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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:
2018-02-07 to 2018-03-012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
Paris Cedex, July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Official Journal of the European Union No. L142, May 2008, including most recent amendments.
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Details on test material:
Physical state: liquid
Appearence: light brown liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16FB3013
- Expiration date of the lot/batch: 2018-06-07 (retest date)
- Purity/correction factor: 1.14

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions.
In order to obtain homogenous samples, the test item was heated at approximately 60ºC for approximately 3 days prior to weighing of the test item. Correction of the purity/composition of the test item was not applicable, since the test method required a logical concentration range rather than specific dose levels to be dosed.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Remarks:
inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 20 female (nulliparous and non-pregnant) mice, CBA/J strain, inbred, SPF-Quality from Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 9 weeks old
- Weight at study initiation: 19.5 - 26.6 grams
- Housing: group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA)were supplied as cage-enrichment. On day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days before the start of treatment, under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C; actual daily mean temperature during the study period was 22°C.
- Humidity (%): 40 to 70%; actual daily mean relative humidity of 40-45%.
- Air changes (per hr): 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
PreScreen Test: 50% and 100% w/w
Main Study: 0, 2%, 10%, 25% w/w
No. of animals per dose:
5 females per group; 4 groups (including control group)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: no data
- At a 100% test item concentration, animals were killed in extremis on Day 2.
- At a 50 and 25% test item concentration, clinical signs of toxicity were noted and variations in ear thickness during the observation period exceeded 25% from Day 1 pre-dose values for the animals.
- At a 10% test item concentration, no signs of systemic toxicity were noted and no irritation was observed. Variations in ear thickness were less than 25%.
Based on these results, the highest test item concentration selected for the main study was a 10% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT - INDUCTION days 1, 2, 3
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer. The results were ev
aluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (G
HS) of the United Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008
of the European Parliament and of the Council of 16 December 2008 on classification, labelling and
packaging of substances and mixtures, including all amendments. Consideration was given to the
EC3 value (the estimated test item concentration that will give a SI =3).

Classification of results:
SI value UN-GHS 2015; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer -
SI ≥ 3 Cat 1 Skin sensitizer H317: May cause an allergic skin reaction
EC3 value ≤ 2%: sub-category 1A
EC3 value ≥ 2%: sub-category 1B
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data

Results and discussion

Positive control results:
The SI values calculated for the substance concentrations 2, 5 and 10% were 1.4, 2.2 and 3.5 respectively. An EC3 value of 19.2% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.2, 14.1, 17.3, 9.8, 17.8, 18.0, 14.7 and 13.2%.
Based on the results, it was concluded 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:
SI
Value:
1.9
Variability:
+/- 0.2
Test group / Remarks:
based on 5 animals of 2% w/w in acetone/olive oil 4:1 group
Parameter:
SI
Value:
8.2
Variability:
+/- 1.1
Test group / Remarks:
based on 5 animals of 5% w/w in acetone/olive oil 4:1 group
Parameter:
SI
Value:
16.4
Variability:
+/- 1.4
Test group / Remarks:
based on 5 animals of 10% w/w in acetone/olive oil 4:1 group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
0% w/w group: mean DPM ± SEM: 713 ± 287
2% w/w group: mean DPM ± SEM: 1333 ± 158
5% w/w group: mean DPM ± SEM: 5812 ± 800
10 %w/w group: mean DPM ± SEM: 11655 ± 973
SEM = Standard Error of the Mean

EC3 CALCULATION
an EC3 value (the estimated test item concentration that will give a SI =3) of 2.5% was calculated

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: No irritation of the ears was observed in any of the animals examined.
- Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.
- Macroscopy of the auricular lymph nodes and surrounding area: The majority of auricular lymph nodes of the test item treated groups were considered enlarged. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

BODY WEIGHTS
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:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 2.5% was calculated.

Based on these results:
- according to the recommendations made in the test guidelines (including all amendments), T001596 would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), T001596 should be classified as skin sensitizer (Category 1B).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), T001596 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.