Morpholinium sulphamate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-02-27 to 2013-03-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD/EU guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Morpholinium sulphamate

EC Number:

258-061-6

EC Name:

Morpholinium sulphamate

Cas Number:

52636-67-6

Molecular formula:

C4H9NO.H3NO3S

IUPAC Name:

morpholin-4-ium sulfamate

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90., Hungary
- Weight at study initiation: 16.5 -21.0 g
- Housing: Grouped caging (4 animals/cage)
- Diet: ssniff® SM R/M-Z+H complete diet ad libitum
- Water: tap water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 2013-02-27 To: 2013-03-04

Study design: in vivo (LLNA)

Vehicle:

other: Aqueous 1 % Pluronic®PE9200 (aqueous 1 % Pluronic)

Concentration:

50, 25, 10, 5 % (w/v)

No. of animals per dose:

4 animals per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Solubility of the test item was evaluated at concentration range of 100 % (i.e. 1 g/mL) to 25 % (w/v). Highest concentrations were achieved in aqueous 1 % Pluronic with a maximum of 50 % (w/v). No or inappropriate solubility was achieved with the other vehicles (Acetone:Olive oil 4:1 mixture (v/v), Dimethylformamide, Methyl ethyl ketone, Dimethyl sulfoxide). Based on the results aqueous 1 % Pluronic was used as vehicle for the test item.
- Irritation: No signs of systemic toxicity or local irritation were observed at the treatment site (ears) at the tested concentrations.
- Lymph node proliferation response: not examined

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (Pooled Treatment Group Approach)
- Criteria used to consider a positive response: Exposure to at least one concentration (non-irritating, non-toxic) of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION:
Test item formulations (solutions) were prepared with aqueous 1 % Pluronic. Test item was weighed and formulations prepared daily on a weight: volume basis (this was specified in the preparation record as w/v %). Test item was formulated in the vehicle at concentrations of 50 %, 25 %, 10 % and 5 % in a final volume of 1 mL using calibrated volumetric vials and mechanical agitation. Formulations were freshly prepared just before the treatments.
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, of the positive control substance (positive control group) or of the vehicles (AOO or aqueous 1 % Pluronic as negative control groups). The formulations were applied, with a pipette, on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

PROLIFERATION ASSAY:
On Day 6 each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS containing 20 µCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
Five hours (± 30 minutes) after the intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes can be removed using forceps. Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and resuspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloracetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % (w/v) TCA at 2-8°C overnight (approx. 18 hrs) the samples were centrifuged at approximately 190 x g for 10 minutes at 4°C, the supernatants were decanted and the pellets were re-suspended in 1 mL of 5 % (w/v) TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and stored at room temperature protected from light until measurement.
For the measurement of incorporated radioactivity the vials were loaded into a beta-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The beta-counter expresses the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % TCA.

Positive control substance(s):

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

Statistics:

not applicable

Results and discussion

Positive control results:

The lymph nodes in the positive control group were larger than the relevant control (AOO). The positive control substance induced the appropriate stimulation compared to the control (SI = 7.5). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed sensitivity and validity of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

other: disintegrations per minute (DPM)

Value:

1

Test group / Remarks:

Negative (vehicle) control for the positive control: 6896 Positive control (25% HCA in AOO): 51174, Details on test item see below

Remarks on result:

other: Test item (50% (w/v)): 2050 Test item (25% (w/v)): 1395 Test item (10% (w/v)): 1465 Test item (5% (w/v)): 2826 Negative (vehicle) control for the test item: 2956

Results 2

Parameter:

SI

Value:

> 0.5 - < 1

Test group / Remarks:

Test item (50% (w/v)): 0.7 Test item (25% (w/v)): 0.5 Test item (10 % (w/v)): 0.5 Test item (5 % (w/v)): 1.0

Results 3

Parameter:

SI

Value:

7.5

Test group / Remarks:

Positive control (25% HCA in AOO)

Results 4

Parameter:

SI

Value:

1

Test group / Remarks:

Negative (vehicle) control for the positive control and Negative (vehicle) control for the test item.

Any other information on results incl. tables

Body Weight Measurement

No significant, treatment related effect on the body weights was observed in any treatment group.

 

Clinical Observations and Signs of Irritation

No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score higher than 3) or any other local effect was observed in any treatment groups.

 

Proliferation Assay

The lymph nodes in the positive control group were larger than the relevant control (AOO). Visual appearance of the lymph nodes was normal in the negative (vehicle) control groups (both AOO and aqueous 1 %Pluronic) and in the test item treated groups (compared to the relevant control of aqueous 1 % Pluronic). No significant lymphoproliferation (SI ≥ 3) was observed for the test item at the tested concentrations. The corresponding stimulation index values were 0.7, 0.5, 0.5 and 1.0 at treatment concentrations of 50 %, 25 %, 10 % and 5 %, respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No significant dose-related response was observed (p = 0.77, r = 0.23).

 

Interpretation of Observations

Selection of test item concentrations based on the results of a formulation evaluation and also results of a preliminary irritation/toxicity test according to the relevant guidelines.

Solubility of the test item was evaluated at concentration range of 100 % (i.e. 1 g/mL) to 25 % (w/v) in different vehicles recommended by the relevant guidelines. Highest concentrations were achieved in aqueous 1 % Pluronic with a maximum of 50 % (w/v). Based on this and results of a preliminary irritation/toxicity test sensitization potential of the test item morpholinium sulphamate was examined at 50 % (as the maximum attainable concentration based on solubility) and at 25 %, 10 % and 5 % formulations in the selected vehicle (aqueous 1 % Pluronic) in the LLNA.

 

Since the test was valid and no sign of systemic toxicity or irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to not cause lymphoproliferation in the Local Lymph Node Assay.

 

No sign of significant irritation (indicated by erythema ≥ 3 on the ears) or any other local effect was observed in the test item treated groups. No significant lymphoproliferative response indicated by an SI ≥ 3 of morpholinium sulphamate was observed at the applied concentrations. The corresponding SI values were 0.7, 0.5, 0.5 and 1.0 at concentrations of 50 %, 25 %, 10 % and 5 %, respectively. No dose-related response was observed.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present Local Lymph Node Assay, morpholinium sulphamate tested at the maximum attainable concentration (based on solubility) of 50 % (w/v) and at concentrations of 25 %, 10 % and 5 % as formulations in a suitable vehicle (aqueous 1 % Pluronic) was shown to have no sensitization potential.

Executive summary:

The aim of this study was to determine the skin sensitization potential of morpholinium sulphamate in the Local Lymph Node Assay (LLNA). Pooled treatment group approach was used in this test.

Selection of test item concentrations based on the results of a formulation evaluation and also results of a preliminary irritation/toxicity test according to the relevant guidelines. The maximum concentration in a suitable vehicle was 50 % (w/v) in aqueous 1 % Pluronic®PE9200 (aqueous 1 % Pluronic). Based on the preliminary test results the test item was examined in the LLNA at 50 % (as the maximum attainable concentration) and at 25 %, 10 % and 5 % as formulations in aqueous 1 % Pluronic.

In the main test, 28 female CBA/Ca mice were allocated to seven groups of four animals each:

- four groups received morpholinium sulphamate at 50 %, 25 %, 10 % and 5 %,

- one control group (used as negative control for the test item) received the vehicle of the test item (aqueous 1 % Pluronic) only,

- one control group (used as negative control for the positive control substance) received the vehicle of the positive control (AOO) only,

- the positive control group received 25 % a-Hexylcinnamaldehyde (HCA) in AOO.

Each substance was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. Auricural lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI).

 

The positive control item (25 % HCA in AOO) induced the appropriate stimulation compared to the relevant control (AOO, the SI value was 7.5) thus confirming the validity of the assay.

No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity was observed in any treatment group. No sign of irritation or any other local effect was observed in any treatment group. Visually larger lymph nodes than the relevant vehicle control were observed in the positive control group only. No significantly increased lymphoproliferation (SI ≥ 3) was observed in any test item treated groups. The observed SI values were 0.7, 0.5, 0.5 and 1.0 at concentrations of 50 %, 25 %, 10 % and 5 %, respectively. No dose-related response was observed (linear regression, p = 0.77, r = 0.23).

 

Since the test was valid and no sign of systemic toxicity or irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to not cause lymphoproliferation in the Local Lymph Node Assay. According to evaluation criteria of the relevant guidelines, since no proliferation value above 3 was observed up to the maximum attainable concentration (based on solubility) of 50 % and the lack of a dose-related response is considered as evidence that morpholinium sulphamate is not a sensitizer.