Sodium nitrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 April 2010 - 07 June 2010

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

Sodium nitrate

EC Number:

231-554-3

EC Name:

Sodium nitrate

Cas Number:

7631-99-4

Molecular formula:

HNO3.Na

IUPAC Name:

sodium nitrate

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Natriumnitrat HQ unbehandelt (non-food grade)
- Substance type: White crystalline powder
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France.
- Age at study initiation: Young adult animals (approx. 10 weeks old) were selected.
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Health inspection: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0– 23.3ºC
- Humidity (%): 37.2 - 83%
Temporary deviations from the minimum level of temperature and maximum relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

Study design: in vivo (LLNA)

Vehicle:

other: 1% watery pluronic L92. Trial formulation results showed that no suitable suspension could be prepared using the guideline vehicle. 1% watery pluronic L92 was selected as suitable vehicle based on trial formulations performed at NOTOX. L92 provides good s

Concentration:

0, 10, 25, 50%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data
supplied by the sponsor.
-Irritation: A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2; see section 6.6) at the highest concentration. Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids).
The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (8-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.
-Lymp node proliferation response: Not determined in the preliminary irritation study.

MAIN STUDY
ANIMAL ASSIGMENT AND TREATMENT
- Name of the test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM . The results were evaluated according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labeling and packaging of substances and mixtures. Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3)

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations were prepared within 4 hours prior to each treatment. No adjustmeent was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.

Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The vehicle and positive control animals were treated the same as the experimental animals, except that the vehicle and/or positive control substance was administered instead of the test substance.

Excision of the nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised.
The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) stored in the refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On Days 1 (pre-treatment) and 6.
Necropsy: No necropsy was performed according to protocol. Indien van toepassing:\\The moribund animals were sacrificed by intraperitoneal injection with Euthasol® 20%. \The \moribund animal\s \or animal\s found dead was\were subjected to necropsy for gross macroscopic examination.
Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
0: No erythema
1: Slight erythema (barely perceptible)
2: Well-defined erythema
3: Severe erythema (beef redness) to slight eschar formation (injuries in depth)

Oedema formation:
0: No oedema
1: Slight oedema (barely perceptible)
2: Moderate oedema
3: Severe oedema

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The positive control group added to the study showed that the vehicle is suitable for eliciting an SI>3 in this batch of animals and with the procedures used for this study used.

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

50%

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Since the within group variation was relatively high, median values were used to interpret the data. Median DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 704, 338 and 576 DPM respectively. The median DPM/animal value for the vehicle control group was 363 DPM and a median DPM/animal value of 1163 DPM was obtained for the positive control group.

Any other information on results incl. tables

Tables and figures have been included in the attached document "LLNA tables and figures".

Initially, three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle and another group of five animals was treated with the positive control substance. Since the DPM values for control animals were generally well below the range expected, the study was considered invalid. The results of that study are not reported; all data are retained in the study records. The study was therefore repeated with the same concentrations, including a vehicle and positive control group.

Other results: Main study:

No irritation of the ears was observed in any of the animals treated with the test substance or vehicle. Slight to well defined erythema was observed on both ears of all animals treated with the positive control substance, which was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined. No macroscopic abnormalities of the surrounding area were noted in any of the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Preliminary irritation study:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

See also Table 1 in the attached document LLNA tables and figures

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) 1272/2008

Conclusions:

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.9, 0.9 and 1.6 respectively.

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%, Natriumnitrat HQ unbehandelt (non-food grade) was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 50%.

Based on these results Natriumnitrat HQ unbehandelt (non-food grade) would not be regarded as skin sensitizer according to the recommendations made in the test guidelines and does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.