Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 July - 24 August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD Guideline 429 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J strain
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France.
- Age at study initiation: Approximately 9 weeks
- Weight at study initiation: 20-25 g
- Housing: Animals were housed individually in labeled Macrolon cages (Ml type; height 12.5 cm) containing sterilized sawdust as bedding material.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C (actual range: 21.5-24.1 °C)
- Humidity: 40-70 % (actual range: 41-78 %)
- Air changes: Approximately 15 air changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 29 July 2009 To: 24 August 2009
Vehicle:
other: Water with 1 % pluronic L92
Concentration:
Preliminary irritation study: 25 and 50 % w/w
Main study: 10, 25 and 50 % w/w
No. of animals per dose:
Preliminary irritation study: One animal/dose
Main study: Five animals/dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Solubility in vehicle (water): 62.9 g/100 g
- One animal/dose was treated at concentrations of 25 and 50 % w/w on three consecutive days. Approximately 3-4 h 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.
- Irritation: No irritation was observed in any of the animals examined. Based on the results, the highest test substance concentration selected for the main study was a 50 % concentration.

MAIN STUDY
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: If the results indicate a Stimulation Index (SI) ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM. 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 (w/w) were prepared within 4 h prior to each treatment. Homogeneity was obtained to visually acceptable levels by adjusting the pH (pH 3 - 5). 25 µL of control or test item were applied to the dorsal surface of both ears on Days 1, 2 and 3. 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 on Day 6. After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol 20 % (AST Farma BV, Oudewater, The Netherlands) and 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. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 mm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic acid (TCA) stored in the refrigerator until the next day. On Day 7, precipitates were recovered by centrifugation, re-suspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail 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).

DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data.
Positive control results:
The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at the laboratory is an appropriate model for testing for contact hypersensitivity. At 25 % Hexylcinnamaldehyde, DPM/group and SI/group were 740 and 3.2, respectively.
Parameter:
SI
Remarks on result:
other: - The SI values calculated for the substance concentrations 10, 25 and 50 % were 0.9, 1.6 and 0.5, respectively. - SI values calculated for vehicle and positive control groups were 1.0 and 3.2, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: - Median DPM values for the experimental groups treated with test substance concentrations 10, 25 and 50 % were 202, 378 and 109, respectively. - Median DPM values for the vehicle and positive control groups were 231 and 740, respectively.

Main study:

Skin reactions/irritation: No irritation of the ears was observed in the control animals, the animals treated at 10 % and 25 % and in three animals treated at 50 %. The slight irritation as seen in two animals treated at 50 % and the slight to well-defined irritation as displayed 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:

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for one extremely large left node in one experimental animal treated at 10 %. Since this animal also showed abnormalities of the left eye (dull appearance), this animal was rejected from the study and the results were not used for interpretation. No macroscopic abnormalities of the area surrounding the nodes 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 except for the slight body weight loss as shown by the rejected animal with the abnormality at the left eye.

Toxicity and mortality:

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

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under these test conditions, Aluminium sulphate is not classified as a skin sensitiser according to the CLP Regulation and the Directive 67/548/EEC.
Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, groups of CBA/J mice (5 females/dose) were treated with Aluminium sulphate at concentrations of 10, 25 and 50 % on three consecutive days, by open application on the ear (25 µL/ear). Five vehicle control animals were treated with vehicle alone (1% aqueous L92) and five positive controls were treated with Hexylcinnamaldehyde (25 %). 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. 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. Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary irritation study at 25 and 50 % using one female/dose. 

No mortality and no clinical signs were observed during the observation period. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. No irritation of the ears was observed in the control animals, the animals treated at 10 % and 25 % and in three animals treated at 50 %. The slight irritation was seen in two animals treated at 50 % and the slight to well-defined irritation as displayed by the positive control animals was considered not to have a toxicologically significant effect on the activity of the nodes. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for one extremely large left node in one experimental animal treated at 10 %. Since this animal also showed abnormalities of the left eye (dull appearance), this animal was rejected from the study and the results were not used for interpretation. No macroscopic abnormalities of the area surrounding the nodes were noted. The median DPM values for the experimental groups treated with test substance concentrations 10, 25 and 50 % were 202, 378 and 109, respectively. The median DPM values for the vehicle and positive control groups were 231 and 740, respectively. The SI values calculated for the substance concentrations 10, 25 and 50 % were 0.9, 1.6 and 0.5, respectively. SI values calculated for vehicle and positive control groups were 1.0 and 3.2, respectively. The SI of the positive control (α- hexylcinnamaldehyde) was > 3; this experiment was therefore considered valid.

Under these test conditions, Aluminium sulphate is not classified as a skin sensitiser according to the CLP Regulation and the Directive 67/548/EEC.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A key study was identified (van Huygevoort, 2009, Kr. 1). The study was conducted according to the OECD guideline No 429 and in compliance with GLP. Following a preliminary screening test, three groups were treated with Aluminium sulphate at concentrations of 10, 25 and 50 %. A further group of five animals was treated with Water with 1 % pluronic L92 alone.

The irritant potential of Aluminium sulphate was assessed in parallel by measurement of ear thickness on days 1 to 6.

The Stimulation Index values were 0.9, 1.6 and 0.5 for the concentrations 10, 25 and 50% respectively .

The SI values calculated for vehicle and positive control groups were 1.0 and 3.2, respectively. This experiment was therefore considered valid.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. No irritation of the ears was observed in the control animals, the animals treated at 10 % and 25 % and in three animals treated at 50 %. The slight irritation was seen in two animals treated at 50 % and the slight to well-defined irritation as displayed by the positive control animals was considered not to have a toxicologically significant effect on the activity of the nodes. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Under the test conditions of this study, Aluminium sulphate is not classified as a skin sensitiser.


Migrated from Short description of key information:
LLNA, Not sensitising (OECD 429, GLP, Kr: 1).

Justification for selection of skin sensitisation endpoint:
Only one study available, GLP-compliant and of high quality (Klimisch rate of 1).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 including ATP3.

Self classification:

- Skin sensitisation: based on the available data, no additional self-classification is proposed according to the Annex VI of the Regulation (EC)

No. 1272/2008 (CLP) and of the Directive 67/548/EC criteria.

- Respiratory sensitisation: no self-classification is proposed due to lack of data.