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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:
01 November 2017 - 29 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
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:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(July 2012)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(March 2003)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd. 3 November 2015
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:
Physical appearance: pink 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. 10 weeks old)
- Weight at study initiation: 19.0 - 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 - 55 )
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 November 2017 - 27 November 2017

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:
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 indicate 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
Key result
Parameter:
SI
Value:
3
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
5.5
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
4.2
Test group / Remarks:
50%

Any other information on results incl. tables

Results Pre-screen test:

At a 25% and 50% test item concentration, no signs of systemic toxicity were noted and no excessive irritation was seen.

Other results - main study:

Desintegrations per minute (DPM):

Mean DPM/animal values for the experimental groups treated with test item concentrations 0, 10, 25, and 50% were 215, 648, 1191 and 911 DPM, respectively.

Skin reactions / Irritation:

No irritation was observed in any of the animals. Pink test item remnants were present on the dorsal surface of the ears of all test item treated animals throughout the study 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. Pink discoloration of urine, feaces and uncovered body parts was noted for all test item treated animals between Days 2 and 6, this was considered to be due to the staining properties of the test item and was considered to be not toxicologically relevant.

Macroscopic examinations of the lymph nodes and surrounding area:

The majority of auricular lymph nodes were considered normal in size, except for the nodes of one animal treated at 25% and one animal treated at 50%, which were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on an LLNA performed according to OECD guideline 429 and under GLP principles, C.I. Pigment Red 81:2 was considered to be a skin sensitiser (category 1B) with an EC3 value of 10%.
Executive summary:

An LLNA skin sensitisation study was performed according to OECD 429 /EC B.42 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), the highest concentration as required by the test guidelines. In the main study, no irritation was observed in any of the animals. Pink test item remnants were present on the dorsal surface of the ears of all test item treated animals throughout the study 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. Pink discoloration of urine, feaces and uncovered body parts was noted for all test item treated animals between days 2 and 6, this was considered to be due to the staining properties of the test item and was considered to be not toxicologically relevant. The majority of auricular lymph nodes were considered normal in size, except for the nodes of one animal treated at 25% and one animal treated at 50%, which were considered enlarged. 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 10%, 25% and 50% were 648, 1191 and 911 DPM, respectively. The mean DPM/animal value for the vehicle control group was 215 DPM. The SI values calculated for the test item concentrations 10%, 25% and 50% were 3.0, 5.5 and 4.2, respectively These results show that the test item elicits a SI ≥ 3. The EC3 value was established to be 10%. According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), C.I. Pigment Red 81:2 is classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.