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Description of key information

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:
10 October to 01 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Bx 20170320
- Expiration date of the lot/batch: 20 March 2018
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°C, ≤70% Relative Humidity, RH), protected from humidity (tight closed container), protected from light.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
Test system:
artificial membrane barrier model
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM (SM)
- Tissue batch number(s): Manufacturer: SkinEthic, France, Batch No.:17-EKIN-042, Expiry Date: 23 October 2017
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing:

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
- Incubation time:
- Spectrophotometer:
- Wavelength:
- Filter:
- Filter bandwidth:
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES:

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 substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
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): 20 mg
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit): 10 µL distilled water
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 5% w/v
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
15 m
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
72.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
73.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
80.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in this in vitro EPISKIN TM (SM) model test with p-toluic acid (Batch number: 20170320), the results indicate that the test item is non-irritant to skin.
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:
09 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CCI Corp.; Bx 20170320
- Expiration date of the lot/batch: 20 March 2018
- Purity test date: 20 march 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70% Relative Humidity, RH), protected from humidity (tight closed container), protected from light.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

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: not applicable
- Final preparation of a solid: ground to fine powder using a mortar and pestle

Species:
chicken
Strain:
other: ROSS 308 and COBB 500
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary
- Number of animals: 4 to 5
- Characteristics of donor animals (e.g. age, sex, weight): Approx. 7 weeks old; no infromation on sex or weight.
- 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.
- Time interval prior to initiating testing: within 2 hours
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: none
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): 30mg

Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Number of animals or in vitro replicates:
2
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Selection; After removing the head from the plastic box, it was put on soft paper. The eyelids 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. The fluorescein-treated cornea was then 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 undamaged and in good condition, the eyeball was carefully removed from the orbit.
Preparation; The eye ball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting the optic 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
Equilibration; The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to clearly see the physiological saline 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 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.
Baseline; 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-point. No changes in thickness (0.0%) were observed in the eyes in each experiment. 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
2

NEGATIVE CONTROL USED
Name: Physiological saline (Salsol solution, 0.9% (w/v) NaCl)
Batch numbers: 70323Y05-1, 72034Y05-1
Manufacturer: B. Braun Pharmaceuticals SA
Expiry dates: 31 December 2019, 30 April 2020
Storage condition: Room temperature


SOLVENT CONTROL USED (if applicable)

POSITIVE CONTROL USED
Name: Imidazole
CAS Number: 288-32-4
Manufacturer: Sigma-Aldrich Co.
Batch number: SLBK9670V
Expiry date: 30 November 2017
Storage conditions: Room temperature

APPLICATION DOSE AND EXPOSURE TIME
30mg for 10 seconds

OBSERVATION PERIOD
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature,
- Indicate any deviation from test procedure in the Guideline
No data

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Cornea opacity was calculated according to the following formulae:


ΔCO at time t = CO at time t – CO at t=0

Mean ΔCOmax = FECOmax(30min to 240min)+ SECOmax(30min to 240min)3 + TECOmax(30min to 240min)

- Damage to epithelium based on fluorescein retention:
Fluorescein retention was calculated according to the following formulae:


ΔFR at time t = FR at time t – FR at t=0

Mean ΔFR = FEFR (30min) + SEFR3(30 min) + TEFR(30min)

- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting:
- Macroscopic morphological damage to the surface:
- Others (e.g, histopathology):

SCORING SYSTEM:
- Mean corneal swelling (%) ; 1.5%
mean maximum opacity score ; 0.42
- Mean fluorescein retention score at 30 minutes post-treatment ; 0.5

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. No data
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
0.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
2
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
2
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
up to 75 minutes.
Run / experiment:
1
Value:
1.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
up to 240 minutes
Run / experiment:
1
Value:
1.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
up to 75 minutes
Run / experiment:
2
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
up to 240 minutes
Run / experiment:
2
Value:
2.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: none
Interpretation of results:
GHS criteria not met
Conclusions:
The test item p-toluic acid showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of OECD guideline No. 438. The second experiment confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes with p-toluic acid, the test item was 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

Additional information

Justification for classification or non-classification

The results of the studies show that the test substance is not irritating to skin or eyes.