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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:
04 April 2018 - 23 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
No conclusion on skin sensitisation can be drawn based on the DEREK (518729), DPRA (517830) and KeratinoSens (518731). See justification 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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
other: EC No 640/2012, Part B: Skin sensitization: "Local Lymph Node Assay"
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Off-white powder
- Storage condition of test material: 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. 9 weeks old)
- Weight at study initiation: 16.9 - 22.2 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
- Temperature (°C): 22 – 23
- Humidity (%): 41 - 48
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 04 April 2018 - 23 April 2018

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 10, 25, 50% (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Two test item concentrations were tested: a 50% and 25% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10 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 (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.

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 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 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 and on test substance data supplied by the sponsor. The vehicle was chosen from the vehicles specified in the test guideline.


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 within 1 hour after dosing) according to a numerical scoring system (see table 1 below). 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 is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.9
Test group / Remarks:
50%
Remarks on result:
other: Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 50%, the substance was not considered to be a skin sensitizer
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
10%
Cellular proliferation data / Observations:
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 592, 761 and 716 DPM, respectively. The mean DPM/animal value for the vehicle control group was 373 DPM.

Any other information on results incl. tables

Results Pre-screen test:

At a 50 and 25% test item concentration, no signs of systemic toxicity were noted and no irritation was observed and therefore the 50% 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. White test item remnants were present on the dorsal surface of the ears of the test item treated animals between days 1 and 4, which did not hamper scoring of the skin reactions.

Macroscopic  Examination of the Lymph Nodes and Surrounding Area

The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 10%, for which the nodes were considered to be smaller. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Systemic Toxicity and bodyweight

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.

Applicant's summary and conclusion

Interpretation of results:
other: Not skin sensitizing
Remarks:
according to EC No 1272/2008
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, PREDIAC-Z was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 50% (w/w).
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 10%, 25% and 50% w/w. No irritation was observed in any of the animals. White test item remnants were present on the dorsal surface of the ears of the test item treated animals between days 1 and 4, 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 10%, for which the nodes were considered to be smaller. 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 10, 25 and 50% were 592, 761 and 716 DPM, respectively. The mean DPM/animal value for the vehicle control group was 373 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.6, 2.0 and 1.9, respectively. As the SI appeared not to be ≥ 3 when tested up to 50% w/w, PREDIAC-Z was considered not to be a skin sensitiser.