[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 July to 04 November 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dimethylnon-6-en-1-ol

Cas Number:

41972-59-2

Molecular formula:

C11H22O

IUPAC Name:

3,7-dimethylnon-6-en-1-ol

Test material form:

liquid

In vitro test system

Test system:

human skin model

Remarks:

The EpiDerm Model (EPI-200)

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDerm™ Model incorporates several features which make it advantageous in the study of potential dermal corrosivity. First, the test system uses a serum-free medium which eliminates the possibility of serum protein and test article interaction (Shopsis and Eng, 1988}. Secondly, the target cells are epithelial, derived from human skin (Cannon et al., 1994}. Third, since the tissue has a functional stratum corneum, the test materials are applied directly to the tissue surface, at air interface, so that undiluted and/or end use dilutions can be tested directly (Harbell et al., 1994). Prior to use, each 6-, 24- and 96-well plate will be uniquely identified with a number written in permanent
marker with the test substance identification or control treatment group.

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDerm Model (EPI-200) (MatTek Corporation, Ashland, USA) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipud layers arranged in patterns analogous to those found in vivo.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was used as supplied, no vehicle used.

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours in an MTT dye solution to determine the degree of cytotoxicity (cell death) caused by exposure to the test material.

Number of replicates:

The test and control articles will be tested by treating 4 EpiDerm tissues per material.
Two tissues will be used to assess viability after the 3-minute exposure and two will be used to assess viability after the 60-minute exposure.

Test system

Details on study design:

The test and control substances will be tested by treating 4 EpiDerm™ tissues per material. Two tissues will be used to assess viability after the 3-minute exposure and two will be used to assess viability after the 60-minute exposure. Fifty microliters (50 μl) of control or liquid test substances or approximately 25 mg of solid test substances (measured using a 25 mg sharp spoon) will be applied to the apical side of the EpiDerm™ tissue. Each EpiDerm™ tissue treated with a solid test substance will receive 25 μl of sterile, deionized water applied directly onto the test substance. The 3-minute exposure time will begin as soon as the test substance is spread
onto the tissue (either directly or with the dosing device). The 3-minute exposure groups will be held at room temperature during the treatment incubation, while the 60-minute exposure groups will be placed in the incubator at standard culture conditions during treatment.
A 10X stock of MTT prepared in PBS (filtered at time of batch preparation) will be thawed and diluted in warm MTT Addition Medium to produce a 1.0 mg/ml solution and used within 2 hours. Three hundred microliters of the MTT solution will be added to each designated well of a labelled 24-well plate.
At the end of the test substance incubation period, each tissue will be rinsed and transferred to the MTT solution. Rinsing will be accomplished using warmed (approximately 37°C sterile CMF-DPBS delivered from a plastic squeeze bottle. The tip of the bottle shall be cut so as to enlarge the orifice to at least 3 mm (interior diameter) that will allow a gentle stream of CMF-OPBS to be delivered to the surface of the tissue.
After the appropriate exposure time, each EpiDerm™ tissue will be thoroughly rinsed on both sides of the tissue insert with CMF-DPBS. The CMF-DPBS will be decanted and the cell culture insert will be blotted on sterile paper towels. The EpiDerm™ tissues will be
transferred to the appropriate wells on the labelled 24-well MTT plate after rinsing. The 24-well plates will be incubated at standard culture conditions for 3 ± 0.1 hours. After 3 ± 0.1 hours, the cell culture insert will be removed from the MTT solution, the bottom blotted on sterile paper towels, cleared of excess liquid, and the cell culture insert transferred to a labelled 24-well plate containing 2.0 ml of isopropanol in each designated well. The plates will be covered with paraffin film and stored in the refrigerator (2 to 8 °C) until all of the tissues have been placed into the isopropanol. If necessary, the plate may be sealed and stored overnight before the extraction step is begun.
To extract the reduced MTT, the plates will be shaken for 2 to 3 hours at room temperature. The shaking should be sufficiently vigorous to move the isopropanol around the cell culture insert. At the end of the extraction period, the liquid within the cell culture inserts will be decanted into the well from which the cell culture insert was taken. The extracted MTT solution will be mixed and 200 μL will be
transferred to the appropriate wells of a labelled 96-well plate. Two hundred microliters of isopropanol will be added to the 2 wells designated as blanks. The absorbance at 570 nm (OD570) of each well will be measured with a Molecular Devices VersaMax plate reader with the shaking function selected.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

After 3 minutes

Value:

118.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

After 3 minutes: 9.58% +/- 13.55%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

After 60 minutes

Value:

88.88

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

After 60 minutes: 6.28% +/- 0.90%

Other effects / acceptance of results:

The assay was accepted since:
1. the positive control resulted in a corrosive classification (i.e., < 50% cell viability compared to negative controls, after a 3‐minute exposure and/or < 15% cell viability compared to negative controls after a 60‐minute exposure)
2. the mean OD570 value of the negative control tissues was ≥ 0.8 and ≤ 2.8. The mean OD570 value of the tissues treated with the negative control at 3‐ and 60‐minutes was 2.216.
3. in the range of 20 to 100% viability, the Coefficient of variation (CV) between 2 identically treated replicates of the negative and positive control per each exposure time are ≤ 30%.

Any other information on results incl. tables

The results were evaluated according to the prediction model presented in OECD TG 431 (2019) in the table below. A test article was determined to be Corrosive or Non-Corrosive using the 3- and/or 60-minute exposures; and when corrosive, an optional sub-classification may be determined using the % relative viability after the 3-minute exposure as presented below:

Viability after exposure times (3 & 60 minutes)	Prediction (OECD TG 431)	Viability after exposure time (3 minutes)	Optional Sub-Classification
< 50% after 3-minutes	Corrosive	<25% after 3-minutes	Sub-category 1A*
≥ 50% after 3-minutesAND < 15% after 60-minutes		≥25% after 3-minutes	Combination of Sub-categories 1B & 1C
≥ 50% after 3-minutesAND ≥ 15% after 60-minutes	Non-corrosive	Not Applicable	Not Applicable

 

Summary of results for the skin corrosion assay:

Test Article Number	Sponsor’s Designation	Conc.	Exposure Time	% Viability	*Corrosive?	Subcategory	pH
22AA19	GR-89-1114 (Rosabloom)	Neat	60 minutes 3 minutes	88.88 +/- 1.06 118.30 +/- 10.95	Non-corrosive	NA	4.5
Positive Control	8N KOH	Neat	60 minutes 3 minutes	6.28 +/- 0.90 9.58 +/- 13.55	Corrosive	1A

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

According to the prediction model presented above, the test article was considered to be non-corrosive.

Executive summary:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential skin corrosivity of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after test article exposure. The protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”. According to the prediction model presented above, the test article was considered to be non-corrosive.