal was also examined and used for comparison.
- Any unusual effects such as pannus, blistering of the conjunctiva, ulceration or other effects indicative of corrosive action were also noted.
- Fluorescein dye was used to evaluate corneal ulceration and irritation starting at the 1-day observation and at each subsequent observation. A sketch was drawn to indicate areas of stain retention.
- Biomicroscopic examinations fpr corneal injury were conducted at the 1-day observation and at each subsequent observation. Treated eyes were scored according to the system presented in Table II (attached).

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

unwashed rabbit eye

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other:

Remarks:

not applicable

Remarks on result:

other: Animal 22148

Remarks:

fluorescein examinations negative for corneal injury at all intervals

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

unwashed rabbit eye

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other:

Remarks:

not applicable

Remarks on result:

other: Animal 22148

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Remarks:

unwashed rabbit eye

Time point:

24/48/72 h

Score:

2.33

Max. score:

3

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: Animal 22148

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

unwashed rabbit eye

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7d

Remarks on result:

other: Animal 22148

Irritation parameter:

cornea opacity score

Remarks:

washed rabbit eye

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other:

Remarks:

not applicable

Remarks on result:

other: Animal 22149

Remarks:

fluorescein examinations negative for corneal injury at all intervals

Irritation parameter:

iris score

Remarks:

washed rabbit eye

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other:

Remarks:

not applicable

Remarks on result:

other: Animal 22149

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Animal 22149

Irritation parameter:

chemosis score

Remarks:

washed rabbit eye

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: Animal 22149

Irritant / corrosive response data:

- Individual eye irritation scores are given in Tables III and IV (attached).
- Slight corneal opacity and moderate iritis were seen in both the washed and unwashed eye.
- The test item also produced severe conjunctival redness, moderate chemosis and copious blood-tinged discharge (confirmed with Heamastix reagent strips) in the unwashed eye.
- Fluorescein and biomicroscopic examinations revealed no damage to the cornea throughout the study.
- All ocular irritation had resolved by day 2 in the washed eye and by day 7 in the unwashed eye.

Interpretation of results:

study cannot be used for classification

Conclusions:

Slight corneal opacity and moderate iritis were seen in both the washed and unwashed eye. The test item also produced severe conjunctival redness, moderate chemosis and copious blood-tinged discharge (confirmed with Heamastix reagent strips) in the unwashed eye. Fluorescein and biomicroscopic examinations revealed no damage to the cornea throughout the study. All ocular irritation had resolved by day 2 in the washed eye and by day 7 in the unwashed eye and the test item was considered to be a severe eye irritant under the conditions of the study. However, the reversibility of the effects was not confirmed by exposing a third animal to test material for 7 days without rinsing of the treated eye, and these data cannot be used to classify for eye irritation under the terms of Regulation (EC) No 1272/2008 in the absence of supporting information.

Executive summary:

The test material was evaluated for acute eye irritation in two female rabbits. An aliquot (0.01 mL) of test material was introduced into the lower conjunctival sac of the right eye in both rabbits. The left eyes served as controls. The treated and control eyes of one animal remained unwashed. Approximately 20 seconds after the test item was administered, both eyes of the second animal were washed with lukewarm tap water for one minute. Rabbits were examined for evidence of eye irritation after approximately 1 hour and 4 hours then 1, 2 and 3 days after treatment. The rabbit that had been exposed to test material without rinsing of the eyes was also examined 7 days after treatment. Treated eyes were examined using illumination, fluorescein plus biomicroscopic examination and the effects were scored. Slight corneal opacity and moderate iritis were seen in both the washed and unwashed eye. The test item also produced severe conjunctival redness, moderate chemosis and copious blood-tinged discharge (confirmed with Heamastix reagent strips) in the unwashed eye. Fluorescein and biomicroscopic examinations revealed no damage to the cornea throughout the study. All ocular irritation had resolved by day 2 in the washed eye and by day 7 in the unwashed eye. The test item was considered to be a severe eye irritant under the conditions of the study. However, the reversibility of the effects was not confirmed by exposing a third animal to test material for 7 days without rinsing of the treated eye, and these data cannot be used to classify for eye irritation under the terms of Regulation (EC) No 1272/2008 in the absence of supporting information.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5th September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

-Batch/Lot number: 7675229
-Manufacture date: August 2015
-Expiry date: August 2018
-Storage conditions: Controlled room temperature (15-25°C, below 70 RH%)

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
- Characteristics of donor animals (e.g. age, sex, weight): Approx. 7 weeks old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box).
- Time interval prior to initiating testing: processed within approximately 2 hours of collection.
- indication of any existing defects or lesions in ocular tissue samples: Only eyes in good condition were used.
- Indication of any antibiotics used: Not reported

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: An additional negative control ('Parafilm control') treated with 30 μL physiological saline was used.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 g

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

up to 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

-Test item: 3 eyes
-Postive control: 3 eyes
-Negative ocntrol: 1 eye
- Parafilm control: 1 eye

Details on study design:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (26 July 2013).

After the zero reference measurements, the eye was held in horizontal position and ~2g of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered (alternative treatment way was used: a thin even layer of test item was spread on a disk of Parafilm and then placed onto the cornea surface to ensure a suitable exposure). After 10 seconds, the surface was rinsed with physiological saline.
Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution).
Additional negative control (one eye) was also used in the experiment to show that the alternative treatment way had no impact on the results. In the study, three test item treated eyes, three positive control treated eyes, one negative control treated eye and one parafilm control eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the experiment was considered to be valid.

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum corneal swelling at up to 75 min

Value:

1.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum corneal swelling at up to 240 min

Value:

2.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I; the test item is not classified as a severe irritant and not classified as non-irritant.

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean fluorescein retention

Value:

3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class IV

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum corneal opacity

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Other effects / acceptance of results:

The positive control (Imidazole) was classified as severely irritating, UN GHS Classification: Category 1.

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

The additional negative control (“Parafilm” control) was using the alternative treatment way was also classified as non-irritating, UN GHS Classification: No Category.

TEST ITEM

 

Based on this in vitro eye irritation in the isolated chicken eyes test with Phosphoric acid, butyl ester, potassium salt the test item is not classified as a severe irritant and not classified as non-irritant.

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	11.0%	II
Mean maximum corneal swelling at up to 240 min	26.4%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck on all cornea surfaces after the post­treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rmse.
Overall ICE Class	IxIII: 2xTV

POSITIVE CONTROL

The positive control (Imidazole) was classified as severely irritating, UN GHS Classification: Category 1.

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	11.0%	II
Mean maximum corneal swelling at up to 240 min	26.4%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck on all cornea surfaces after the post­treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rmse.
Overall ICE Class	IxIII: 2xTV

NEGATIVE CONTROL

The negative control Physiological saline was classified as non-irritating. UN GHS Classification: No Category.

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3x1

ADDITIONAL NEGATIVE CONTROL

The additional negative control ("Parafilin'’ control) was using the alternative treatment way was also classified as non-irritating. UN GHS Classification: No Category.

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3x1

Conclusions:

Based on this in vitro eye irritation in the isolated chicken eyes test with Phosphoric acid, butyl ester, potassium salt, the test item is not classified as a severe irritant and not classified as non-irritant.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (26 July 2013).

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the experiment was considered to be valid.

No significant corneal swelling change (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 1) was observed on three eyes. Severe fluorescein retention change (severity 3) was noted on three eyes. No other corneal effect was observed.

Based on this in vitro eye irritation in the isolated chicken eyes test with Phosphoric acid, butyl ester, potassium salt, the test item is not classified as a severe irritant and not classified as non-irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion in vivo:

Two animal studies, similar to OECD 404 study designs, are available. In the key study (Brock, 1987), test material was evaluated for acute skin irritation in six male rabbits after 24 hours exposure to the substance under occlusive conditions. Severe erythema with moderate edema was seen in one rabbit and moderate erythema with slight edema in five rabbits at 24 hours after treatment. By 48 hours, one rabbit exhibited moderate erythema with slight edema and five rabbits exhibited slight or mild erythema with no edema. Following grading at 24, 48 and 72 hours, the mean scores for erythema were ≥ 2.3 in 1/6 rabbits and the mean scores for edema were < 2.3 in 6/6 animals. The results do not provide evidence of skin corrosivity, but study duration did not permit demonstration that skin irritation would be fully reversible in all animals. In a supporting pre-GLP study (Morrow, 1973), the test material was evaluated for acute skin irritation in 5/6 male rabbits after 24 hours exposure to the substance under occlusive conditions because one animal died in the stocks due to strangulation (not compound related). No skin reactions were observed in the 5/6 surviving animals at 24, 72 or 144 hours after treatment.

 

Skin irritation in vitro:

In the key investigation, performed in accordance with the OECD 439 guideline, a reconstructed human epidermis model (EPISKIN (SM) designed to predict and classify the irritation potential of chemicals by measuring cytotoxic effect as reflected in the MTT (3 -(4,5 -dimethylthiazol-2 -yl)-2,5 -diphenyltetrazolium bromide) assay was used to assess the test material. Disks of EPISKIN (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 % CO₂and protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS solution treated epidermis was used as the negative control and 5 % w/v sodium dodecyl sulphate (SDS) solution treated epidermis was used as the positive control (three units/control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a percentage relative to the negative control. The test item was irritant to skin if the mean viability after 15 minutes exposure and 42 hours post-exposure incubation was ≤ 50 % of the negative control. Following exposure to test material, the mean cell viability was 66.4 % compared to the negative control. This value is above the threshold of 50 % and the test item was non-irritant to skin. The experiment met the validity criteria.

 

Eye irritation in vivo:

One animal study, similar to an OECD 405 study, is available. The test material was evaluated for acute eye irritation in two female rabbits. An aliquot (0.01 mL) of test material was introduced into the lower conjunctival sac of the right eye in both rabbits. The left eyes served as controls. The treated and control eyes of one animal remained unwashed. Approximately 20 seconds after the test item was administered, both eyes of the second animal were washed with lukewarm tap water for one minute. Rabbits were examined for evidence of eye irritation after approximately 1 hour and 4 hours then 1, 2 and 3 days after treatment. The rabbit that had been exposed to test material without rinsing of the eyes was also examined 7 days after treatment. Treated eyes were examined using illumination, fluorescein plus biomicroscopic examination and the effects were scored. Slight corneal opacity and moderate iritis were seen in both the washed and unwashed eye. The test item also produced severe conjunctival redness, moderate chemosis and copious blood-tinged discharge (confirmed with Heamastix reagent strips) in the unwashed eye. Fluorescein and biomicroscopic examinations revealed no damage to the cornea throughout the study. All ocular irritation had resolved by day 2 in the washed eye and by day 7 in the unwashed eye. The test item was a severe eye irritant under the conditions of the study. However, the reversibility of the effects was not confirmed by exposing a third animal to test material for 7 days without rinsing of the treated eye, and these data cannot be used to classify for eye irritation under the terms of Regulation (EC) No 1272/2008 in the absence of supporting information.

Justification for classification or non-classification

Skin irritation / corrosion: Data from one of the available in vivo studies provided evidence of skin irritation rather than skin corrosion after an exposure period of 24 hours under occlusive conditions. However, this in vivo study did not definitively permit classification, because exposure to the test material took place for longer than 4 hours and reversibility was not demonstrated. A contemporary in vitro investigation (OECD 439), that used a more appropriate exposure period and allows distinction between irritants and non-classified substances, concluded that the test item was not irritant to skin.

 

Eye irritation:

Reversible effects on the cornea, iris and conjunctivae were seen in one washed and one unwashed rabbit eye and the test item was a severe eye irritant under the conditions of the study. However, the reversibility of the effects was not confirmed by exposing a third animal to test material for 7 days without rinsing of the treated eye. An in vitro test, using chicken eyes, did not demonstrate severe eye irritation. These data could not, therefore, be definitively used to classify for eye irritation under the terms of Regulation (EC) No 1272/2008 in the absence of supporting information.

 

Considering a weight of evidence approach to classification for the test substance, these data would suggest that skin or eye irritation may result after topical exposure (skin or eye) to the test substance. Classification as H315, Skin irritant, Cat. 2 and H319, Eye irritant, Cat. 2. may be appropriate.