Fir, Abies balsamea, ext.

Endpoint summary

Description of key information

Skin irritation (OECD TG 439): irritant

Skin corrosion (OECD TG 431): not corrosive

Eye irritation (OECD TG 437): not irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Nov 2017 - 17 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142

Version / remarks:

31 May 2008

Deviations:

no

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: Botanical source and Lot# 67919
- Expiration date of the lot/batch: 13 septembre 2018
- Purity test date: UVCB, considered 100% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70 RH%), protected from light, avoided contact with iron.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: No dilution performed. Test item applied as-is
- Final preparation of a solid: not applicable/test item is a liquid

Test system:

human skin model

Remarks:

EPISKIN Small Model(TM), 0.38cm2, Batch# 17-EKIN-042,Manufactured by SkinEthic, France

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult Donors

Source strain:

other: Strain no:09-KERA-007 + 09-KERA-010

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recommended by the relevant OECD guideline for corrosivity

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used:
Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994).
- Quality control for skin discs:
EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (20.8 - 24.5 deg C) for 4 hours
- Temperature of MTT test (if applicable): 37 deg C for 3 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Not specified
- Observable damage in the tissue due to washing: None

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm


NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
- Two replicates test item, two negative controls and two positive controls were run. additionally, as the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used.


PREDICTION MODEL / DECISION CRITERIA
- The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2016).
- 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)

For more than 2 disks:
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.

Classification Packing group: Criteria for In Vitro interpretation:
UN GHS / EU-CLP Sub Category 1A If corrosive after 3 min exposure
Sub Category 1B If not corrosive after 3 min exposure and corrosive after 1 hour exposure
Sub Category 1C If not corrosive after 1 hour exposure and corrosive after 4 hours exposure
Non corrosive If not corrosive after 4 hours exposure

Control samples:

yes, concurrent negative control

yes, concurrent positive control

other: As the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): Physiological saline (0.9% (w/v) NaCl solution)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): undiluted Glacial Acetic Acid by VWR Lot#15A160011


The same treatment steps were followed in case of the killed tissues as in case of the living tissues.

Duration of treatment / exposure:

4 hours (±10 min) at room temperature (20.8-24.5°C)

Duration of post-treatment incubation (if applicable):

After the incubation times, all test item treated tissues or also the positive control tissues or also the additional control tissues (as mentioned in 3.6.2.) 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.
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (±15 minutes), protected from light, in a >95% humidified atmosphere.

Number of replicates:

2

Details on test animals or test system and environmental conditions:

Not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4 hours exposure

Value:

109.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean OD 0.8 = 100% RV

Positive controls validity:

valid

Remarks:

Mean OD 0.006 = 0.8% RV

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: NSMTT% = -0.9
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not Specified

ACCEPTANCE OF RESULTS:
- 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.

-Range of historical values if different from the ones specified in the test guideline: SEE TABLE

The Non-Specific Colour % (NSC_living%) was determined in the In Vitro Skin Irritation Test. This value was below 5% and additional data calculation was not necessary, therefore check-method for colouring potential of test-items the use of additional controls for the non specific OD evaluation was not necessary in this study

Negative values of additional controls (e.g.: NSMTT%) were excluded from the calculation, but this did not change the result or integrity of the study.

Interpretation of results:

other: Not corrosive to the skin

Remarks:

Based on CLP criteria (Annex I 1272/2008/EC)

Conclusions:

In conclusion, in this in vitro EpiSkin™(SM) model test with Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) (Batch number: 67919), the results indicate that the test item is non-corrosive to the skin in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

An in vitro skin corrosivity test of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext)test item was performed in a reconstructed human epidermis model. EPISKIN^TM(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^TM(SM) (two units) were treated with Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) 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). The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units.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 Fir Needle Oil Canadian (Fir, Abies Balsamea, ext), the mean cell viability was 109.1% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) (Batch number: 67919), the results indicate that the test item is non-corrosive to the skin. The test item was not corrosive after the exposure time of 4 hours, therefore additional experiment with additional exposure times were not necessary to the classification.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Oct 2017 - 20 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Botanical source and Lot# 67919
- Expiration date of the lot/batch: 13 septembre 2018 (retest date)
- Purity test date: UVCB, considered 100% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70 RH%), protected from light, avoided contact with iron.

Test system:

human skin model

Remarks:

EPISKIN Small Model(TM), 0.38cm2, Batch# 17-EKIN-042,Manufactured by SkinEthic, France

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Source strain:

other: Strain no:09-KERA-007 + 09-KERA-010

Details on animal used as source of test system:

Not relevant

Justification for test system used:

Performing an in vitro test before considering the in vivo test is mandatory as per the REACH regulation and guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin Small Model 0.32cm2
- Tissue batch number(s): 17-EKIN-042
- Production date: Not specified
- Shipping date: Not specified
- Expiry date: 23 October 2017
- Date of initiation of testing: 18 October 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (26.2-26.8 deg C)
- Temperature of post-treatment incubation (if applicable): 37 deg C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinse with Phosphate buffered Saline (PBS)
- Observable damage in the tissue due to washing: Not reported
- Modifications to validated SOP: Not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3hrs at 37 deg C
- Spectrophotometer:
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is less or equal (≤) to 50% of the mean viability of the negative controls.
- In case the test item is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test item is considered to be irritant to skin in accordance with UN GHS Category 2.

- The test item may be considered to be non-irritant to skin in accordance with UN GHS (No Category), if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than (˃) to 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of PBS (Sigma-Aldrich Lot#BCBT9380)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of SDS (lach:ner Lot#PP/2016/11637)
- Concentration (if solution): 5% (w/v) of SDS in water

Duration of treatment / exposure:

15 min (± 0.5 min)

Duration of post-treatment incubation (if applicable):

After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.
After the 42 hours incubation, all EPISKINTM (SM) units (except the two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere.

Number of replicates:

3

Details on test animals or test system and environmental conditions:

Not applicable

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean OD value 0.255

Run / experiment:

mean (percentage of control)

Value:

30.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean OD value 0.833.

Positive controls validity:

valid

Remarks:

Mean tissue viability 6.8%. Mean OD value of 0.056

Remarks on result:

positive indication of irritation

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 item 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

- 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 tissues was 0.017, Non Specific Colour % was calculated as 2.0%. This value was below 5%, therefore additional data calculation was not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not specified

ACCEPTANCE OF RESULTS:

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the three negative control tissues was in the recommended range (0.833). Standard deviation of the viability results for negative control samples was 2.4%.
The positive control treated tissues showed 6.8% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.0%.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 0.7%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.

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

Interpretation of results:

other: Skin Irritant

Remarks:

Cat 1 or 2. In the case the test item is found to be non-corrosive, the test item is considered to be irritant to skin in accordance with UN GHS Category 2

Conclusions:

After treating the EPISKIN (SM) with Fir Needle Oil Canadian, the relative cell viability (when compared to the negative controls cell viabilities), decreased to 30.6%, which is below the threshold of irritancy of 50%. Therefore, Fir Needle Oil Canadian is irritant to skin in accordance with UN GHS (Category 1 or Category 2).

Executive summary:

An in vitro skin irritation test of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext)test item was performed in a reconstructed human epidermis model. EPISKIN^TM(SM) is designed to predict and classify the irritation potential of chemicals by measuring their 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^TM(SM) (three units) were treated withthe test itemand incubated for 15 minutes at room temperature. Exposure to 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, in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution atin an incubator with 5% CO₂protected from light, in a >95% humidified atmosphere. 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). Two additional disks were used to provide an estimate of colour contribution (NSC_living) 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 to Fir Needle Oil Canadian (Fir, Abies Balsamea, ext), the mean cell viability was 30.6% compared to the negative control. This is below the threshold of 50%, therefore the test item was considered as being irritant to skin in accordance with UN GHS (Category 1 or Category 2).

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

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP

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)

Deviations:

no

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: Botanical source and Lot# 67919
- Expiration date of the lot/batch: 13 septembre 2018
- Purity test date: UVCB, considered 100% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70 RH%), protected from light, avoided contact with iron.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: No dilution performed. Test item applied as-is
- Final preparation of a solid: not applicable/test item is a liquid

Species:

chicken

Strain:

other: chicken: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Commercial abattoir of chickens for human consumption
- Number of animals: Several (or not relevant)
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were transported at ambient temperature wrapped with tissue paper moistened with saline.
- Time interval prior to initiating testing: Max 2 hours
- indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: Information is not available

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 µL for test item and both controls

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

One eye was treated with physiological saline (negative control), three eyes with the test item and another three eyes with powdered 5% (w/v) Benzalkonium chloride solution (positive control).

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids on the heads were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the Haag-Streit BP 900® slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
One eye was treated with the negative control. Three eyes were treated with the test item and another three eyes with the positive control.

NEGATIVE CONTROL USED
Physiological saline (Salsol solution, 0.9% w/v NaCl)

POSITIVE CONTROL USED
Benzalkonium chloride solution, 50 % in water diluted to obtain a 5% (w/v) solution

APPLICATION DOSE AND EXPOSURE TIME
30 µL was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly for 10 seconds

OBSERVATION PERIOD
Prior to treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse (fluorescein retention is determined only prior to treatment and 30 minutes after test chemical exposure)

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual of the test item if possible.
- Indicate any deviation from test procedure in the Guideline: No deviation

EVALUATION
Corneal thickness or swelling determination: Corneal swelling is determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope with the following settings: Slit length/full length/position 8 and Slit width/Fully open/Setting 9.5
Corneal opacity determination: Corneal opacity is scored using the area of the cornea that is most densely opacified with the following settings for the slit lamp microscope: Slit length/full length/position 8 and Slit width/Fully open/Position on indefinite
Fluorescein retention determination: The fluorescein retention will be measured on two occasions, baseline (t=0) and 30 minutes after the post-treatment rinse. the setting for the slip lamp microscope is identical to the opacity assessment, except with the green light filter


SCORING SYSTEM:
- Mean corneal swelling (%) and ICE Class
0-5 I (No Swelling)
>5 to 12 II (Slight Swelling)
: :
>32 IV (Severe Swelling)

- Mean maximum opacity score and ICE Class
0.0 - 0.5 I (No opacity)
0.6 - 1.5 II (Slight opacity)
1.6 - 2.5 III (Moderate opacity)
2.6 - 4.0 IV (Severe or total opacity)

- Mean fluorescein retention score at 30 min post-treatment and ICE class
0.0 - 0.5 I (No fluorescein retention)
0.6 - 1.5 II (Slight fluorescein retention)
1.6 - 2.5 III (Moderate fluorescein retention)
2.6 - 4.0 IV (Severe fluorescein retention)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Run / experiment:

See 'Remarks on results'

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Overall ICE Class 3xI, hence, the negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

Positive controls validity:

valid

Remarks:

Overall ICE Class : 1xIII 2xIV, hence, the positive control 5% (w/v) Benzalkonium chloride solution was classified as severely irritating, UN GHS Classification: Category 1.

Remarks on result:

no indication of irritation

Remarks:

Overall ICE Class of test item: 3xI, hence, the test item is non-irritant, UN GHS Classification: No Category. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.

Other effects / acceptance of results:

Morphological effects: In the positive control group, severe loosening of epithelium was observed in one eye at 120 minutes and in one eye at 240 minutes after the post-treatment rinse.
No other morphological effect was observed in the study.

RESULTS:

Test item

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	0.50	I
Mean fluorescein retention	0.17	I
Other Observations	None
Overall ICE Class	3xI

Positive Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	7.5%	II
Mean maximum corneal swelling at up to 240 min	23.4%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Severe loosening of epithelium was observed in one eyes at 120 minutes and in one eyes at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

Negative Control

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	3xI

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditons of this study, Fir Needle Oil Canadian, was found to have an overall ICE Classification of 3xI. Therefore, based on this in vitro eye irritation in the isolated chicken eyes test with Fir Needle Oil Canadian (Fir, Abies Balsamea, ext), the test item is non-irritant, UN GHS Classification: No Category.

Executive summary:

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 (26 July 2013).

After the zero reference measurements, the eye was held in horizontal position and 30 µL of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 µL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated 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 in good correlation with the historical control data. Thus, the experiment was considered to be valid.

No corneal swelling was observed during the four-hour observation period on test item treated eyes. No significant corneal opacity change (severity 0.5) was noted on the three eyes. No significant fluorescein retention change (severity 0.5) was noted on one eye and no fluorescein retention change was noted on two eyes. No other corneal effect was observed.

Based on this in vitro eye irritation in the isolated chicken eyes test, Fir Needle Oil Canadian is non-irritant, UN GHS Classification: No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the results of the irritation and corrosive tests, Fir Needle Oil Canadian is a skin irritant but not a skin corrosive. Therefore, it is irritant to skin in accordance with UN GHS Category 2.

It is not an eye irritant as per the results of the eye irritation test.