Aluminate(2-),[[N,N'-1,2-ethanediylbis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON] (oxidoborate-µ-oxido)(-1),disodium

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: 040802#
- Expiration date of the lot/batch: 06.08.2016
Purity: 99.17%
- Purity test date: 08/01/2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable in water, hygroscopic
- Solubility and stability of the test substance in the solvent/vehicle: Different vehicles were tested to solubilize the test item but remained less to non-soluble compared to solubility in water. Therefore aqua ad inject containing 2% CMC was used as vehicle.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Yes, incompatibilities exist between the substance and the CMC

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dilution into the vehicule
- Final dilution of a dissolved solid, stock liquid or gel: 12.5, 25 and 50%

FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories GmbH, Venray, The netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: controlled full-barrier maintained breeding system (SPF)
- Age at study initiation:8-9 weeks
- Weight at study initiation:
- Housing: 5 mice/IVC cages
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period:at least 5 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3°C
- Humidity (%):55 +/- 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, 12 hours light/ 12 hours dark

Study design: in vivo (LLNA)

Vehicle:

other: water with 2% carboxymethylcellulose (CMC)

Remarks:

AOO, DMSO, PG were tested but found insoluble.

Concentration:

12.5% - 25% - 50%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: a solubility test is performed to define the vehicle and the maximum concentration which is technically applicable to the animals
- Irritation: Tested on 2 animals by topical application on 3 consecutive days, by measurement of the thickness of both ears (without assessment of lymph node proliferation).
+1 animal treated with the vehicle and served as negative control.


- Systemic toxicity: Body weights recorded + daily observation for any clinical signs
- Ear thickness measurements: performed on day 1 (pre-dose), day 3 (48 hours after the 1st dose) and day 6.
Criteria used to consider irritation: Excessive local irritation is indicated by an erythema score ≥ to 3 and/or swelling of ≥ 25%.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- animal assignment: The animals were randomly selected using the validated departmental computerised system E WorkBook (version 9.4.0, ID Business Solutions Ltd.)+ identification (tail)

- Criteria used to consider a positive response: a substance is regarded as a “sensitizer” in the LLNA ifat least 1 concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine – incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (stimulation index equal or greater than 3)

TREATMENT PREPARATION AND ADMINISTRATION:
- Topical application of 25 µl to the entire dorsal surface of each ear.
- Application once daily over 3 consecutive days (first treatment day = day 1).
- Administration of 3H-Methyhl thymidine : on day 5, 20µCi by IV
- Sacrifice: by cervical dislocation, approx.. 5 hours after 3H-methyl thymidine injection.
- Excision of the draining auricular lymph node and collection in PBS.
- Multiple washing procedures.
- Suspension in 1 ml 5% TCA at 4°C for 18 hours.
- Re-suspension in 1 ml 5% TCA and 7 ml scintillation fluid after 1 washing and then storage at room temperature overnight.
- Determination of incorporated 3H-methyl thymidine with a Betacounter and expressed as the number of desintegrations per minute (DPM)

Positive control substance(s):

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

Results and discussion

Positive control results:

The stimulation index of the positive control was 9.1. Test is considered valid

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

not determinable

Any other information on results incl. tables

All animals survived throughout the test period without showing any clinical signs.

Incompatibility exists between CMC and chemical containing aluminium. The test was invalidated.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In the prescreen test vehicles acetone/olive oil, polyethylene glycol and dimethylsulfoxyde were tested but the test item was found to be less to non-soluble compared to solubility in water. Threrefore, aqua ad inject containing 2% CMC was applied as vehicle. CMC was added in order to reduce surface tension of water, which is required to cover the dermis of the ear with the test item formulation. Incompatibilities of the test item with CMC was detected afterwards. Therefore the results obtained in this study are considered invalid. EC3 value and classification cannot be obtained.

Executive summary:

X330 was tested according to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) for skin sensitisation. A prescreen test was performed in order to define the vehicle which will allow to test a minimum of three concentrations of the test substance and in order to keep the test substance in contact with the mouse ears. Following the guideline, acetone/olive oil, polyethylene glycol and dimethylsulfoxyde were tested but the test item was found to be less to non-soluble compared to solubility in water. Threrefore, aqua ad inject containing 2% CMC was applied as vehicle. CMC was added in order to reduce surface tension of water, which is required to cover the dermis of the ear with the test item formulation. Incompatibilities of the test item with CMC was detected afterwards. Therefore the results obtained in this study are considered invalid. EC3 value and classification cannot be obtained. This study cannot be used for classification.