Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-10-10 to 2016-11-21
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
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: liquid (viscous)
- Appearance: clear viscous liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16DB1991
- Expiration date of the lot/batch: 2018-06-07 (retest date)
- Purity/composition correction factor: 1.23
- Purity (GC): 98% w/w

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- 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: Test item preparation were prepared with 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 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: young adults (approx. 11 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- 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 Spezialdiaten 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
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 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:
pre-screen test: 100 and 50%, followed by 10, 5, 2, and 1% w/w
main study: 0, 0.25, 0.5 and 1% w/w
No. of animals per dose:
5 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:
- A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
- Two test item concentrations were tested; a 100% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
- The test system, procedures and techniques were identical as those used in the main study except that the animals were approximately 10 weeks 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 (Kroeplin C110T-K) prior to dosing on days 1 and 3, and on Day 6.
- Animals were sacrificed after the final observation.
- Based on results, eight additional animals were treated in a similar manner with four lower concentrations (1%, 2%, 5% and 10%) at a later stage.
- At 100%, both animals sacrificed for humane reasons on Day 2 due to severe systemic toxicity.
- At 50%, piloerection, hunched posture, lethargy, shallow respiration and/or hypothermia wre noted for animals between Days 2 and 6. Scabs on the ears were noted for both animals between Days 4 and 6.
- At 10%, piloerection and/or hunched posture were noted for both animals between Days 2 and 5. Additionally, bald skin spots were noted for one animals on Day 6.
- At 5%, hunched posture was noted for both animals between Days 2 and 5.
- At 2%, hunched posture and/or piloerection were noted for both animals between Days 1 and 3.
- Based on this systemic toxicity, these concentrations were considered not suitable for the main study.
- At 1%, very light erythema was noted for both animals on Day 2 and scaliness was noted for one animal at Day 3. These findings were considered not to have a toxicologically significant effect on the activity of the nodes. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.
- Based on these results, the highest test item concentration selected for the main study was a 1% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
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. 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 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 statistics performed

Results and discussion

Positive control results:
At concentrations 5%, 10% and 25% SI values of the positive control item were 1.4, 1.5, and 4.3 respectively. An EC3 value of 18.0% 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 16.5, 14.5, 13.4, 14.1, 17.3 and 9.8%
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:
1.6
Variability:
+/- 0.2
Test group / Remarks:
Based on 5 animals in 0.25% w/w in acetone/olive oil 4:1 group
Parameter:
SI
Value:
1.5
Variability:
+/- 0.3
Test group / Remarks:
Based on 5 animals in 0.5% w/w in acetone/olive oil 4:1
Parameter:
SI
Value:
2
Variability:
+/- 0.5
Test group / Remarks:
Based on 5 animals in 1% w/w in acetone/olive oil 4:1
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
0% w/w group: mean DPM/animal ± SEM: 634 ± 37
0.25% w/w group: mean DPM/animal ± SEM: 1005 ± 114
0.5% w/w group: mean DPM/animal ± SEM: 948 ± 166
1% w/w group: mean DPM/animal ± SEM: 1282 ± 291

In test group 0.25%, the DPM value (3344) of animal no.10 was considered an outlier and was therefore excluded from interpretation. Sufficient data was available to warrant study integrity.
SEM = Standard Error of the Mean

EC3 CALCULATION
There was no indication that the test item elicits a SI ≥ 3 when tested up to 1%. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 1%.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: No erythema 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: 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.

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:
GHS criteria not met
Conclusions:
Based on clinical signs, ear thickness and skin irritation at higher concentrations, the highest test item concentration selected for the main study was a 1% concentration. There was no indication that the test item elicits a SI ≥ 3 when tested up to 1%. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 1%.

Based on these results,the test item at concentrations up to 1% was not regarded as a skin sensitizer according to the recommendations made in the test guidelines.