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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:
2010-05-10 to 2010-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, 2002-04-24
Deviations:
no
GLP compliance:
yes
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 chlordie di-hydrate
- EC No.: 233-788-1
- Molecular weight: 244.26
- Physical state: White crystalline powder (determined at NOTOX)
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperture in the dark
- - pH: 5.5 - 7.5 (100 g/L at 20 °C)
- Solubility in water: 400 g/L (20 °C)
No further information on the test material was stated.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: approx. 11 weeks old
- Weight at study initiation: 23 g - 27 g
- Housing: During the study, 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). During the acclimatization period, the animals were group housed in Macrolon cages (MIII type; height 18 cm).
- Diet (ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days;

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.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.7 - 22.9ºC
- Relative humidity: 43 - 62%
- Air changes: approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test material was stated.

Study design: in vivo (LLNA)

Vehicle:
other: 1 % L92 (Elix, Millipore S.A.S., Molsheim, France) with 1 % pluronic L92 (BASF, New Jersey, U.S.A.)
Concentration:
5 %, 10 %, and 25 % of the test substance (w/w)
No. of animals per dose:
5 female mice
Details on study design:
RANGE FINDING TESTS:
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 at the highest concentration.
Two test substance concentrations were tested; a 10% and 25% concentration. The highest concentration was the highest concentration that could be prepared homogeneously. 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 (Source: Harlan, Horst, The Netherlands; Age: 8-14 weeks old; Harlan, Horst, The Netherlands). 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.

Results:
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 25% concentration.

MAIN STUDY
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.

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 nodes - Day 6
All animals:
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 prcessing 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) and 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: Om days 1 (pre-treatment) and 6
- Necropsy: No necrops was performed
- Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to a modified Draize scoring system (see " Any other infromation on materials and methods incl. tables" below). Furthermore descriptions of all other (local) effects were recorded.
INTERPRETATION:
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 (Reference: National Institute of Environmental Health Sciences, The Murine Lymph Node Assay: A test method for assessing the allergic contact dermatitis potential of chemicals/compounds. Independent peer review by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods (NICEATM), NIH publication; No 99-4494, February 1999).

Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3) (Reference: Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I. A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999;19:261-266.).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Please refer to "Details on study design (LLNA)" above

Results and discussion

Positive control results:
Stimulation Index of the positive control (25 % Alpha- hexylcinnamaldehyde) was 7.7 +/- 2.2. This showed that the vehicle is suitable for eliciting an SI>3 in this batch of animals and with the procedures used for this study.
A mean DPM/animal value of 2613 +/- 660 DPM was determined for the positive control group.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 5 %: 1.3 +/- 0.3 10 %: 1.5 +/- 0.3 25 %:: 1.2 +/- 0.3 Vehicle control: 1.0 +/- 0.2
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 427 +/- 81, 511 +/- 59 and 401 +/- 88 DPM respectively. The mean DPM/animal value for the vehicle control group was 340 +/- 44 DPM.

Any other information on results incl. tables

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 25%, Barium Chloride Di-Hydrate 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 25%.

Skin reaction/irritation:

No irritation of the ears was observed for any of the animals treated with the test substance or for vehicle control animals. White test substance remnants on one ear were observed for two animals treated at 25%, but the remnants did not hamper scoring for skin irritation. The positive control animals showed slight erythema, but no oedema was observed. The slight irritation of the ears as shown by the positive control animals was considered not to have a toxicologically significant effect on the activity of the nodes.

Macroscopy of the auricular lymph nodes and surrounding area:

The majority of auricular lymph nodes were considered normal in size, except for the enlarged nodes in the majority of animals treated at 25% test substance and in all positive control animals. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Body weights:

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss, noted among the majority of animals, occurred in the absence of a treatment-related distribution and was therefore considered to be of no toxicological significance.

Toxicity and mortality:

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on these results Barium Chloride Di-Hydrate would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. It does not have to be classified for sensitization by skin contact according to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.