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Description of key information

Under the conditions of the study, the test material was shown to have sensitization potential in the Local Lymph Node Assay. The calculated EC3 value of the test material is 3.0 % (w/v).

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:
31 August 2016 to 06 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 9 weeks old (age-matched, within one week). In the preliminary experiment the mice were 10 weeks of age.
- Weight at study initiation: 20.4 – 23.2 g. The weight variation in animals in the study did not exceed ± 20 % of the mean weight. In the preliminary experiment the mice weighed 22.7 - 24.8 g.
- Housing: The animals were housed in groups in Type II polypropylene / polycarbonate cages.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 14 days

FOOD AND WATER QUALITY
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Water quality control analysis was performed once every three months and microbiological assessment was performed monthly

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.8 - 25.9 °C
- Humidity (%): 30 - 88 %
- Air changes (per hr): 15-20 air exchanges/hour.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Vehicle:
dimethylformamide
Remarks:
N,N-dimethylformamide (DMF)
Concentration:
25, 10, 5 and 2.5 % (w/v)
No. of animals per dose:
4 animals per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: AOO (acetone:olive oil 4:1 (v:v) mixture), N,N-dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic® PE9200. The formulations using the listed vehicles at 100 % (w/v) concentrations were not achievable. The best vehicle taking into account the test item characteristics, its usage and the requirements of the relevant OECD guideline was considered to be DMF. The formulation at 25 % (w/v) using DMF as vehicle was the highest concentrations which were suitable for the test.
The 25 % (w/v), 10 % (w/v), 5 % (w/v) and 2.5 % (w/v) formulations appeared to be in solution by visual examination in the main study.
The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)).
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.
Based on the observation of the solubility test, the maximum available concentration was 25 % (w/v).
Test item precipitate or minimal amount of test item precipitate was observed for both animals of the 25 % (w/v) dose group on Days 1-5 and for both animals of the 10 % (w/v) dose group on Days 1-4.

- Preliminary Irritation: The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 25 % (w/v) and 10 % (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. There were no indications of any irritancy at the site of application.

- Systemic toxicity: 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.
During the Preliminary Irritation / Toxicity Test, no mortality was observed.
Marked body weight loss (>5 % decrease) was detected for both animals in the 25 % (w/v) and 10 % (w/v) dose groups. The mean body weight loss of these groups was more than 5 %.
The draining auricular lymph nodes of the animals were visually examined: They were larger than normal for both animals of the 25 % (w/v) dose group and they were slightly enlarged than normal for both animals of the 10 % (w/v) dose group (subjective judgement by analogy with observations of former experiments).
Taking into account these results, the body weight difference was close to the 5 % weight loss in 2 animals of the 25 % (w/v) and in 2 animals of the 10 % (w/v) dose groups. With the small group sizes in the preliminary test, it is difficult to be certain where the threshold for systemic toxicity exists; hence, an additional fourth dose group was also included to ensure that there were three analysable concentrations in the main test. Therefore, 25 % (w/v), 10 % (w/v), 5 % (w/v) and 2.5 % doses were examined in the main test.

- Ear thickness measurements: Ear thickness was measured using a thickness gauge on Day 1 (pre-dose), Day 3 and Day 6, and by ear punch weight determination after the euthanasia of the experimental animals. The ear thickness values and ear punch weights were within the acceptable range.

- Erythema scores:
No erythema = 0
Very slight erythema (barely perceptible) = 1
Well-defined erythema = 2
Moderate to severe erythema = 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema = 4

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- 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.
- Criteria used to consider a positive response: Erythema Scoring above. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25 % on any day of measurement.

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 (phosphate buffered saline) 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 4 °C), 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.

OBSERVATIONS
- 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 records were maintained.

- Measurement of Body Weight
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
RELIABILITY OF THE TEST
The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (DMF) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 6.9) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.
Key result
Parameter:
EC3
Value:
3
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
The appearance of the lymph nodes was slightly enlarged than normal in the 25 % (w/v) and 10 % (w/v) dose groups and they were normal in the negative control group and in the 5 % (w/v) and 2.5 % (w/v) dose groups. Larger than normal lymph nodes were observed in the positive control group.
The stimulation index values were 9.3, 8.5, 4.2 and 2.7 at concentrations of 25 % (w/v), 10 % (w/v), 5 % (w/v) and 2.5 % (w/v), respectively.

EC3 CALCULATION
EC3 means the effective chemical concentration required for SI=3. The calculated EC3 of the test material is 3.0 % (w/v).

CLINICAL OBSERVATIONS: No mortality or signs of systemic toxicity were observed during the main study. Test item precipitate or minimal amount of test item precipitate was observed for all animals of the 25 % (w/v), for all animals of the 10 % (w/v) and for two animals of the 5 % (w/v) dose groups on Days 1-3 and for two animals of the 5 % (w/v) dose group on Days 2-3. There were no indications of any irritancy at the site of application.

BODY WEIGHTS
Marked body weight loss (>5 % reduction of body weight) was observed for one animal of the 25 % (w/v) dose group, for one animal of the 10 % (w/v) dose group and for one animal of the positive control group; and for two animals of the 5 % (w/v) dose group, however the mean body weight loss of these groups was less than 5 %. These changes were considered as individual variability.

Table 1: DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Measured

DPM / group

DPM

Number

of lymph

nodes

DPN

Stimulation

Index

Background

(5 % (w/v) TCA)

32

33

-

-

-

-

Negative control

(DMF)

3596

3563.5

8

445.4

1.0

Reactive Yellow 2

25 (w/v) %

in DMF

33196

33163.5

8

4145.4

9.3

Reactive Yellow 2

10 (w/v) %

in DMF

30380

30347.5

8

3793.4

8.5

Reactive Yellow 2

5 (w/v) %

in DMF

14843

14810.5

8

 

1851.3

4.2

Reactive Yellow 2

2.5 (w/v) %

in DMF

9610

9577.5

8

1197.2

2.7

Positive control

(25 % (w/v) HCA

in DMF)

 

24735

24702.5

8

3087.8

6.9

Interpretation of results:
other: Classified under EU Criteria
Conclusions:
Under the conditions of the study, the test material was shown to have sensitization potential in the Local Lymph Node Assay. The calculated EC3 value of the test material is 3.0 % (w/v).
Executive summary:

The potential of the test material to act as a sensitiser was investigated in a GLP study in accordance with the standardised guidelines OECD 429 and EU Method B.42.

Based on the results of the Preliminary Compatibility Test and the test item characteristics, the test item was tested for formulation compatibility in N,N-dimethylformamide (DMF). The highest achievable concentration based on the regulatory requirements and the physical characteristics of the test item was 25 % (w/v). The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 25 % (w/v) and 10 % (w/v) in DMF. Based on the observations recorded in the preliminary test, the 25 % (w/v) was selected as top dose for the main test.

In the main assay, twenty four female CBA/CaOlaHsd mice were allocated to six groups of four animals each. Four groups received the test material (formulated in DMF) at 25 % (w/v), 10 % (w/v), 5 % (w/v) and 2.5 % (w/v) concentrations. T

he negative control group received the vehicle (DMF).

The positive control group received 25 % (w/v) HCA (dissolved in DMF).

The test item solutions 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. 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 study. Test item precipitate or minimal amount of test item precipitate was observed for all animals of the 25 % (w/v), for all animals of the 10 % (w/v) and for two animals of the 5 % (w/v) dose groups on Days 1-3 and for two animals of the 5 % (w/v) dose group on Days 2-3. Marked body weight loss (≥5 %) was observed in the 25 % (w/v), 10% (w/v) and 5 % (w/v) dose groups and in the positive control group.

There were no indications of any irritancy at the site of application.

The stimulation index values were 9.3, 8.5, 4.2 and 2.7 at concentrations of 25 % (w/v), 10 % (w/v), 5 % (w/v) and 2.5 % (w/v), respectively. The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

Under the conditions of the study, the test material was shown to have sensitization potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of the test material is 3.0 % (w/v).

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

The potential of the test material to act as a sensitiser was investigated in a GLP study in accordance with the standardised guidelines OECD 429 and EU Method B.42. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Based on the results of the Preliminary Compatibility Test and the test item characteristics, the test item was tested for formulation compatibility in N,N-dimethylformamide (DMF). The highest achievable concentration based on the regulatory requirements and the physical characteristics of the test item was 25 % (w/v). The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 25 % (w/v) and 10 % (w/v) in DMF. Based on the observations recorded in the preliminary test, the 25 % (w/v) was selected as top dose for the main test.

In the main assay, twenty four female CBA/CaOlaHsd mice were allocated to six groups of four animals each. Four groups received the test material (formulated in DMF) at 25 % (w/v), 10 % (w/v), 5 % (w/v) and 2.5% (w/v) concentrations. T

he negative control group received the vehicle (DMF).

The positive control group received 25 % (w/v) HCA (dissolved in DMF).

The test item solutions 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. 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 study. Test item precipitate or minimal amount of test item precipitate was observed for all animals of the 25 % (w/v), for all animals of the 10 % (w/v) and for two animals of the 5 % (w/v) dose groups on Days 1-3 and for two animals of the 5 % (w/v) dose group on Days 2-3. Marked body weight loss (≥5 %) was observed in the 25 % (w/v), 10 % (w/v) and 5 % (w/v) dose groups and in the positive control group.

There were no indications of any irritancy at the site of application.

The stimulation index values were 9.3, 8.5, 4.2 and 2.7 at concentrations of 25 % (w/v), 10 % (w/v), 5 % (w/v) and 2.5 % (w/v), respectively. The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

Under the conditions of the study, the test material was shown to have sensitization potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of the test material is 3.0 % (w/v).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is classified with respect to skin sensitisation.