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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:
From 04 September 2013 (Start of in-life phase) to 20 December 2013 (GLP compliance statement)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
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: particulate/powder
Details on test material:
- Name of test material : Lithium cryolite
- Molecular formula : Li3AlF6
- Molecular weight : 162
- Physical state: White powder
- Storage condition of test material: At room temperature in the dark

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: young adult, approx. 9 weeks old
- Weight at study initiation: from 20 to 23 grams, 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): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions

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

IN-LIFE DATES: From: 04 September 2013 To: 30 September 2013

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
Pre-screen test : 25% and 50% concentration
Main study : 10%, 25% and 50% concentration
The test substance formulations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
No. of animals per dose:
Pre-screen test : 2 per concentration
Main study : 5 per concentration, and 5 for the vehicle control
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.
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 in the main study except that the application method may have been different and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 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).
Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

No irritation was observed in any of the animals examined. At a 50% test substance concentration, one of the animals showed signs of systemic toxicity (flat- and hunched posture, thin, hypothermia, closed eyes and piloerection) and significant body weight loss. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.
Since systemic toxicity was seen in only one animal dosed at 50% and based on the severity of the effects, it was considered that this was not test substance related and therefore the highest test substance concentration selected for the main study was a 50% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test.
One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Induction - Days 1, 2 and 3 : The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle 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) containing 20 μCi of 3H-methyl thymidine. After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20%. 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 : Following excision of the nodes, 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) 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 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.
Body weights : On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs : Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation : Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.
Grading Irritation Reactions: 0 = no erythema; 1 = Very slight erythema (barely perceptible); 2 = Well-defined erythema; 3 = Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth); 4 = Severe erythema (beet redness) to eschar formation preventing grading of erythema.
Necropsy : No necropsy was performed according to protocol.

Interpretation :
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.



Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Positive control substance:
The results of a reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures are summarized as follows:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.6 and 7.7 respectively. An EC3 value of 13.4% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 16.0, 11.9, 16.9, 14.4, 16.5, 14.5 and 16.5%.
Based on these results it was concluded that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

For both scientific and animal welfare reasons, no concurrent positive control group was added to the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.7
Variability:
Standard error of the mean = 0.5
Test group / Remarks:
10 % (w/w)
Key result
Parameter:
SI
Value:
1
Variability:
Standard Error of the mean = 0.4
Test group / Remarks:
25% (w/w)
Key result
Parameter:
SI
Value:
1.6
Variability:
Standard Error of the mean = 0.4
Test group / Remarks:
50% (w/w)
Cellular proliferation data / Observations:
Radioactivity measurements
Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 396, 234 and 383 DPM respectively. The mean DPM/animal value for the vehicle control group was 239 DPM.

Any other information on results incl. tables

Skin reactions / Irritation:

No irritation of the ears was observed in any of the animals examined. White staining by the 50% concentration on Day 3 did not prevented scoring for erythema.

 

Systemic toxicity (Body weights):

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

The body weight was between 20 and 23 g at test initiation and between 18 and 25 g at termination of the study. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

 

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.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Lithium cryolite is not a skin sensitizer according to the recommendations made in the test guidelines.
Executive summary:

The skin sensitisation study with Lithium cryolite in the mouse was investigated in a study performed according to the OECD Testing Guideline No.429 (Local Lymph Node Assay) and under GLP.

Test substance concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Propylene glycol).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation of the ears was observed in any of the animals examined.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

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.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 396, 234 and 383 DPM respectively. The mean DPM/animal value for the vehicle control group was 239 DPM.

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.7, 1.0 and 1.6 respectively.

Since there was no indication that the test substance elicited an SI ≥ 3 when tested up to 50%, Lithium cryolite was considered to be a non skin sensitizer

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 Lithium cryolite does not have to be classified as a skin sensitizer according to the CLP and the UN GHS.