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Skin irritation / corrosion

Link to relevant study records

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Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
The Corrositex® - InVitro International, Placentia, CA
Principles of method if other than guideline:
The Corrositex® (InVitro International, Placentia, CA) test is a standardized and reproducible method that can be employed to determine the potential corrosivity and the Packing Group classification of specified
categories of chemical compounds under the hazardous materials transportation regulations administered by the U.S. Department of Transportation (DOT) and international dangerous goods codes. The
Corrositex® test predicts1,2 the in vivo corrosive potential of a chemical compound or mixture by using as an endpoint the amount of time it takes for a chemical to permeate or destroy a synthetic biobarrier.
A color change in a proprietary liquid Chemical Detection System (CDS) is used to indicate that the chemical has passed through the biobarrier. This assay system is depicted below.

The Corrositex® test is a three step procedure. First, each test article was qualified to ensure that it was compatible with the Corrositex® system, and then it was categorized according to pH to determine cut-off
times. Finally, they were classified based on the mean time each test article took to penetrate the biobarriers.
Qualification
For each test article, 150 μl or 100 mg of the test article were added to the CDS reagent in a Qualify vial, and the vial was observed for any notable color change. An observable color change indicates that the
test article is compatible with the Corrositex® system.
Categorization
Next, each test article was categorized, which determined cut-off times for the Packing Group designations. A 10% formulation of each test article was prepared and its pH was measured. If the pH of the 10%
formulation was  7.0, 150 μl or 100 mg of the neat test article were added to Tube A. The pH of Tube A was then measured, and if it was  5.0, the test article was assigned to Category 1, and if it was  5.0, the
test article was assigned to Category 2. If the pH of the 10% formulation was  7.0, 150 μl or 100 mg of the neat test article were added to Tube B. The pH of Tube B was measured, and if the final pH was  9.0, the
test article was Category 1, and if it was  9.0, the test article was Category 2.
Classification
Finally, each test article was classified by adding 500 μl or 500 mg of the test article to four test vials containing biobarriers to determine the Packing Group. If the test article mixture permeates or destroys
the biobarrier, it will come in contact with the CDS reagent in the vial and induce a color change similar to that observed in the Qualify tube. The amount of time required for the test article to permeate or destroy
each biobarrier was recorded and the mean time of the four replicates is used to designate the United Nations (U.N.) Packing Group classification. Classifications are as follows: Packing Group I (severe
corrosivity), Packing Group II (moderate corrosivity), Packing Group III (mild corrosivity), or Non-Corrosive. A positive control was performed using 1.0 N Sodium Hydroxide . The result for the Positive
Control is considered valid if it falls within the range of the MB Research historical mean ± 2 standard deviations. A negative control was performed using 1% citric acid; the result is considered valid if the
breakthrough time is greater than 60 minutes.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Lot# 141125
Test system:
artificial membrane barrier model
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The crushed test article was used neat.
Irritation / corrosion parameter:
penetration time (in minutes)
Run / experiment:
1
Value:
> 67
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
penetration time (in minutes)
Run / experiment:
2
Value:
> 66.92
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
penetration time (in minutes)
Run / experiment:
3
Value:
> 66.83
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
penetration time (in minutes)
Run / experiment:
4
Value:
66.58
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
1-Chloro-6-Hydroxyhexane was found to be non-corrosive in Corrositex study.
Executive summary:

1-Chloro-6-Hydroxyhexane was found to be non-corrosive in Corrositex study.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 February - 02 September 2020
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:
adopted 18 June 2019
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted 6 July 2012.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source: Sponsor; Batch: 141125
- Purity, including information on contaminants, isomers, etc.: 98.86%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed human epidermis model
Cell source:
other:
Justification for test system used:
The test method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermal keratinocytes as cell source, representative tissue and cyto-architecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: three-dimensional reconstructed human epidermis model EpiDermTM, comprised of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis
- Tissue batch number(s): 30847
- Production date: Febraury 12, 2020


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution (1 mg/mL)
- Incubation time: 60 min
- Spectrophotometer: Tecan Infinite 200Pro; Lot no. 071119ALD; MatTek Corporation, 200 Homer Avenue Ashland, MA 01721, USA
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 19.968 +- 0.158
- Barrier function: 5.05 h

NUMBER OF REPLICATE TISSUES: 3


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

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 vehicle
yes, concurrent positive control
Amount/concentration applied:
30 µL
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1. experiment
Value:
ca. 5.2
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Assay acceptance criterion 1: Negative control
The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean ODof the NC tissues is ≥ 0.8 and ≤ 2.8.

Assay acceptance criterion 2: Positive control
A 5% SDS (in H2O) solution was used as a positive control (PC) and tested concurrently with the test chemicals. Concurrent means here that the PC has to be tested in each assay, but only one PC is required per testing day.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%.

Assay acceptance criterion 3: Standard deviation
Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low.
The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%.

Three tissues were used for each treatment and concurrent control group. Cell viability was determined by using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

The test item was applied as liquid test item topically undiluted to the model skin surface. Dulbecco’s phosphate buffered saline (DPBS) was used as the negative control, 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium. The mean viability of cells exposed to the test item was 5.2% of the negative control and, hence, was below the cut-off percentage cell viability value of ≤ 50% that distinguishes irritant from non-irritant test items. The test item was considered to be cytotoxic and predicted to be irritant to skin in accordance with UN GHS category 1 or 2 (H314 or H315). The mixture of test item with water or isopropanol did not show any discolouration nor did the test item itself directly interact with MTT. Therefore, no quantitative correction of assay results was necessary. The mean optical density (OD) of 3 negative control tissues was 1.705 and was well within the acceptable range of ≥ 0.8 to ≤ 2.8. The viability of cells treated with the positive reference item, 5% SDS, was 5.1% of the negative control and fulfilled the acceptance criterion of ≤ 20%.

The standard deviation calculated from individual % tissue viabilities of the 3 identically treated replicates was below the limit of acceptance of 18%. Hence, all acceptance criteria required were fulfilled. Historical data of negative and positive controls are presented in "Overall remarks, attachements" under Attachments.

Table 1 Summarized data of in vitro Skin irritation

   Optical density [OD 540] DPBS  Mean Viability (n=3) [%]  SD Viability %
 Negative control DPBS  1.705  100.0  2.1
 Test item  0.088  5.2  0.3
 Positive control 5% SDS  0.087  5.1  0.0

SD: Standar deviation

CV: Coefficient of variation

SDS: Sodium dodecyl sulphate

D-PBS: Dulbecco´s phosphate buffered saline

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the present test conditions, the test item tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was cytotoxic and, hence, predicted to be irritant to skin in an experiment employing an artificial three-dimensional model of human skin in accordance with UN GHS category 1 or 2 (H314 or H315).
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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)
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
The bovine eyes, in a Hank's Balanced Salt Solution with penicillin-streptomycin, were received from Spear Products on 11 Aug 2016 and transported to MB Research in a refrigerated container.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
A volume of 0.75 ml of ethanol, MEM or test article was applied to the epithelium of each of the three positive controls, three negative controls or three test article-treated corneas in a manner, which ensured the entire cornea was covered. All corneas were dosed via the closed-chamber method.
Duration of treatment / exposure:
All holders and corneas were placed in a horizontal position (anterior side up) in the 32ºC (± 1º) incubator. After 10 (± 1) minutes, the test article, ethanol or MEM solution was removed from the epithelium of the
cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were
refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill. All corneas were incubated at 32ºC (± 1º) for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the
OP-KIT. This reading was used in the final IVIS calculations. Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution in Dulbecco's Phosphate Buffered Saline (PBS). Each holder was returned to the 32ºC (±1º) incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea.
After 90 (± 5) minutes, the fluid from the posterior chamber was removed and the amount of dye, which passed through the cornea (permeability), was measured as the optical density at 490 nm by a
spectrophotometer. A 1:1000 dilution of the fluorescein was prepared and measured in the spectrophotometer as a measure of consistency.
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
See Details on study design
Number of animals or in vitro replicates:
Not applicable
Details on study design:
The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded.
Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS.
The dissected corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects.
The entire holder was incubated at 32ºC (± 1º) and allowed to equilibrate for at least one hour but not longer than two hours.
A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied with the opacitometer. Any cornea with a value greater than 7 units was discarded.
Following the pretest observations, the MEM solution was removed from the anterior chamber. A volume of 0.75 ml of ethanol, MEM or test article was applied to the epithelium of each of the three positive controls, three negative controls or three test article-treated corneas in a manner, which ensured the entire cornea was covered. The closed-chamber method of dose administration was used.
All holders and corneas were placed in a horizontal position (anterior side up) in the 32ºC (± 1º) incubator. After 10 (± 1) minutes, the test article, ethanol or MEM solution was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.
All corneas were incubated at 32ºC (± 1º) for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the
OP-KIT. This reading was used in the final IVIS calculations.
Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution in Dulbecco's Phosphate Buffered Saline (PBS). Each holder was returned to the 32ºC (±1º) incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea.
After 90 (± 5) minutes, the fluid from the posterior chamber was removed and the amount of dye, which passed through the cornea (permeability), was measured as the optical density at 490 nm by a spectrophotometer. A 1:1000 dilution of the fluorescein was prepared and measured in the spectrophotometer as a measure of consistency.
Data analysis
Individual corrected opacity scores were calculated by subtracting the pretest score from the ten-minute and two-hour scores. Corrected mean opacity scores were calculated by averaging the individual two-hour corrected opacity scores for a given dose group and subtracting the mean opacity score for the negative control group. A corrected mean opacity score was not calculated for the negative control, rather only the mean of the individual two-hour corrected opacity scores were calculated (with no subtraction of mean opacity score for negative control.)
Individual corrected optical densities were calculated by subtracting the mean optical density for the negative control group from the individual optical density values. Individual corrected optical densities were not calculated for the negative control group. Corrected mean optical densities were calculated by averaging the individual corrected optical density values for a given dose group. A corrected mean optical density was not calculated for the negative control, rather only the mean of the individual optical densities was calculated.
The In Vitro Irritancy Score (IVIS) for the test article and Vehicle control was calculated by adding the corrected mean opacity score to fifteen times the corrected mean optical density, as shown by the equation below. The calculation to obtain an IVIS for the positive control was performed in the same manner as the test article.
In Vitro Irritancy Score (IVIS) = Corrected Mean Opacity Score + (15 x Corrected Mean Optical Density Score)
The calculation to obtain an IVIS for the negative control was performed by adding the mean opacity score to fifteen times the mean optical density, as shown by the equation below.
In Vitro Irritancy Score (IVIS) = Mean Opacity Score + (15 x Mean Optical Density Score)

Based on the IVIS score, the test article was classified according to the prediction model described in DB-ALM Protocol No. 1271, a modification of the prediction model suggested by Gautheron, et al. (1994). Results from test situations should be compared to known materials tested under similar conditions. This classification system is the most consistent with recent regulatory reviews of the assay performance.
IVIS CLASSIFICATION
0 to 3 Non-Irritant
3.1 to 25 Mild Eye Irritant
25.1 to 55 Moderate Eye Irritant
55.1 and above Severe/Corrosive Eye Irritant
OECD Guideline #437 defines a substance that produces an IVIS of > 55 as Category 1, a substance that causes “Serious eye damage.”
IVIS UN GHS2
≤ 3 No Category
>3 to ≤55 No prediction can be made
>55 Category 1
The assay was accepted because the positive control had an IVIS that fell between two standard deviations of the historical mean.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean value for 3 experiments
Value:
23.06
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Mean of 3 experiments
Value:
14.34
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
Category 2B (mildly irritating to eyes) based on GHS criteria
Conclusions:
Based on an In Vitro Irritatancy Score between 3 and 55, no prediction can be made for the UN GHS categorization of the test article as defined in OECD Guideline #437.
According to EURL ECVAM DB-ALM Protocol No. 127, 6-Chloro-1-hexanol, Lot No. 141125 is considered to be a mild eye irritant.
Executive summary:

Based on an In Vitro Irritatancy Score between 3 and 55, no prediction can be made for the UN GHS categorization of the test article as defined in OECD Guideline #437.

According to EURL ECVAM DB-ALM Protocol No. 127, 6-Chloro-1-hexanol, Lot No. 141125 is considered to be a mild eye irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Justification for classification or non-classification