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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-11-11 - 2017-02-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

OECD Guideline for the testing of chemicals 429. Skin Sensitisation: Local Lymph Node Assay (Adopted: 22 July 2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

by Slovak National Accreditation Service

Type of study:

mouse local lymph node assay (LLNA)

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 vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: AnLab Prague, Czech Republic
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-10 weeks
- Weight at study initiation:
- Housing: The animals were housed in polypropylene cages (5 animals per cage) suspended on stainless steel racks. Bedding was Lignocel S3/4, Lufa - ITL GmbH, Germany.
- Diet (e.g. ad libitum): A laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was served ad libitum, each day approximately at the same time.
- Water (e.g. ad libitum): The animals received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is periodically monitored (including microbiological control) and recorded.
- Acclimation period: The animals were acclimated in identical conditions as during the experiment for 6 days prior to the start of treatment.
- Indication of any skin lesions: The health condition of animals was examined by a veterinarian before initiation of the study, no leasions were stated.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 - 60 %
- Air changes (per hr): Animals were housed in a room equipped with central air-conditioning.
- Photoperiod (hrs dark / hrs light): 12-hour light / 12-hour dark cycle
- IN-LIFE DATES: From: 2016-11-09 (Delivery)

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

For the test item; Acetone/Olive Oil, 4:1, v/v was used for positive control

Concentration:

100% (pre-screen test)
100%, 50%, 25% (main test)
100% concentration was obtained by mixing of 1g of test item with 1mL of vehicle

No. of animals per dose:

5 females – control (vehicle)
5 females – positive control
15 females – test item
4 females - pre-screen test plus spare animals

Details on study design:

PRE-SCREEN TESTS:
The pre-screen test was conducted under the same conditions as the main LLNA study, except there was no assessment of lymph node proliferation. The test item was tested at a concentration of 100%.
- Compound solubility: The test item was insoluble in the vehicle; therefore, a homogeneous suspension was obtained.
- Irritation: Both ears of each mouse were assessed for any sign of irritation. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%.
- Systemic toxicity: All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6).
- Ear thickness measurements: Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6
- Erythema scores: Erythema scores were scored according to the guideline.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: random
- Criteria used to consider a positive response: A substance is regarded as sensitizer in the LLNA test when SI≥3.

TREATMENT PREPARATION AND ADMINISTRATION:
Day 1:
Each animal was identified and the body weight was recorded. To the dorsum of each ear 25µL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6:
The body weight of each animal was recorded. 250µL of phosphate-buffered saline (PBS) containing 2 µCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein.
Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group, (pooled treatment group approach) and weighted.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

For calculation of mean, S.D. and t-test values of body weights MS Excel was used.

Results and discussion

Positive control results:

Si = 8.75

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.23

Test group / Remarks:

Test item, 100%

Results 2

Parameter:

SI

Value:

2.9

Test group / Remarks:

Test item, 50%

Results 3

Parameter:

SI

Value:

1.06

Test group / Remarks:

Test item, 25%

Results 4

Parameter:

SI

Value:

8.75

Test group / Remarks:

Positive control

Results 5

Key result

Parameter:

EC3

Test group / Remarks:

Test item

Remarks on result:

not determinable

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Lymph Node Proliferation
In comparison with the control group, an increase of the pooled lymph node weight was not recorded at used concentrations. The pooled lymph node weights of treated groups were 0.0292g for 25% concentration, 0.0359g for 50% concentration and 0.0320g for 100% concentration of tested item. The lymph node weight of control group and positive control group were 0.0457g and 0.0764g, respectively. The DPM values for the three treated groups were 749 (25%), 2048 (50%) and 1576 (100%), respectively. The EC3 value could not be calculated, as all measured points were below the stimulation index of three. The lymph node weights, DPM and SI values are shown below.

DETAILS ON STIMULATION INDEX CALCULATION
see below

EC3 CALCULATION
Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance a sensitizer. Therefore, it was not possible to calculate an EC3 value.

CLINICAL OBSERVATIONS:
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.

BODY WEIGHTS
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. The decrease of body weight was observed in all treated groups and at concentrations of 50% and 100%, the decrease was statistically significant.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Substance is not a skin sensitizer

Conclusions:

The study was conducted under GLP according to OECD guideline 429 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 sensitizing potential of N,N-Dimethyl-N-(2-methacryloyloxyethyl)-N-(3-sulfopropyl)-ammonium betaine; (SPE) in mice. All animals survived throughout the test period without showing any clinical signs of toxicity or local irritation. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance a sensitizer.
N,N-Dimethyl-N-(2-methacryloyloxyethyl)-N-(3-sulfopropyl)-ammonium betaine; (SPE) was so identified as a non-sensitizing agent in the Local Lymph Node Assay. Hence, no classification as skin sensitizer is triggered.

Executive summary:

The sensitization potential of N,N-Dimethyl-N-(2-methacryloyloxyethyl)-N-(3-sulfopropyl)-ammonium betaine; (SPE) was evaluated using the Local Lymph Node Assay (LLNA) in a OECD 429 guideline study under GLP. The LLNA has been developed to determine the allergic contact sensitization potential of chemicals.

Based on the recommendations of the OECD Guideline 429, the test item was dissolved in Dimethyl sulfoxide (DMSO). The positive control (alpha-Hexyl cinnamic aldehyde) (25%) was dissolved in acetone:olive oil 4:1 (v/v).

The Pre-screen test was performed using a dose of 100 % (w/v). Based on the observations recorded in the preliminary test, the concentration of 100 % was selected as top dose for the main test.

Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 25%, 50% and 100%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive ¹²⁵I-iododeoxyuridine and 10^-5 M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (25%, 50% and 100% w/v) the animals did not show visible clinical symptoms of either local irritation or systemic toxicity.

In this study Stimulation Indices (SI) of1.06, 2.90 and 2.23 were determined with the test item at concentration of 25, 50, and 100% in dimethyl sulfoxide, respectively. The EC3 value could not be calculated, as all measured points were below the Stimulation Index of three.

The test item N,N-Dimethyl-N-(2-methacryloyloxyethyl)-N-(3-sulfopropyl)-ammonium betaine; (SPE) is not considered a skin sensitizer under the test conditions of this study.