oligomeric reaction product of 2-hydroxybenzaldehyde with 1-chloro-2,3-epoxypropane and phenol

Endpoint summary

Description of key information

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, EPICLON EXA-7250 does not require classification as a skin irritant. According to the classification system based on the scheme devised by Draize (1959), EPICLON EXA-7250 is a "non-irritant".

Following exposure with EPICLON EXA-7250, the mean relative viability was 73.0% compared to the negative control value. 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 conclusion, in this in vitro EPISKIN model test with EPICLON EXA-7250, the results indicate that the test item is non-irritant to skin (No Category).

The test item EPICLON EXA-7250, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.   According to Regulation (EC) No 1272/2008, EPICLON EXA-7250 does not require classification as an eye irritant.   According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, EPICLON EXA-7250 does not require classification as an eye irritant.

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:

10 November 2015 - 16 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In vitro skin corrosivity and irritation studies were performed prior to treatment on any animals. Based on the results from the In Vitro Skin Corrosivity Test
(CiToxLAB code: 15/289-039B) in the EPISKIN model test and In Vitro Skin Irritation Test in the EPISKIN Model (CiToxLAB code: 15/289-043B) with EPICLON EXA-7250 concluded that the test item is not corrosive or irritant to the skin. It was concluded that an in vivo study is required for proper classification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

These deviations are considered to have no impact on the outcome of the study and interpretation of the results.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:B006
- Expiration date of the lot/batch:02 March 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature 15-25°C, below 70 RH%

FORM AS APPLIED IN THE TEST: yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, HUNGARY
- Age at study initiation: 18 weeks old
- Weight at study initiation: 3316 – 3825 g
- Housing:Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
- Diet (e.g. ad libitum): Animals received UNI diet for rabbits produced by Cargill Takarmány Zrt., H- 5300 Karcag, Madarasi út, Hungary, ad libitum.
- Water (e.g. ad libitum): The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system.
- Acclimation period:51 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 – 23.2 °C
- Humidity (%): 25 – 64 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light):12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 21 September 2015 To: 16 November 2015

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: The untreated skin of each animal served as a control.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test item was administered as a single dose of 0.5 g, applied to the test area.

Duration of treatment / exposure:

4 hours.

Observation period:

Animals were examined for signs of erythema and oedema, and the responses scored at 60 minutes and then at 24, 48 and 72 hours after patch removal.

Number of animals:

3 rabbits

Details on study design:

TEST SITE
Patch testing was used to detect primary irritating effects of the test item. Three male animals in acceptable health condition were selected for this test. Approximately 24 hours prior to the test, the hair was clipped from the back of the animals. Removal of hair was performed in two steps. The majority of hair
was clipped with an electronic hair clipper and the remaining hair was moistened with water and shaved with a razor. The test item was applied to an approximately 6 cm² area of intact skin as follows:

• A single layer of a fine medical gauze (open-weave with large holes) ofapproximately 5x5 cm was placed over the application area,
• The appropriate amount of test item was carefully spread over the application area (the gauze helped maintain the test item in place),
• Three more layers of gauze were placed over the test item,
• These gauze patches were kept in contact with the skin by a patch of clear plastic with a surrounding adhesive hypoallergenic plaster to ensure continued good contact between the moistened test item and the shaved skin.
• The entire trunks of the animals were wrapped with plastic wrap for 4 hours.
• Medical elastic tubing was placed over the plastic to keep it in place. An initial test was performed using one animal. One hour after application of the
test item, the application site was examined. No severe irritation or corrosive effect was found in the initial test, therefore the bandage was replaced and the
exposure continued for a further 3 hours (a total 4 hours exposure). Two additional animals were then included in the study.

REMOVAL OF TEST SUBSTANCE
Since the test item had adhesive properties it could not be executed. Any expressive force could have cause the damage of the skin, thus the test item was left on the skin until the termination time.

OBSERVATION TIME POINTS
60 minutes and then at 24, 48 and 72 hours after patch removal.

SCORING SYSTEM:
- Method of calculation: The dermal irritation scores were evaluated according to the scoring system by Draize (1959) shown in Appendix 1. The animals were observed for 72 hours and the duration of the study was sufficient to evaluate fully the reversibility or irreversibility of the effects observed.

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

24/48/72 h

Score:

0

Irritant / corrosive response data:

MORTALITY
There was no mortality observed during the study.
BODY WEIGHTS
There was no test item related effect on body weight.
CLINICAL OBSERVATION
General Daily Examination
There were no test item related clinical signs noted.

Examination of Skin-Irritancy
At observation 1, 24, 48 and 72 hours after patch removal, there were no adverse signs noted on the skin of the treated animals; however the test item
adhered to the skin and could not be removed after 4 hours. Any expressive force could have cause damage of the skin thus it was decided to leave the test item on the skin. The test item did not disturb the observation of clinical signs. At 72 hours after patch removal, remaining test item was still present on the skin and there were no adverse signs noted. The termination of the experiment was executed on the 6th day after a discussion and confirmation of the Sponsor.
The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema were 0.00, 0.00 and 0.00 respectively.
The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for oedema were 0.00, 0.00 and 0.00 respectively.
The Primary Irritation Index (considering readings at 24, 48 and 72 hours after patch removal) was calculated as 0.00.

Results were presented and interpreted according to (I) Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 and the (II) UN Globally Harmonised System of Classification and Labelling of Chemicals.

Interpretation of results:

GHS criteria not met

Conclusions:

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, EPICLON EXA-7250 does not require classification as a skin irritant. According to the classification system based on the scheme devised by Draize (1959), EPICLON EXA-7250 is a "non-irritant".

Executive summary:

An acute skin irritation study was performed with EPICLON EXA-7250 in three New Zealand White rabbits. Parameters monitored during this study included mortality, body weight measurements and clinical observations. The irritancy of the test item was evaluated according to the Draize method (OECD No.: 404, 2015).

A weight of 0.5 g of test item was applied to the skin of the experimental animals. The test item was applied as a single dose. Sufficient water to damp the material was used to ensure good contact with the skin. Sterile gauze pads were placed on the skin of rabbits. These gauze pads were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The trunk was wrapped in clear plastic with medical tubing used to hold the patch in place. The untreated skin of each animal served as control.

After 4 hours, the remaining test item was removed with water at body temperature. To assess skin irritation, animals were examined at 1, 24, 48, and 72 hours after the patch removal. Additional general examinations were performed daily.

There was no mortality during the observation period. There was no test item related effect on body weight. The test item adhered to the skin during the whole experiment and could not be removed, however this did not disturbed the observation and at observation 1, 24, 48, 72 hours after patch removal, there were no observed adverse effect noted on the skin of the treated animals. Since the test adhered to the skin the experiment was terminated only after discussion with the sponsor on the 6th day. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema were 0.00, 0.00 and 0.00 respectively. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for oedema were 0.00, 0.00 and 0.00 respectively.

The Primary Irritation Index (considering readings at 24, 48 and 72 hours after patch removal) was calculated as 0.00.

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, EPICLON EXA-7250 does not require classification as a skin irritant. According to the classification system based on the scheme devised by Draize (1959), EPICLON EXA-7250 is a "non-irritant".

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 September 2015 - 30 September 2015

Reliability:

1 (reliable without restriction)

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)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Units: EPISKIN-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: EPISKIN-SM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” (Batch No.: 15 MAIN3 039; Exp. Date: 07 October 2015)
A flask of sterile “Assay Medium” (Batch No.: 15 ESSC 040; Exp. Date: 07 October 2015)

Justification for test system used:

The EPISKIN-SM model has been validated for irritation testing in an international validation study [9] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.:15-EKIN-039, Expiry Date: 05 October 2015) is a three-dimensional human epidermis model. Adult humanderived 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) [7]. 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 73 mg of test item was applied for each skin unit


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of negative control (PBS) 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).


POSITIVE CONTROL
- Amount(s) applied (volume or weight):50 µL of 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).

Duration of treatment / exposure:

After the 42 hours incubation, all EPISKIN-SM units (except of one colour control unit) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

Duration of post-treatment incubation (if applicable):

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.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

73.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

72.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

72.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: As no colour change (yellow colour) was observed after three hours of incubation of the test items in MTT working solution, thus the test materials did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:The mean OD value of the three negative control tissues was in the recommended range (0.940). Standard deviation of the viability results for negative control samples was 0.2.
- Acceptance criteria met for positive control:The positive control treated tissues showed 6.5% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.7.
- Acceptance criteria met for variability between replicate measurements:The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 0.3.

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with EPICLON EXA-7250, the mean relative viability was 73.0% compared to the negative control value. 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 conclusion, in this in vitro EPISKIN model test with EPICLON EXA-7250, the results indicate that the test item is non-irritant to skin (No Category).

Executive summary:

An in vitro skin irritation test of EPICLON EXA-7250 test item was performed in a reconstructed human epidermis model. EPISKIN-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 [1].

 

Disks of EPISKIN (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). An additional disk was used to provide an estimate of colour contribution from the test item. 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 EPICLON EXA-7250, the mean cell viability was 73.0% 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 conclusion, in this in vitro EPISKIN model test with EPICLON EXA-7250, the results indicate that the test item is non-irritant to skin (No Category).

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 August 2015-07 August 2015

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)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.: 15-EKIN-031, Expiry Date: 10 August 2015) is a three-dimensional human epidermis model. Adult humanderived 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.

Justification for test system used:

The EPISKIN-SM model has been validated for corrosivity testing in an international trial (Fentem, 1998) 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:

SKIN DISC PREPARATION
- Procedure used:EPISKIN-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.
In this assay, two replicates per test item per time point were used. Two negative controls and two positive controls were also run in the assay. As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.

- Quality control for skin discs: EPISKIN-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 dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkin-SM Test Kits used in the present study).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable):The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (21.9-22.4°C) covered with the plate lids.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps:After the incubation time (4 hours), all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with approximately 25 mL 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).

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):Due to the specific physical nature of the test item, higher amount than 20 mg was necessary to properly cover the entire surface of the Episkin units and meet the requirements the relevant OECD guideline (approximately 43 mg of test item was applied evenly to each of two test units and two additional colour control skin units) and then 100 µL physiological saline was added to the test item to ensure good contact with the epidermis.


NEGATIVE CONTROL
- Amount(s) applied (volume or weight):50 µL of physiological saline was added to each of the two negative control skin units;


POSITIVE CONTROL
- Amount(s) applied (volume or weight):50 µL of glacial acetic acid was added to each of the two positive control skin units.

Duration of treatment / exposure:

4 hours

Number of replicates:

2 replicates per test

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

97.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

82.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: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.


DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:The mean OD value of the two negative control tissues was in the recommended range (1.004).
- Acceptance criteria met for positive control:The positive control treated tissues showed 1.6% viability demonstrating the proper performance of the assay.
- Acceptance criteria met for variability between replicate measurements:The difference of viability between the two test item-treated tissue samples in the MTT assay was 16.5%.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with EPICLON EXA-7250, the mean cell viability was 90.0% 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.

In conclusion, in this in vitro EPISKIN model test with EPICLON EXA-7250, the results indicate that the test item is not corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test of EPICLON EXA-7250 test item was performed in a reconstructed human epidermis model. EPISKIN-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 EPISKIN (two units) were treated with EPICLON EXA-7250 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 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 EPICLON EXA-7250, the mean cell viability was 90.0% 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.  

In conclusion, in this in vitro EPISKIN model test with EPICLON EXA-7250, the results indicate that the test item is not corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 October 2015 - 10 October 2015

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:

yes

Remarks:

This deviation is considered to have no impact on the outcome of the study and interpretation of the results.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:S&K-LAP Kft. 2173 Kartal, Császár út 135, Hungary
- Age at study initiation:13 weeks old (young adult)
- Weight at study initiation:3584 g – 3841 g
- Housing:Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages
- Diet (e.g. ad libitum):Animals received UNI diet for rabbits produced by Cargill Takarmány Zrt., H-5300 Karcag, Madarasi út 0399, Hungary, ad libitum
- Water (e.g. ad libitum):The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system.
- Acclimation period:at least 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19.4 –22.6 °C
- Humidity (%):36 – 80 %
- Air changes (per hr):15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light):12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From:06 October 2015 To:10 October 2015

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

An amount of the 0.1 g of test item EPICLON EXA-7250 was administered to the animals.

Duration of treatment / exposure:

As the solid test item remained in the eye sac at the one hour observation time point, the treated eye of test animals was rinsed with physiological saline solution in all animals.

Observation period (in vivo):

The eyes were examined at 1, 24, 48 and 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated eye of test animals was rinsed with physiological saline solution in all animals.
- Time after start of exposure: 1 hour

SCORING SYSTEM:The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (02 October 2012).

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Other effects:

MORTALITY
There was no mortality observed during the study.
BODY WEIGHTS
The body weight of the animals was considered to be within the normal range of variability.
CLINICAL OBSERVATION
General daily examination
There were no clinical signs observed that could be related to treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item EPICLON EXA-7250, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.
According to Regulation (EC) No 1272/2008, EPICLON EXA-7250 does not require classification as an eye irritant.
According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, EPICLON EXA-7250 does not require classification as an eye irritant.

Executive summary:

An acute eye irritation study of the test item EPICLON EXA-7250 was performed in three New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 405, 2012). Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification.

 

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A single amount of 0.1 g of the test item was administered as a single dose.  

The eyes were examined at 1, 24, 48 and 72 hours after application.  

No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.  

Animal 1 (No: 837) clinical observation  

At one hour after the application, conjunctival redness (score 1) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.

At 24 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.  

At 48 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.  

At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.  

Animal 2 (No: 835) clinical observation  

At one hour after the application, conjunctival redness (score 1) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.

At 24 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.  

At 48 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.  

At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.  

Animal 3 (No: 832) clinical observation  

At one hour after the application, conjunctival redness (score 1) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.

At 24 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.  

At 48 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.  

At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

The general state and behaviour of animals were normal throughout the study period.  No mortality occurred during the study. The body weights of all rabbits were considered to be within the normal range of variability.  

The animals’ individual mean scores for all aniamls (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

Chemosis 0.00

Discharge 0.00

Redness 0.00

Cornea 0.00

Iris 0.00

The test item EPICLON EXA-7250, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.  According to Regulation (EC) No 1272/2008, EPICLON EXA-7250 does not require classification as an eye irritant.  According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, EPICLON EXA-7250 does not require classification as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)