Aluminium tribenzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2017 - 09 Januari 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Aluminium tribenzoate is a salt which dissociates to benzoate and aluminium ions in aqueous solution. The available information on benzoate does not indicate that this ion has skins sensitising properties. For aluminium, the available information was less conclusive. Both DEREK assessment and the DPRA assay are not suitable to address skin sensitizing potential of metals, so in this case these tests were not considered relevant since the aluminium ion was the main focus of the assessment. A KeratinoSensTM assay was performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a. Aluminium tribenzoate is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction, no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) were observed at test concentrations <1000 μM and cell viability >70%. Performance of an additional in vitro test was considered, however both a negative result and a positive outcome would result in the necessity to further address the skin sensitizing properties in vivo. It was considered scientifically justified to omit further in vitro test work and to proceed with an in vivo test. The full assessment is attached.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(July 2010)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(March 2003)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium tribenzoate

EC Number:

209-091-3

EC Name:

Aluminium tribenzoate

Cas Number:

555-32-8

Molecular formula:

C7H6O2.1/3Al

IUPAC Name:

aluminium tribenzoate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White powder
- Storage conditions: At room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Young adult animals (approx.12 weeks old)
- Weight at study initiation: 18.8 - 24.4 g
- Housing: Animals were group (max 5) housed in labeled Makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 – 22
- Humidity (%): 42 - 55
- Air changes (per hr): ten or more
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 13 December 2017 - 08 Januari 2018

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 5, 10, 25% (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
Two test item concentrations were tested; a 10% and 25% concentration. The highest concentration was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: 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, based on the test guideline and recommendations done by ICCVAM.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.

Rationale for vehicle: The vehicle was selected based on trial formulations performed at Charles River Den Bosch and on test substance data supplied by the sponsor.


Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.


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 immediately after dosing) according to a numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy was performed, since all animals survived until the end of the observation period.

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde shows that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.4

Remarks on result:

other: Test item concentration: 5%

Results 2

Key result

Parameter:

SI

Value:

1.5

Remarks on result:

other: Test item concentration: 10%

Results 3

Key result

Parameter:

SI

Value:

0.6

Remarks on result:

other: Test item concentration: 25%, the highest concentration technically achievable.

Any other information on results incl. tables

Results Pre-screen test:

At a 10% and 25% test item concentration, no erythema or signs of toxicity were noted. Variation in ear thickness during the observation period were more than 25% from day 1 pre-dose values for one animal at 10%. Since ear thickness measurements were not conclusive, 25% was selected as the highest concentration to be used in the main study and ear thickness measurements were added to the main study.

Other results - main study:

Very slight erythema was noted for one animal treated at 25% on day 3, this was considered not to have a toxicologically significant effect on the activity of the nodes.

White test item remnants were present on the dorsal surface of the ears of the test item treated animals between days 1 and 6, which did not hamper scoring of the skin reactions.

The majority of auricular lymph nodes were considered normal in size, except for one of the nodes in one animal treated at 5%, which was considered to be enlarged. 

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Mean DPM/animal values: concentrations 0, 5, 10 and 25% were 333, 785, 514 and 215 DPM, respectively.  The SI values calculated for the test item concentrations 5, 10 and 25% were 2.4, 1.5 and 0.6, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD test guide 429, aluminium tribenzoate was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 25% (w/w), the highest concentration technically achievable.

Executive summary:

An LLNA skin sensitisation study was performed according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 5%, 10% and 25% (w/w), the highest concentration technically possible. In the main study a very slight erythema was noted for one animal treated at 25% on day 3. This was considered not to have a toxicologically significant effect on the activity of the nodes. White test item remnants were present on the dorsal surface of the ears of the test item treated animals between Days 1 and 6, which did not hamper scoring of the skin reactions.The majority of auricular lymph nodes were considered normal in size, except for one of the nodes in one animal treated at 5%, which was considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 785, 514 and 215 DPM, respectively. The mean DPM/animal value for the vehicle control group was 333 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.4, 1.5 and 0.6, respectively. As the SI appeared not to be ≥ 3 when tested up to 25% (w/w), aluminium tribenzoate was considered not to be a skin sensitiser.