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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 10, 2016 to October 25, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/CaOlaHsd mice
Source: Envigo
Hygienic level: SPF at arrival; standard housing conditions during the study
Number of animals: 4 animals / group
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 11 weeks old in Experiment I and 8 weeks old in Experiment II (age-matched, within one week)
Body weight range at starting: 20.8 – 22.7 grams in Experiment I and 19.1 – 19.8 grams in Experiment II
(The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 28 days in Experiment I and 7 days in Experiment II

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunneltubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.0 - 25.7 °C
Relative humidity: 30 - 88 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.

Food and feeding
Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable complete diet for rats/mice” produced by ssniff Spezialdiäten, ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum. Water quality control analysis was performed once every three months and microbiological
assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal
Bedding and certified nest building material were produced by J. Rettenmaier & Söhne GmbH + Co.KG.

Identification and randomisation: a unique number written on the tail with a permanent marker identified each animal.

Vehicle:
dimethylformamide
Concentration:
Preliminary test: 100 and 50 % (w/v) of the test substance in DMF
Experiment I: three groups (4 animals each) received the test substance at 100, 50 and 25 % (w/v) concentrations in DMF
Experiment II: one dose group 2 % (w/v) of the test substance in DMF
No. of animals per dose:
Preliminary test: 2 animals/dose

Experiment I:
twenty female mice were allocated to five groups:
- three groups (4 animals each) received the test substance
- the negative control group (4 animals)
- the positive control group (4 animals)

Experiment II:
twelve mice were allocated to three groups of four animals each:
- one group received the test substance
- the negative control group
- the positive control group
Details on study design:
Solubility test

The solubility of the test substance was examined in a short preliminary compatibility test. The following standard OECD vehicles were assessed: AOO (acetone:olive oil 4:1 (v:v) mixture) and N,N-dimethylformamide (DMF).

The Preliminary Irritation/Toxicity Test
Two doses were used (2 animals/dose) at test substance concentrations of 100 and 50 % (w/v) in DMF. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on day 6 and the radioactive proliferation assay was not performed.

In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse
were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

Topical application
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (days 1, 2 and 3). There was no treatment on days 4, 5 and 6.

Experiment I:
twenty female mice were allocated to five groups:
- three groups (4 animals each) received the test substance at 100, 50 and 25 % (w/v) concentrations in DMF
- the negative control group (4 animals)
- the positive control group (4 animals)

Experiment II:
twelve mice were allocated to three groups of four animals each:
- one group received the test substance at 2 % (w/v) of the test substance in DMF
- the negative control group
- the positive control group

PROLIFERATION ASSAY

Injection of Tritiated Thymidine (3HTdR)
On day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed
before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation
of macromolecules. After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β- scintillation counter and 3HTdR incorporation was measured (10-minute measurement). The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

Clinical Observations
During the study (day 1 to day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice
a day (before and after treatments) on days 1, 2 and 3 and once daily on days 4, 5 and 6.

Individual body weights were recorded on day 1 (beginning of the test) and on day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g. Ear thickness of the experimental animals in the Experiment I was determined by ear punch weight determination which was performed after the animals were humanely killed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
conc. 2 % (w/v)
Key result
Parameter:
SI
Value:
3.1
Test group / Remarks:
conc. 25 % (w/v)
Key result
Parameter:
SI
Value:
3.1
Test group / Remarks:
conc. 50 % (w/v)
Key result
Parameter:
SI
Value:
5
Test group / Remarks:
conc. 100 % (w/v)
Cellular proliferation data / Observations:
Resluts from two main experiments:

CLINICAL OBSERVATION
- No mortality or signs of systemic toxicity were observed during the Experiment I and II.
- Alopecia around the ears was observed in the 100 % (w/v) dose group on days 3-6.
- Test substance precipitate or minimal amount of test substance precipitate was observed on the ears of the experimental animals in the 100 % (w/v) dose group on days 1-6, in the 50 % (w/v) group on days 1-4, in the 25 % (w/v) group on days 1-3 and in the 2 % (w/v) group on day 1.
- No erythema was detected on the experimental animals in the experiments.

BODY WEIGHT MEASUREMENT
No marked body weight loss (≥5 %) was observed on the mean body weight change during Experiment I and II; however the body weight loss was slightly more than 5 % for one animal in each of the 100, 25 % (w/v) and positive control group.

EAR THICKNESS MEASUREMENT
The ear punch weights measured in Experiment I of the animals were within the acceptable range.

PROLIFERATION ASSAY
In Experiment I and II the appearance of the lymph nodes was normal in the negative control groups and in the 50, 25 and 2 % (w/v) test substance treated dose groups. Slightly enlarged
lymph nodes were observed in the 100 % (w/v) dose group. Larger than normal lymph nodes were observed in the positive control groups in both experiments.
The stimulation index values obtained in Experiment I and II were 5.0, 3.1, 3.1 and 1.6 at concentrations of 100, 50, 25 and 2 % (w/v), respectively.

The test substance showed no sensitisation potential at the concentration of 2 % (w/v) which means that EC3 value > 2 % (w/v); therefore the test substance is classified as skin sensitizer Category 1 (sub-category 1B).

Solubility test

The best vehicle taking into account the test substance characteristics, its usage and the requirements of the relevant OECD guideline was considered to be DMF. The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test substance was 100 % (w/v).

Preliminary Irritation / Toxicity Test

Based on these observations, 100 % (w/v) dose was selected as top dose for the main test.

- No mortality or signs of systemic toxicity were observed.

- Alopecia around the ears of the animals was observed in the 100 % (w/v) dose group on Days 4-6.

- Test substance precipitate was observed on the ears of the animals in the 100 % (w/v) dose group on days 1-5 or 1-6 and in the 50 % (w/v) dose group on days 1-5.

- No marked body weight loss (≥5 %) was detected on the mean body weight values of the groups; however one animal had slightly more than 5 % body weight loss in the 100 % (w/v) dose group .

- Increased ear thickness values were observed on day 3 in the 100 % (w/v) dose group; however test substance precipitate was present on the ears of the animals which may have interfered with the measurement. Additionally on Day 6 all the ear thickness data was acceptable. The ear punch weights of all animals were within the acceptable range.

- The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the study conditions, the test substance was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. As the SI is below 3 when a concentration of 2 % (w/v) is used, the test substance is considered a moderate sensitizer and should be classified as Category 1 (sub-category 1B) under Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance in CBA/CaOlaHsd mice according to OECD Guideline 429, EU Method B.42 and OPPTS 870.2600 (Local Lymph Node Assay), in compliance with GLP. The substance was tested for formulation compatibility in N,N-dimethylformamide (DMF) before further tests were carried out. The Preliminary Irritation/Toxicity Test was performed using two doses (2 animals/dose): 100 and 50% (w/v) in DMF. Based on the observations recorded in the preliminary test, 100% (w/v) was selected as top dose for the main test. In the Experiment I, twenty female mice were allocated to five groups of four animals each: three groups received the test substance at 100, 50 and 25% (w/v) concentrations, the negative control group received the vehicle only (DMF), the positive control group received 25% (w/v) HCA (dissolved in DMF). In order to sub-categorize the test substance Experiment II was performed to examine the 2% (w/v) dose. In the Experiment II, twelve mice were allocated to three groups of four animals each: one group received the test substance at a concentration of 2% (w/v), the negative control group received the vehicle only (DMF), the positive control group received 25% (w/v) HCA (dissolved in DMF). The test substance formulations were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. During the study, each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Additionally body weights were checked on Days 1 and 6 (prior to 3HTdR injection). Ear thickness of the experimental animals in the Experiment I was determined by ear punch weight determination which was performed after the animals were humanely killed. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or signs of systemic toxicity were observed during the Experiment I and II. Alopecia around the ears was observed in the 100% (w/v) dose group on Days 3-6. Test substance precipitate or minimal amount of test substance precipitate was observed on the ears of the experimental animals in the 100% (w/v) dose group on Days 1-6, in the 50% (w/v) group on Days 1-4, in the 25% (w/v) group on Days 1-3 and in the 2% (w/v) group on Day 1. No erythema was detected on the experimental animals in the experiments. No marked body weight loss (≥5%) was observed on the mean body weight change during Experiment I and II. The ear punch weights measured in Experiment I of the animals were within the acceptable range. The stimulation index values were 5.0, 3.1, 3.1 and 1.6 at concentrations of 100, 50, 25 and 2% (w/v), respectively in Experiment I and II. The result of the positive control substance HCA was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historical positive control data was noted for the positive control chemical. This result confirmed the validity of the assay. Under the study conditions, the test substance was shown to have a sensitisation potential in the Local Lymph Node Assay. As the SI is below 3 when a concentration of 2% (w/v) was used, the test substance is considered a moderate sensitizer (Váliczkó, 2017).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A study was conducted to determine the skin sensitisation potential of the test substance in CBA/CaOlaHsd mice according to OECD Guideline 429, EU Method B.42 and OPPTS 870.2600 (Local Lymph Node Assay), in compliance with GLP. The substance was tested for formulation compatibility in N,N-dimethylformamide (DMF) before further tests were carried out. The Preliminary Irritation/Toxicity Test was performed using two doses (2 animals/dose): 100 and 50% (w/v) in DMF. Based on the observations recorded in the preliminary test, 100% (w/v) was selected as top dose for the main test. In the Experiment I, twenty female mice were allocated to five groups of four animals each: three groups received the test substance at 100, 50 and 25% (w/v) concentrations, the negative control group received the vehicle only (DMF), the positive control group received 25% (w/v) HCA (dissolved in DMF). In order to sub-categorize the test substance Experiment II was performed to examine the 2% (w/v) dose. In the Experiment II, twelve mice were allocated to three groups of four animals each: one group received the test substance at a concentration of 2% (w/v), the negative control group received the vehicle only (DMF), the positive control group received 25% (w/v) HCA (dissolved in DMF). The test substance formulations were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. During the study, each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Additionally body weights were checked on Days 1 and 6 (prior to 3HTdR injection). Ear thickness of the experimental animals in the Experiment I was determined by ear punch weight determination which was performed after the animals were humanely killed. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or signs of systemic toxicity were observed during the Experiment I and II. Alopecia around the ears was observed in the 100% (w/v) dose group on Days 3-6. Test substance precipitate or minimal amount of test substance precipitate was observed on the ears of the experimental animals in the 100% (w/v) dose group on Days 1-6, in the 50% (w/v) group on Days 1-4, in the 25% (w/v) group on Days 1-3 and in the 2% (w/v) group on Day 1. No erythema was detected on the experimental animals in the experiments. No marked body weight loss (≥5%) was observed on the mean body weight change during Experiment I and II. The ear punch weights measured in Experiment I of the animals were within the acceptable range. The stimulation index values were 5.0, 3.1, 3.1 and 1.6 at concentrations of 100, 50, 25 and 2% (w/v), respectively in Experiment I and II. The result of the positive control substance HCA was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historical positive control data was noted for the positive control chemical. This result confirmed the validity of the assay. Under the study conditions, the test substance was shown to have a sensitisation potential in the Local Lymph Node Assay. As the SI is below 3 when a concentration of 2% (w/v) was used, the test substance is considered a moderate sensitizer (Váliczkó, 2017).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test substance was shown to have sensitisation potential in the Local Lymph Node Assay. As the SI was below 3 when a concentration of 2% (w/v) was used, the test substance is considered a moderate sensitizer and should be classified as Skin Sens. 1B – H317 (May cause an allergice reaction) according to EU CLP (EC 1272/2008) criteria.