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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:
17 October 2017 - 07 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See assessment attached below.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(July 2010)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(July 2012)
Deviations:
no
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

Constituent 1
Reference substance name:
Reaction products of Resin acids and Rosin acids, sodium salts and barium chloride
Molecular formula:
not applicable
IUPAC Name:
Reaction products of Resin acids and Rosin acids, sodium salts and barium chloride
Test material form:
solid: particulate/powder
Details on test material:
Name as cited in the report: Barium salts of resin acids and rosin acids
Appearance: light tan powder
Storage conditions: at room temperature desiccated
Specific details on test material used for the study:
pH (1% in water, indicative range) 6.8 – 7.6 (determined at the test facility)

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. 10 weeks old)
- Weight at study initiation: 18.6 - 23.7 g
- Housing: Animals were group 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 (set conditions)
- Temperature (°C): 18 – 24 (actual: 21-22)
- Humidity (%): 40 - 70 (actual: 42-61)
- Air changes (per hr): approx >= 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 October 2017 to 06 November 2017

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10 and 25%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
In a pre-screen test, two test item concentrations were tested; a 25% and 50% (maximum technically applicable) concentration.
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 animals were treated with one concentration each on three consecutive days. Ear thickness measurements were conducted on days 1 and 3, and on day 6. Animals were sacrificed after the final observation.

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 using the individual SI values. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

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, kept under room temperature until dosing and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were stirred until and during dosing.
Rationale for vehicle: The vehicle was selected based on trial preparations performed at Charles River Den Bosch and on information provided 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 indicates 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)

Resultsopen allclose all
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test item concentrations 0, 5, 10 and 25% were 531, 726, 999 and 1402 DPM, respectively.
Key result
Parameter:
SI
Value:
1.4
Variability:
0.2
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.9
Variability:
0.4
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
2.6
Variability:
0.7
Test group / Remarks:
25%

Any other information on results incl. tables

Results Pre-screen test:

At a 50% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and therefore this concentration did not meet the selection criteria. At a 25% test item concentration, no signs of systemic toxicity were noted and no irritation was observed and therefore this concentration was selected as highest concentration for the main study.

Other results - main study:

Skin reactions / Irritation:

No irritation was observed in any of the animals. Brown test item remnants were present on the dorsal surface of the ears of all animals at 25% throughout the observation period, which did not hamper scoring of the skin reactions.

Systemic toxicity:

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.

Macroscopic examinations of the 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 for any of the animals.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines and GLP principles, Barium salts of resin acids and rosin acids was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 25% v/v.
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% v/v. No irritation was observed in any of the animals. Brown test item remnants were present on the dorsal surface of the ears of all animals at 25% throughout the observation period, which did not hamper scoring of the skin reactions. 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. 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 for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 726, 999 and 1402 DPM, respectively. The mean DPM/animal value for the vehicle control group was 531 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.4, 1.9 and 2.6, respectively. As the SI appeared not to be ≥ 3 when tested up to 25% v/v, Barium salts of resin acids and rosin acids was considered not to be a skin sensitiser.