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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:
2017-11-29 to 2018-01-08
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
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical state: Viscous liquid
- Appearance: Yellow to orange, clear liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I17BB0397
- Expiration date of the lot/batch: 2018-03-14 (retest date)
- Purity/composition correction factor: 1.23
- Purity: 98.2% w/w GC

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: There was no information available regarding the solubility or stability in vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item container was heated at approximately 60°C for 3 days prior to weighing of the test item. 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. 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: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Approximately 10 weeks old
- Weight at study initiation: 19.4 - 23.9 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 of the main study, 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 (deg C): 18 to 24°C; actual daily mean temperature during the study period: 22°C
- Humidity (%): 40 to 70%; actual main relative humidity during the study period: 42 to 46%
- Air changes (per hr): At least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
PreScreen Test: 50% and 100% w/w
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (10% and 25%) at a later stage. At a 10% and 5% test item concentration no signs of toxicity or excessive irritation were noted and therefore 10% was selected as the highest concentration to be used in the main study.
Main Study: 0, 2%, 5%, 10% w/w
No. of animals per dose:
Five females per group; 4 groups (including control group)
Details on study design:
Rationale vehicle selection:
The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at Charles River Den Bosch. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v) (clear solution), N,N-dimethylformamide (not tested according vehicle selection criteria), methylethylketone (not tested according vehicle selection criteria), propylene glycol (not tested according vehicle selection criteria) and dimethylsulfoxide (not tested according vehicle selection criteria).

PRE-SCREEN TESTS:
- Compound solubility: no data
- Irritation: The very slight irritation of the ears as shown by the animals treated at 10% between Days 2 and 5 was considered not to have a toxicologically significant effect on the activity of the nodes.
- Systemic toxicity: At a 10% and 5% test item concentration no signs of toxicity were noted and therefore 10% was selected as the highest concentration to be used in the main study.
- Ear thickness measurements: At a 100%, 50% and 25% test item concentration,variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted (with one animal found dead at 100%). Therefore these concentrations did not meet the selection criteria.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- 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 evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (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 skinreaction
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:
At concentrations 5%, 10% and 25% SI values of the positive control item 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 in the accepable range of 4.8 and 19.5%. The results of the 6 monthly 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 in the laboratory is an appropriate model for testing contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
4.4
Variability:
+/- 0.8
Test group / Remarks:
Based on 5 animals in 2% w/w in acetone/olive oil 4:1 group
Parameter:
SI
Value:
14.3
Variability:
+/- 2.1
Test group / Remarks:
Based on 5 animals in 5% w/w in acetone/olive oil 4:1 group
Parameter:
SI
Value:
39.8
Variability:
+/- 8.1
Test group / Remarks:
Based on 5 animals in 10% w/w in acetone/olive oil 4:1 group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
mean DPM ± SEM:
0% w/w group: mean DPM ± SEM: 455 ± 90
2% w/w group: mean DPM ± SEM: 1992 ± 342
5% w/w group: mean DPM ± SEM: 6502 ± 971
10% w/w group: mean DPM ± SEM: 18090 ± 3670
SEM = Standard Error of the Mean

EC3 CALCULATION
The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between 0 and 2%.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: The very slight irritation of the ears as shown by the animals treated at 10% between Days 2 and 5 was considered not to have a toxicologically significant effect on the activity of the nodes.
- 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 were considered normal in size, except for the nodes in the animals at a concentration of 10%, which were considered enlarged. 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 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
These results show that the test item elicits a SI ≥ 3. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between 0 and 2%.
Based on these results:
- according to the recommendations made in the test guidelines (including all amendments), T001591 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), T001591 should be classified as skin sensitizer (Category 1A).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), T001591 should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.