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Description of key information

Under the conditions of this study, K-Salt was interpreted as a non-irritant to skin (UN GHS No Category) in the EpiDerm irritation assay.

Under conditions described for this experiment, K-Salt was interpreted as non-corrosive (UN GHS Non-Corrosive) in the EpiDerm corrosion assay.

The test substance, K- Salt, resulted in a mean viability of 7.8%, and is predicted to potentially require labelling as an ocular irritant (GHS category 2).

The in vitro studies have not returned a positive result for eye or skin irritation.

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:
2017 (report date 31st October 2017)
Reliability:
2 (reliable with restrictions)
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:
not specified
GLP compliance:
no
Remarks:
Lab was not set up to do GLP for these specific assays. Studies were performed in accordance with the principles of GLP and relevant OECD guideline. They were monitored by QA in accordance with GLP.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source: Mitsuboshi Chemical Co. Ltd. Lot: YY00G7P285
- Expiration date of the lot/batch: Date not specified - "The certificate of analysis lists the test material as having an expiration date of 2 years after shipment date (Miyazaki, 2016)"
- Purity test date: 99.8%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not specified
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test material(s) were tested as neat (100%) or as provided, following the OECD 431 test guideline.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

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

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) n/a

OTHER SPECIFICS: n/a
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Source strain:
not specified
Details on animal used as source of test system:
n/a
Justification for test system used:
The EpiDerm three-dimensional human skin model has been extensively characterised and is approved for identification and classification of non-corrosive and corrosive substances and mixtures in accordance with United Nations Global Harmonised System (UN GHS).
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: Upon receipt, the EpiDerm tissues were stored at 2-8ºC until used. On the day prior to testing, EpiDerm assay medium was warmed to room temperature and an aliquot of 0.9 mL was
dispensed into each well of a 6-well plate. The EpiDerm tissues were transferred from a 24-well shipping plate to a 6-well plate containing the fresh, pre-warmed assay medium. The EpiDerm tissues were incubated at approximately 37ºC in a humidified atmosphere of approximately 5% CO2 for 60 ± 5 min. At the end of the first preincubation period, the inserts were transferred into wells containing fresh warm assay medium and were incubated overnight (18 ± 3 hrs) to acclimate the tissue.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ] : Not specified

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

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

DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:]
- Spectrophotometer:
- Wavelength:
- Filter:
- Filter bandwidth:

NUMBER OF INDEPENDENT TESTING RUNS / 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. if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, but the mean disc dye content is greater than or equal to the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, and the mean disc dye content is well below the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): not specified

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): not specified

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): not specified
Duration of treatment / exposure:
60 minutes +/- 1 minute
Duration of post-treatment incubation (if applicable):
42 hours +/- 2 hours
Number of replicates:
3
Species:
other: EpiDerm
Strain:
other: Human
Details on test animals or test system and environmental conditions:
n/a
Type of coverage:
not specified
Preparation of test site:
other: Administered by topical application to the surface of the EpiDerm tissues: directly dispensed atop the tissue
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): not specified - as supplied by sponsor

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): not specified

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): not specified
Duration of treatment / exposure:
60 minutes +/- 1 minute
Observation period:
42 +/- 2 hours
Number of animals:
n/a
Details on study design:
TEST SITE
- Area of exposure: 0.6 cm^2
- % coverage: not specified
- Type of wrap if used: not specified

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes - tissues rinsed with sterile DPBS to remove test substance after 60 +/- 1 minute treatment period
- Time after start of exposure: 60 +/- 1 minute

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : not specified


SCORING SYSTEM:
- Method of calculation: The mean of the corrected OD570 values for the negative control was calculated using the formula:

Corrected Individual Tissue OD570 = individual tissue OD570 – mean blank OD570

For each individual tissue, % viability relative to negative control was calculated using the following formula:

% Viability= (Corrected Individual OD570 of Test Chemical (or Control) / Corrected Mean OD570 of Negative Control) X 100
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute +/- 1 minute
Value:
ca. 103
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: Not specified
- Direct-MTT reduction: Results from the assessment of direct test method reduction of MTT experiment suggested no direct reduction of MTT dye by K-Salt, as the test material did not turn the MTT solution to a blue/purple color.
- Colour interference with MTT: n/a

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes: The corrected mean OD570 value of the negative control tissues (exposed for 60 minutes) was 1.00 (i.e., ≥ 1.0 and ≤ 2.5; criteria set by the tissue manufacturer).
- Acceptance criteria met for positive control: Yes: The relative mean viability of positive control (1% TRITON™ X-100) was 16.4% (i.e., ≤ 20% compared to negative control; criteria set by the tissue manufacturer).
- Acceptance criteria met for variability between replicate measurements: Not specified
- Range of historical values if different from the ones specified in the test guideline: n/a
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, K-Salt was interpreted as a potential non-irritant to skin (UN GHS No Category) in the EpiDerm irritation assay
Executive summary:

K-Salt was evaluated for skin irritation potential in an in vitro EpiDerm skin irritation assay. In this assay, K-Salt was topically applied to the EpiDerm tissue for 60 minutes, followed by a 42-hour post-exposure recovery. Following recovery, the cell viability was measured in treated and control tissues using MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and the data reported as a percentage of the mean of negative control.

The mean relative cell viability of K-Salt and positive control-treated tissues were 103% and 16.4% (i.e. ≤ 50%), respectively. Therefore, under the conditions of this study, K-Salt was interpreted as a potential non-irritant to skin (UN GHS No Category) in the EpiDerm irritation assay.

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:
9 January to (date to be inserted)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
non-GLP study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:
no
Remarks:
Lab was not set up to do GLP for these specific assays. Studies were performed in accordance with the principles of GLP and relevant OECD guideline. They were monitored by QA in accordance with GLP.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: The Dow Chemical Company
- Lot No.of test material: YY00G7P285
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from exposure to light
- Stability under test conditions: Assumed stable for the durstion of the study
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was administered to the test system as a 20% (w/v) dilution in sterile, deionized water. Stability not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item (white crystalline powder) diluted to 20% (w/v) dilution in sterile, deionized water.
- Final dilution of a dissolved solid: 20% (w/v) dilution in sterile, deionized water.

FORM AS APPLIED IN THE TEST (if different from that of starting material) . As instructed by the Sponsor, the test substance was administered as a 20% (w/v) dilution in sterile, deionized water. The test article dilution was prepared by weighing the test article into a prelabeled conical tube. Sterile, deionized water added until a 20% (w/v) dilution ws acheived, and the vial vortexed mixed prior to application.


Species:
other: Bovine corneas, obtained as a by-product from freshly slaughtered animals.
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals (J.W. TREUTH & SONS, Inc., Baltimore, MD) (United States)
- Number of corneas: Three corneas were used in the presence of the positive or negative control. Five corneas were used in the presence of the test substance.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
- Time interval prior to initiating testing: The eyes were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice packs. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated.

- indication of any existing defects or lesions in ocular tissue samples: The eyes were grossly examined for damage and those exhibiting defects were discarded.
- Indication of any antibiotics used: The eyes were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice packs.
Vehicle:
water
Remarks:
sterile deinionized water
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume with unit): 750 µL of the test substance
- Concentration (if solution): The solid test substance, K-Salt, was tested as a 20% (w/v) dilution in sterile, deionized water.

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL of negative control, sterile, deionized water (Quality Biological).

Duration of treatment / exposure:
4 hour exposure at 32 ± 1ºC
Duration of post- treatment incubation (in vitro):
90 minutes at 32 ± 1ºC
Number of animals or in vitro replicates:
Three corneas were incubated in the presence of the positive or negative control at 32 ± 1ºC for approximately 4 hours.
Five corneas were incubated in the presence of the test substance at 32 ± 1ºC for approximately 4 hours.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS : The eyes were examined for damage, any exhibiting defects were discarded. The tissue surrounding the eyeball was carefully removed and the cornea was excised such that a 2 to 3 mm rim of sclera was present around the cornea. The isolated corneas were then stored in a Petri dish containing HBSS until they were mounted in a corneal holder. The corneas were mounted in holders with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and the screws tightened. Starting with the posterior chamber, the two chambers were then filled with Minimum Essential Medium (EMEM) without phenol red, containing 1% fetal bovine serum and 2 mM L glutamine (Complete MEM (without phenol red)). Each corneal holder was uniquely identified with a number in permanent marker, on both the anterior and posterior chambers. The corneal holders were incubated at 32 ± 1ºC for a minimum of 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS: The eyes were grossly examined for damage and those exhibiting defects were discarded.

NUMBER OF REPLICATES : Three corneas were incubated in the presence of the positive or negative control at 32 ± 1ºC for approximately 4 hours. Five corneas were incubated in the presence of the test substance at 32 ± 1ºC for approximately 4 hours.

NEGATIVE CONTROL USED : Sterile, deionized water (Quality Biological)

POSITIVE CONTROL USED : 20% dilution of imidazole in complete MEM

APPLICATION DOSE AND EXPOSURE TIME : An aliquot of 750 µL of test substance, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea, and incubated at 32 ± 1ºC for approximately 4 hours.
After the 4-hour exposure period, the controls or test substance treatments were removed. The epithelial side of each cornea was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the controls or test substance. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior and the posterior chambers were refilled with fresh Complete MEM (without phenol red) and a final opacity measurement was performed immediately (with no further post-exposure incubation).

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: yes, corneas were then incubated for approximately 90 minutes at 32 ± 1ºC.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After 4-hour exposure, the controls or test substance treatments were removed. The epithelial side of each cornea was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the controls or test substance. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior and the posterior chambers were refilled with fresh Complete MEM (without phenol red) and a final opacity measurement was performed immediately (with no further post-exposure incubation).

- POST-EXPOSURE INCUBATION: No post-exposure incubation after test substance removal.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Opacity measurement: The change in opacity for each cornea (including the negative control corneas) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of each cornea for that treatment condition.

- Corneal permeability:
Permeability Measurement: The mean OD490 value for the blank wells was calculated. The mean blank OD490 value was then subtracted from the raw OD490 value of each well (corrected OD490). Any dilutions that were made to bring the OD490 readings into the linear range of the platereader (OD490 should be less than 1.500), had each diluted OD490 reading multiplied by the dilution factor. The final corrected OD490 values of the test substance and the positive control were then calculated by subtracting the average corrected OD490 value of the negative control corneas from the corrected OD490 value of each treated cornea.

- Others (e.g, pertinent visual observations, histopathology): None

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) : For regulatory purposes, the In Vitro Score (IVIS) cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category I) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are found in the table below (OECD 437, adopted 26 July 2013). This guidance on categorization applies only to test substances evaluated using the appropriate standard protocols as described in OECD 437 .

IVIS UN GHS

<3 No Category
>3; <55 No prediction can be made
>55 Category I

For non-regulatory purposes, the following classification system was established by Sina et al. (1995) based on studies with a wide range of test materials. While this classification system provides a good initial guide to interpretation of these in vitro data, these specific ranges may not be applicable to all classes of materials or other exposure times. Whenever possible, results should be compared to “benchmark” materials tested under similar exposure conditions.

In Vitro Score:

≤ 25 = mild irritant
from 25.1 to 55 = moderate irritant
from 55.1 and above = severe irritant

In addition, Vanparys et al. (1993) proposed a modification of the Sina classification system based on an evaluation of 50 pharmaceutical and commercially available industrial compounds. The modification allows for the identification of a non-irritant classification.

In Vitro Score:
from 0 to 3 = non irritant
from 3.1 to 25 = mild irritant
from 25.1 to 55 = moderate irritant
from 55.1 and above = corrosive/severe irritant


DECISION CRITERIA:
Criteria for Determination of a Valid Test: The BCOP assay was accepted when the positive control (imidazole) caused an in vitro score that fell within two standard deviations of the historical mean. The results of the positive control fell within two standard deviations of the historical mean (within a range of 66.8 to 127.5) and the assay was considered valid.
Irritation parameter:
in vitro irritation score
Run / experiment:
1 run
Value:
80.7
Vehicle controls validity:
not specified
Positive controls validity:
valid
Remarks:
The results of the positive control fell within two standard deviations of the historical mean (range: 66.8 to 127.5). The assay was therefore considered valid.

 Assay Date

 IIVS Test substance No.

 Sponsor’s Designation

 Conc.

 Exposure Time

 Opacity Value

 OD490Value

 In VitroScore

 pH

 9 Jan 18

 17AJ00

 K-Salt  20%  4 hours  -3.0  -0.007  -3.1  2.5
 9 Jan 18  Positive control  Imidazole  20%  4 hours  62.3  1.224  80.7  -
                 
Interpretation of results:
GHS criteria not met
Remarks:
According to the current OECD TG 437 the test article is predicted to not require classification or labelling for ocular irritation according to the Globally Harmonized System (GHS No Category).
Conclusions:
Based on the in vitro score of -3.1, the test substance, K-Salt, would be classified as a non-irritant according to the classification system established by Vanparys et al. (1993), where a substance that induces an in vitro score 0 to 3 would be predicted to be a non-irritant to the eye.
According to the current OECD TG 437 the test article is predicted to not require classification or labelling for ocular irritation according to the Globally Harmonized System (GHS 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 in vitro studies have not returned a positive result for eye or skin irritation.