Dimethyl[2-[(2-methyl-1-oxoallyl)oxy]ethyl](3-sulphopropyl)ammonium hydroxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 June 2016 - 07 September 2016 (study plan to final report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Standard Operating Procedure SOP/T/57: “In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl[2-[(2-methyl-1-oxoallyl)oxy]ethyl](3-sulphopropyl)ammonium hydroxide

EC Number:

222-860-8

EC Name:

Dimethyl[2-[(2-methyl-1-oxoallyl)oxy]ethyl](3-sulphopropyl)ammonium hydroxide

Cas Number:

3637-26-1

Molecular formula:

C11H21NO5S

IUPAC Name:

dimethyl[2-[(2-methyl-1-oxoallyl)oxy]ethyl](3-sulphopropyl)ammonium hydroxide

Test material form:

solid: particulate/powder

In vitro test system

Test system:

isolated skin discs

Source species:

rat

Source strain:

Wistar

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: conventional husbandry of laboratory animals of the Centre for Experimental Medicine at the Medical University in Katowice
- Sex: female
- Age at study initiation (in days): At the beginning of the experiment, the animals were 21 days old. Skin discs used in the experiment were obtained from two 30-day-old animals
- Housing:
During the quarantine period, the animals were kept in air-conditioned rooms under the following conditions:
- air temperature: 19 - 21°C
- relative air humidity: 49 – 93%
- artificial fluorescent lighting; lighting cycle: 12 hours light/12 hours dark
- facility air exchange: about 13 times/hour
Both rats were kept in a plastic cage covered with a wire bar lid. The dimensions of the cage were 58 x 37 x 21 cm.
UV-sterilized wood shavings were used as bedding. The environment of the animals was enriched by placing wooden blocks and nesting materials for laboratory animals in the cages.
- Diet (e.g. ad libitum): The animals had ad libitum access to the “Murigran” standard granulated laboratory fodder
- Water (e.g. ad libitum): tap water
- Acclimation period: They were quarantined and observed daily for 3 days

Vehicle:

other: 150 μL deionized water was added on top of the solid

Details on test system:

SKIN DISC PREPARATION
- Procedure used: After the quarantine, the dorsal and flank hair from young, 25-day-old, female rats was carefully removed with a shaver. Then, the shaven area was washed with an antibiotic solution containing streptomycin (10 mg/mL), penicillin (10 μg/mL), and amphotericin (0.25 mg/mL) to inhibit bacterial growth. The animals were washed with antibiotics again on the third day after the first wash and used in the study within two days of the second wash. The animals were euthanized by intraperitoneal administration of morbital at a dose of 200 mg/kg b.w.. After euthanasia, the dorso-lateral skin of each animal was removed and stripped of excess subcutaneous fat by carefully peeling it away from the skin using a paper towel. The skin discs were cut out using a scalpel. Each skin disc was placed over one of the ends of a PTFE (polytetrafluoroethylene) tube, ensuring that the epidermal surface was in contact with the tube. A rubber ‘O’ ring was press-fitted over the end of the tube to hold the skin in place and excess tissue is trimmed away. The rubber ‘O’ ring was then carefully sealed to the end of the PTFE tube with petroleum jelly. The tube was supported by a spring clip inside a receptor chamber containing MgSO4 solution (154 mM). The skin disc should be fully submerged in the MgSO4 solution. As many as 11 skin discs with a diameter of 20-mm each were obtained from a single rat skin. Two of them were used to control the quality of the procedure, whereas the remaining nine were used for the purpose of the experiment.
The age of the animals was particularly important. The age of 30 days ensures that the hair follicles are in the dormant phase before adult hair growth begins [SOP/T/57].

- Quality control for skin discs: Before the test, the electrical resistance of two skin discs obtained from each rat was measured as a quality control procedure for each animal skin. Both discs should give resistance values greater than 10 kΩ for the remainder of the discs to be used for the test.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21-22°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: the test item and the control items were removed by washing with a jet of tap water at up to 30°C

DYE BINDING METHOD
- Dye used in the dye-binding assay: none - the dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: As many as 11 skin discs with a diameter of 20-mm each were obtained from a single rat skin. Two of them were used to control the quality of the procedure, whereas the remaining nine were used for the purpose of the experiment (per animal: 3 for positive control, 3 for negative control and 3 for test item).

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if:
- the mean TER value obtained for the test item is greater than 5 kΩ, or
- the mean TER value is less than or equal to 5 kΩ, and the skin disc shows no obvious damage.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

EST MATERIAL
- Amount(s) applied (volume or weight with unit): A sufficient amount of the test item (ground to a powder) was applied evenly to the disc to ensure that the whole surface of the epidermis is covered
- Concentration (if solution): After adding 150 μL deionized water on top of the solid, the tube was gently agitated

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 150 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 150 µL

Duration of treatment / exposure:

24 hours

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

For the test item and the control items, three skin discs were used (three replicates). The experiment was performed in duplicate, because the difference of TER means of both skin discs treated with the test item were below 5 ± 0.5 kΩ.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

mean (skin discs from animal no. 1)

Value:

12.54

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

mean (skin discs from animal no. 1)

Value:

15.12

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The concurrent mean values for the positive and negative controls were as follows: for 10M HCl – 0.98 kΩ (animal no. 1) and 0.97 kΩ (animal no. 2), whereas for distilled water – 13.43 kΩ (animal no. 1) and 15.62 kΩ (animal no. 2). These values fell within the acceptable ranges for the method.
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

not corrosive

Conclusions:

The study was conducted under GLP according to OECD guideline 430 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the corrosive potential of the test substance to the skin in vitro.
On the grounds of the study, it may be stated that N,N-Dimethyl-N-methacryl-oxyethyl-N-(3-sulfopropyl)-ammonium-betaine belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.
In a tiered in vitro testing strategy, this result (non-corrosive) may however only give an indication of the skin-damaging potential, but does not allow a definitive classification according to Regulation 1272/2008. So, an additional skin irritation test should be conducted.

Executive summary:

The in vitro skin corrosion: transcutaneous electrical resistance test (TER) according to OECD TG 430 (GLP) was performed in order to obtain information on health hazards resulting from skin contact with the test item N,N-Dimethyl-N-methacryl-oxyethyl-N-(3-sulfopropyl)-ammonium-betaine. Skin discs used in the experiment were obtained from two 30-day-old WISTAR female rats (outbred). In order to control the procedure quality, the electrical resistance of two skin discs obtained from each test animal was measured before the start of the experiment. In each case, the skin disc resistance values were greater than 10 kΩ; therefore, the remainder of the animals’ skin discs could have been used in the experiment. A sufficient amount of the test item (ground to a powder) was uniformly applied to the epidermal surface of the skin disc placed inside a tube. Concurrent positive (10M hydrochloric acid) and negative (distilled water) controls were used. Three skin discs obtained from each animal were used for the test item and three for each control item. The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. Then, they were removed by washing with a jet of tap water. Prior to measuring the electrical resistance, the surface tension of the skin was reduced by adding a sufficient volume of 70% ethanol to cover the epidermis. After a few seconds, the ethanol was removed from the tube, whereas the tissue was then hydrated by the addition of 3 mL of a solution of MgSO4 (154 mM). A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩ by placing the databridge electrodes on either side of the skin disc. After the transcutaneous electrical resistance test (TER), the MgSO4 solution was removed from the tube, whereas the skin discs were subjected to a gross examination in order to reveal possible damage.

The dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

The experiment was performed in duplicate, because the difference of TER means of both skin discs treated with the test item were below 5 ± 0,5 kΩ. The mean TER results for the skin discs treated with the test item were equal to 12.54 kΩ (animal no. 1) and 15.12 kΩ (animal no. 2). They can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the skin discs treated with the test item did not reveal any pathological changes.

The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs and hence, the test item can be considered as not corrosive under the conditions of the test. Further in vitro testing is needed to assess the irritation potential of the substance.