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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-03-02 to 2010-06-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium azide
EC Number:
247-852-1
EC Name:
Sodium azide
Cas Number:
26628-22-8
Molecular formula:
N3Na
IUPAC Name:
sodium azide
Test material form:
solid: crystalline

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: mean weight: 20.7 ± 1.5g (range: 18.5-23.1g)
- Housing: individually in Makrolon Type II cages with wire mesh top and granulated soft wood bedding
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: at least 5 days prior to the start of dosing under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 27-65%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2010-03-02 To: 2010-03-17

Study design: in vivo (LLNA)

Vehicle:
other: ethanol: sterile water (3+7)
Concentration:
0, 5, 10, and 20% in vehicle
No. of animals per dose:
4 female animals per group
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: up to 20% in ethanol : sterile water (3+7)
- Irritation: tested in two animals with concentrations of 10 and 20%.

MAIN STUDY
Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 20% (w/v) in ethanol:sterile water (3+7). The application volume, 25 μl, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear 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
Five days after the first topical application, all mice were administered with 250 μl of 79.7 μCi/ml 3HTdR (corresponds to 19.9 μCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3HTdR
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 (8 nodes per group). 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 scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Interpretation of Raw Data
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; S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
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.
- 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.

Additional observations
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: Once daily (week day) from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Clinical signs (local / systemic): In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. In the main experiment clinical signs were recorded within 1 hour each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:
Treatment with α-Hexylcinnamaldehyde at concentrations of 0, 5, 10, and 25% in acetone : olive oil (4+1) served as positive control for the LLNA assay and was performed twice per year using 5 animals per concentration.
The number of desintegrations per minute (DPM) after treatment with 0, 5, 10, and 25% of the test item were 572.5, 1020.6, 1456.2, and 2792.2, respectively (mean of 10 lymph nodes). The respective stimulation indices (S.I.) were 1.00, 1.78, 2.54, and 4.88.
Using these data, the EC3 (estimated concentration for a S.I. of 3.) was determined to be 12.9%.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.44
Test group / Remarks:
5% (w/v)
Key result
Parameter:
SI
Value:
0.6
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
0.69
Test group / Remarks:
20% (w/v)
Cellular proliferation data / Observations:
EC3 CALCULATION:
EC3 = (a-c) [(3-d)/(b-d)] + c = 12.9 % (w/v)
Test group 3: Test item concentration = 10% (a), S.I. = 2.54 (b)
Test group 4: Test item concentration = 25% (c), S.I. = 4.88 (d)
EC3 = Estimated concentration for a S.I. of 3.
a,b,c,d = Coordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

CLINICAL OBSERVATIONS:
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

Table 1: Results of the LLNA assay with Sodium azide

Test item concentration [%]

Group

Measured DPM

Calculation

Result

DPM – BG a)

number of lymph nodes

DPM per lymph node b)

S.I.

-

BG 1

16

-

-

-

-

-

BG 2

15

-

-

-

-

0

1

4798

4783

8

597.8

1.00

5

2

2129

2114

8

264.2

0.44

10

3

2884

2869

8

358.6

0.60

20

4

2483

2468

6

411.3

0.69

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

1 = Control Group

2-4 = Test Group

S.I. = Stimulation Index

a)=The mean value was taken from the figures BG I and BG II

b)= Since the lymph nodes 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.

 

VIABILITY / MORTALITY

One animal belonging to the group treated with the highest test item concentration (20%) was found dead on study day 4.

 

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.

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Sodium azide was not a skin sensitiser in the LLNA assay.
Executive summary:

In the study the test item Sodium azide dissolved in ethanol:sterile water (3+7) was assessed for its possible skin sensitization potential according to OECD Guidelines for Testing of Chemicals, Updated Guideline 429: Skin Sensitisation:Local Lymph Node Assay (adopted 24 April 2002) and"European Union Method 8.42, Skin Sensitisation: Local Lymph Node Assay-Commission regulation (EC) No 44012008, Official Journal of the European Union (EN), dated May 30, 2008.

For this purpose a local lymph node assay (LLNA) was performed using test item concentrations of 5, 10, and 20% (w/v).

The animals did not show any signs of local irritation or systemic toxicity during the courseof the study. However, one animal of the group treated with the highest test item concentration (20%) was found dead on study day 4.

In this study Stimulation Indices (S.I.) of 0.44, 0.60, and 0.69 were determined with the testitem at concentrations of 5, 10, and 20% in ethanol:sterile water (3+7), respectively. The S.I. for the high dose group was derived from 3 animals only due to the fact that one animal belonging to this group died. Nevertheless, the result obtained for the high dosegroup is considered clearly negative.

Therefore, the test item Sodium azide was not a skin sensitiser in this assay.