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Description of key information

Skin irritation (OECD TG 439): non-irritant (confirmational assay is ongoing)

Eye irritation (OECD TG 438): non-irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 May 2019 - July 2019
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
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN Small Model (EPISKIN-SM, 0.38 cm2, Batch no.: 19-EKIN-019 and 19-EKIN-013), SkinEthic Laboratories, Lyon, France
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, )
- Tissue batch number(s): Batch no.: 19-EKIN-019 and 19-EKIN-013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 36.3 - 37.1°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.
- The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly (acceptability criteria).

NUMBER OF REPLICATE TISSUES: 3

DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25μL undiluted
NEGATIVE CONTROL
- Amount(s) applied: 25μL undiluted
POSITIVE CONTROL
- Amount(s) applied: 25μL
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours at 37°C
Number of replicates:
three
Irritation / corrosion parameter:
% tissue viability
Remarks:
Main experiment
Run / experiment:
1
Value:
80
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
15
Irritation / corrosion parameter:
% tissue viability
Remarks:
Main experiment
Run / experiment:
2
Value:
30
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
15
Irritation / corrosion parameter:
% tissue viability
Remarks:
Main experiment
Run / experiment:
3
Value:
109
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
15
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: yes (7.2%)
- Colour interference with MTT: yes (non-specific, 4.4% of negative control tissues)

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 15%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated with positive or negative control was < 6%, indicating that the test system functioned properly.

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: no - SD to high between test item tissues

Since the individual values of the first experiment were both above and below the criterium of 50% (30, 80 and 109% respectively) the test has to be repeated (planned for July 2019).

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria (Annex I 1272/2008/EC)
Conclusions:
Based on the results obtained, Blue Tansy Oil is not considered a likely skin irritant, and does not have to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC). However, the results were spread over two categories (80%, 30% and 109%), which lead to a high standard deviation and therefore this assay will be repeated.
Executive summary:

The skin irritation potential of Blue Tansy Oil was tested in accordance to OECD TG 439. Undiluted test item was topically applied to EPISKIN-SMTM for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements. Both the positive (15% viability) and the negative control were within the historical control data range and therefore considered valid. The standard deviation value of the percentage viability of three tissues treated with positive or negative control was < 6%, indicating that the test system functioned properly. The tissue viability obtained after 15 ± 0.5 minutes treatment with test item compared to the negative control tissues was 30, 80 and 109%.

Since the mean relative tissue viability for Blue Tansy Oil was above 50% after 15 ± 0.5 minutes treatment Blue Tansy Oil could be considered a non-irritant. Therefore, Blue tansy oil does not have to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC). Results were however spread over two categories (80%, 30% and 109%), which lead to a high standard deviation and therefore this assay will be repeated.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
24 May 2019
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes
Species:
chicken
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Baileys Turkeys Ltd., Cheshire, UK, (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler))
- Number of animals: Multiple
- Characteristics of donor animals (e.g. age, sex, weight): approximately 3 kg and approximately 56 days old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Time interval prior to initiating testing: same day
- indication of any existing defects or lesions in ocular tissue samples: The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 mL to the cornea
- Concentration (if solution): unchanged
Duration of treatment / exposure:
10 seconds
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES: Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected. Eyelids were carefully excised and the integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined for damage of cornea with the use of the Haag-Streit BQ 900 (Switzerland) microscope, An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are or smaller then 0.5.

Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip and examined again for damage. The temperature of the chambers was at 32 ±1.5 °C.

EQUILIBRATION AND BASELINE RECORDINGS
After the approval process the eyes were incubated for approximately 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Sodium chloride 0.9% w/v

POSITIVE CONTROL USED: Benzalkonium chloride 5 v/v

APPLICATION DOSE AND EXPOSURE TIME: 0.3 mL was applied for 10 seconds

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: After 10 seconds of exposure, the surface of the cornea was rinsed with 20 mL isotonic saline.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calculated with the most densely opacified areas for scoring
- Damage to epithelium based on fluorescein retention: calculated at the 30 minute time interval only
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Percentage corneal swelling was assessed from corneal thickness measurements.
- Macroscopic morphological damage to the surface: evaluated with Haag-Streit BQ 900 (Switzerland) microscope

DECISION CRITERIA:
Once each endpoint had been established, ICE classes were determined based on a predetermined range. The overall in vitro irritancy classification of the test item was determined by using all the
results applied it to ICE classification criteria. A test was considered acceptable if the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non-Classified and GHS Category 1, respectively.
Irritation parameter:
cornea opacity score
Remarks:
mean
Run / experiment:
0, 30, 75, 120, 240 minutes
Value:
0.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Remarks:
mean
Run / experiment:
30 minutes
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Remarks:
mean
Run / experiment:
30 minutes
Value:
4.46
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Remarks:
mean
Run / experiment:
75 minutes
Value:
9.41
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Remarks:
mean
Run / experiment:
120 minutes
Value:
8.42
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Remarks:
mean
Run / experiment:
180 minutes
Value:
7.43
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Remarks:
mean
Run / experiment:
240 minutes
Value:
10.89
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: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, valid positive and negative controls.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean corneal opacity 0, mean Fluorescein retention 0, Mean corneal swelling up to 3.70%; 3x ICE class I)
- Acceptance criteria met for positive control: yes (mean corneal opacity up to 4.0, mean Fluorescein retention 2.7, Mean corneal swelling up to 39.60%; 3x ICE class IV)
Interpretation of results:
other: not classified
Remarks:
based on CLP criteria (Annex I of 1272/2008/EC)
Conclusions:
Under the conditions of this test, Blue Tansy oil does not need to be classified for eye irritation, according to the criteria laid down in Annex I of 1272/2008/EC (CLP).
Executive summary:

In an isolated chicken eye test, performed according to the 438 OECD Guideline under GLP conditions, Blue Tansy oil was tested to evaluate the eye hazard potential of the substance. 0.3 mL of the test item was applied to the cornea of each of 3 chicken enucleated eyes and after 10 seconds the corneas were rinsed with isotonic saline. After the exposure, toxic effects were measured by qualitative assessment of opacity (0.3, ICE class I), fluorescein retention (0.5 ICE class I), and corneal swelling (10.89% ICE class II). Valid positive (overall ICE class IV) and negative (overall ICE class I) controls were included. According to the overall in vitro irritancy criteria, the ICE classes combined for teat item lead to result ‘no Category’. Therefore under the conditions of this test, the test item does not need to be classified for eye irritation, according to the criteria laid down in Annex I of 1272/2008/EC (CLP).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation OECD 439

The skin irritation potential of Blue Tansy Oil was tested in accordance to OECD TG 439. Undiluted test item was topically applied to EPISKIN-SMTM for 15 minutes. After a 42 hour postincubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements. Both the positive (15% viability) and the negative control were within the historical control data range and therefore considered valid. The standard deviation value of the percentage viability of three tissues treated with positive or negative control was < 6%, indicating that the test system functioned properly. The tissue viability obtained after 15 ± 0.5 minutes treatment with test item compared to the negative control tissues was 30, 80 and 109%.

Since the mean relative tissue viability for Blue Tansy Oil was above 50% after 15 ± 0.5 minutes treatment Blue Tansy Oil could be considered a non-irritant. Therefore, Blue tansy oil does not have to be classified for skin irritation in accordance with the criteria outlined inAnnex I of the CLP Regulation (1272/2008/EC). Results were however spread over two categories (80%, 30% and 109%), which lead to a high standard deviation and therefore this assay will be repeated.

Eye Irritation OECD 438

In an isolated chicken eye test, performed according to the 438 OECD Guideline, under GLP, Blue Tansy oil was tested to evaluate the eye hazard potential of the substance. 0.3 mL of the test item was applied to the cornea of each of 3 chicken enucleated eyes and after 10 seconds the corneas were rinsed with isotonic saline. After the exposure, toxic effects were measured by qualitative assessment of opacity (0.3, ICE class I), fluorescein retention (0.5 ICE class I), and corneal swelling (10.89% ICE class II). Valid positive (overall ICE class IV) and negative (overall ICE class I) controls were included. According to the overall in vitro irritancy criteria, the ICE classes combined for teat item lead to result no Category. Therefore under the conditions of this test, the test item does not need to be classified for eye irritation, according to the criteria laid down in Annex I of 1272/2008/EC (CLP).

Justification for classification or non-classification

Based on the available data, Blue Tansy oil does not need to be classified as skin or eye irritant in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).