Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 17th to July 9th, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April 24th, 2002

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:

-

EC Number:

480-340-8

EC Name:

-

Cas Number:

156157-97-0

Molecular formula:

C12H30Cl2N2Na2O14

IUPAC Name:

Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst, The Netherlands.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: 18.8 - 23.6 g.
- Housing: single caging. Makrolon Type I, with wire mesh top with granulated soft wood bedding.
- Diet: pelleted standard diet, ad libitum.
- Water: tap water, ad libitum.
- Acclimatisation: at least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C.
- Humidity: 30 - 70 %.
- Photoperiod: 12 hrs dark / 12 hrs light; artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

other: ethanol:deionised water, 7+3

Concentration:

10 %, 25 %, 50 %.

No. of animals per dose:

five animals per dose group.

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: the highest test item concentration which can be technically used was a 50 % solution in ethanol:deionised water (7+3).
- Dose selection test: to determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 5, 10, 25 and 50 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application.
- Rationale for dose selections: at the tested concentrations the animals did not show any signs of irritation or systemic toxicity. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

TEST ITEM PREPARATION: the test item was placed into a volumetric falsk on a tared balance and the vehicle was quantitavely added. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

TOPICAL APPLICATION: each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 10, 25, and 50 % (w/v) in ethanol:deionised water (7+3). The application volume, 25 μl, was spread over the entire dorsal surface ( ̴ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION of 3H-METHYL THYMIDINE (3HTdR): five days after the first topical application, all mice were administered with 250 μl of 81.1 μCi/ml 3HTdR (corresponds to 20.3 μCi 3HTdR per mouse) by intravenous injection via the tail vein.

LYMPH NODES POOLED: approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital - Natrium. The draining lymph nodes were rapidly excised and pooled per group (10 nodes per group besides of the vehicle control group where only 8 nodes per group could be evaluated due to the death of animal).

PREPARATION OF CELL SUSPENSIONS: single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately 4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.

DETERMINATION OF CELLULAR PROLIFERATION: the level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml - aliquots of 5 % trichloroacetic acid. The β - scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

OBSERVATIONS: the animals were observed for mortality / viability once daily (week day) from experimental start to necropsy. Body weights were measured prior to the first application and prior to treatment with 3HTdR. Clinical signs (local / systemic) were observed once daily (week day). The treatment sites were observed carefully.

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE:
- Criteria used to consider a positive response: a test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: first, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index and second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for the body weights.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.74

Test group / Remarks:

High dose group (50 %)

Remarks on result:

other: EC3 not determined.

Results 2

Parameter:

SI

Value:

0.85

Test group / Remarks:

Medium dose group (25 %)

Remarks on result:

other: EC3 not determined.

Results 3

Parameter:

SI

Value:

1.05

Test group / Remarks:

Low dose group (10 %)

Remarks on result:

other: EC3 not determined.

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION: the proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

EC3 CALCULATION: EC3 value (estimated concentration of test item required to produce a S.I. of 3) could not be calculated, since all S.I.´s are below 3.

OBSERVATIONS
- Mortality/viability: one animal from control group was found dead on the day of preparation.
- Clinical signs: no symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.
- Body weights: the body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

The results and the Stimulation Index (SI) for each tested group are presented in the table below.

Table: stimulation index per tested group.

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG ^a)	number of  lymph nodes	DPM per lymph node ^b)	S.I.
-	BG I	24	-	-	-	-
-	BG II	23	-	-	-	-
-	control	3243	3220	8	402.4
10	low dose	4244	4221	10	422.1	1.05
25	mid dose	3462	3439	10	343.9	0.85
50	high dose	3003	2980	10	298	0.74

BG = background (1 ml 5 % trichloroacetic acid) in duplicate.

S.I. = stimulation index.

^a) the mean value was taken from the figures BG I and BG II.

^b) since the lymph noded of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

Positive Control results

Positive control substance was tested in February 2008 and its S.I at test concentration 10 % was found to be 1.84 whereas at concentration 25 % was found to be 4.87. The EC3 calculated was equal to 15.7 % (w/v).

Deviation from Study Plan

In the vehicle control group one animal was found dead on the day of preparation. Therefore, only 8 instead of 10 lymph nodes could be evaluated for this group. This deviation does not affect the validity of the study.

Applicant's summary and conclusion

Interpretation of results:

other: not classified as skin sensitiser according to the CLP Regulation (EC) No.1272/2008

Conclusions:

The substance is not a skin sensitiser.

Executive summary:

The sensitising potential of the substance was assessed in the LLNA test according to the OECD Guideline 429. Groups of five female mice were treated daily for three consecutive days with the test item at concentrations of 10, 25, and 50 % (w/v) by topical application to the dorsum of each ear lobe. A control group was treated with the vehicle only. Five days after the first topical application the mice were injected intravenously with radio-labelled thymidine (³HTdR). Five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured in a β- scintilltion counter.

The animals did not show any clinical signs during the course of the study and no cases of local irritation or test item-related mortality were observed. One animal of the vehicle control group (animal 1) was found dead on the day of preparation. In this study Stimulation Indices (S.I.) of 1.05, 0.85, and 0.74 were determined with the test item at concentrations of 10, 25, and 50 % respectively. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study the EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

The substance is not a skin sensitiser under the tested conditions.