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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD Guideline 429, EU Method B42 and EPA OPPTS 870.2600.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Barium nitrate
- Substance type: White crystalline solid
- Physical state: Solid
- Analytical purity: 99.52%
- Lot/batch No.: 120724
- Expiration date of the lot/batch: 07 September 2013
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature in the dark
- Other:
pH 5.0-8.0
Stability at higher temperatures: Yes, maximum 70°C for maximum 30 minutes

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J strain, inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 9 weeks
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- 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): ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 degrees C
- Humidity (%): 40-70%
- Air changes (per hr): 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
Range-finding tes 25%, 50%

Main test: 10, 25, 50%
No. of animals per dose:
Range-finding test: 1

Main test: 5
Details on study design:
RANGE FINDING TESTS:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should not cause no systemic toxicity, may give well-defined irritation at the most (maximum grade 2 and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.
Two test substance concentrations were tested; a 50% and 25% concentration. The highest concentration was the maximum concentration as required in the test guideline (undiluted for liquids, 50% for solids).
The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to 14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Animals were sacrificed after the final observation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
DPM (disintegrations per minute) 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.

Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI=3).

TREATMENT PREPARATION AND ADMINISTRATION:
- 25 uL test item to dorsal ear surface days 1, 2, and 3 at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
- Day 6: injection via tail vein of 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi 3H-methyl thymidine
- 5 h post-injection, all animals killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20%
- Auricular lymph nodes excised and pooled for each animal in 3 mL PBS
- 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) and stored in the refrigerator until the next day.
-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).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.4
Test group / Remarks:
Test item: 10%
Parameter:
SI
Value:
1.5
Test group / Remarks:
Test item: 25%
Parameter:
SI
Value:
1.3
Test group / Remarks:
Test item: 50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Disintegrations per minutes (DPM):
- Vehicle: 396 dpm
- Test item 10%: 571 dpm
- Test item 25%: 602 dpm
- Test item 50%: 498 dpm

DETAILS ON STIMULATION INDEX CALCULATION

EC3 CALCULATION
Since there was no indication that the substance elicits an SI >= 3 when tested up to 50%, barium nitrate 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%.

CLINICAL OBSERVATIONS:
Range finding test:
No signs of systemic toxicity were noted and very slight erythema was observed. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

Main study - Observations
- Clinical Observations: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.
- Body weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- Ear thickness: Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Comparable variations were observed between the vehicle control group and the experimental groups.
- Skin reactions: No erythema on the ears was observed in any of the animals examined.
- Macroscopy of the auricular lymph nodes and surrounding area:
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

The six-month reliability check with alpha-hexylcinnamicaldehyde indicates that the local lymph node assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

Based on these results barium nitrate would not be regarded as skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirements for sensitization by skin contact according to GHS of the United Nations (2011) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these results, Barium Nitrate would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally
Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011)
and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and
mixtures.