Registration Dossier

Diss Factsheets

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:
16 March 2016 to 12 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Benzaldehyde, hydroxy-, polymer with phenol
Cas Number:
106466-55-1
Molecular formula:
(C7H6O2.C6H6O)x
IUPAC Name:
Benzaldehyde, hydroxy-, polymer with phenol
Test material form:
solid: flakes
Details on test material:
- Appearance: Red – reddish brown solid flake
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH %)
Specific details on test material used for the study:
- Analytical determination of the test material concentration, stability and homogeneity was not performed because of the character and the short period of study.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 weeks old (age-matched, within one week)
- Weight at study initiation: 17.3 – 18.9 grams (the weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
- Only healthy animals were used for the study. Health status was certified by the veterinarian.
- Housing: Group caging in Type II. polypropylene / polycarbonate cages, mice were provided with glass tunnel-tubes. Bedding was available to animals during the study.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.1 - 25.8 °C
- Humidity: 28 - 78 %
- Air changes: 15-20 air exchanges/hour
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
5, 10, 25 and 50 % (w/v)
No. of animals per dose:
Four females per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test material 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, Propylene glycol, Dimethyl sulfoxide and 1% aqueous Pluronic® PE9200. The best vehicle taking into account the test material 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 material was 50 % (w/v). The 50, 25, 10 and 5 % (w/v) formulations appeared to be solutions by visual examination. The test material was weighed and formulations prepared daily on a weight: volume basis (as % (w/v)) in the Pharmacy of the testing facility.
- 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 material concentrations of 50 and 25 % (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 using Table 1. 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.
- During the Preliminary Irritation / Toxicity Test, no mortality was observed. Extensive alopecia was observed on the animals in the 50 % (w/v) dose group on Days 2-6 and alopecia (around the ears only or extensive) was also observed in the 25 % (w/v) group on Days 3-6. Test material precipitate or minimal amount of test material precipitate was observed on the ears of the animals in 50 % (w/v) dose group on Days 1-6 and in the 25 % (w/v) dose group on Days 1-4. No marked body weight loss (>5 %) was detected in the experimental animals.
- Increased ear thickness values (≥25 % increase) were observed in the 50 % (w/v) dose group; however test material precipitate was present on the ears of the animals during the experiment which may have interfered with the measurement. The ear thickness values of the 25 % (w/v) dose group and ear punch weights of both groups were within the acceptable range.
- The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in both dose groups (subjective judgement by analogy with observations of former experiments).
- Based on these observations, the 50 % (w/v) dose was selected as top dose for the main test, however due to the observed increased ear thickness values in this group, an additional dose group (a total of four) was used in the main experiment to ensure that there are three acceptable dose groups for evaluation. Therefore, 50, 25, 10 and 5 % (w/v) doses were examined in the main test. Additionally, ear thickness of the experimental animals in the main test was measured by using a thickness gauge on Days 1 and 6 and by ear punch weight determinations, which was performed after the animals are humanely killed.

MAIN STUDY
- 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.

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 euthanised 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.
- Measurement of Ear Thickness: Ear thickness of the experimental animals in the main test was determined by using a thickness gauge on Days 1 and 6 and by ear punch weight determination which was performed after the animals were humanely killed.

EVALUATION OF THE RESULTS
- DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

- Interpretation of Results:
The test material is regarded as a sensitiser if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Since the test material gave a positive response and data permitted, the EC3 value of the test material was calculated (EC3 means the effective chemical concentration required for SI=3). The calculation of the EC3 value was conducted by log-linear interpolation according to the equation:
EC3ex = 2^{log^2(c) + [(3 – d)/(b- d)] x [log^2(a) – log^2(c)]
Where the lowest data point (lying immediately above the SI value of 3 on the LLNA dose-response plot) has the co-ordinates (c,d), and the next higher data point has the co-ordinates (a,b).

- Acceptability of the test:
The Local Lymph Node Assay is considered valid if it meets the following criteria:
- the DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,
- the positive control substance produces a significant lymphoproliferative response increases (SI > 3),
- each treated and control group includes at least 4 animals,
- the test material does not cause serious systemic or local toxicity.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
- 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 5.7) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
4.3
Remarks on result:
other: % (w/v)
Key result
Parameter:
SI
Value:
15.1
Test group / Remarks:
50 % (w/v)
Key result
Parameter:
SI
Value:
13.3
Test group / Remarks:
25 % (w/v)
Key result
Parameter:
SI
Value:
8.1
Test group / Remarks:
10 % (w/v)
Key result
Parameter:
SI
Value:
3.9
Test group / Remarks:
5 % (w/v)
Cellular proliferation data / Observations:
CLINICAL OBSERVATION
-No mortality or signs of systemic toxicity were observed during the study. Alopecia or extensive alopecia was observed on the animals in the 50 % (w/v) dose group on Days 2-6 and in the 25 % (w/v) dose group on Days 3-6. Test material precipitate or minimal amount of test material precipitate was observed on the ears of the animals in the 50 and 25 % (w/v) dose groups on Days 1-6 and in the 10 % (w/v) dose group on Days 2-3.

BODY WEIGHT MEASUREMENT
-No treatment related effects were observed on animal body weights.

EAR THICKNESS MEASUREMENT
-Increased ear thickness values (≥25 % increase) were observed in the 50 % (w/v) dose group; the mean ear thickness increase of this group was 26.4 %; however test material precipitate was present on the ears of the animals during the experiment which may have interfered with the measurement. Slightly increased ear thickness values were also observed in the 25 % (w/v) dose group (mean increase of the group was 17.3 % which is well below the limit of 25 %); however minimal amount of test material precipitate was present on the ears which may have interfered with the measurement. The amount of the precipitate on the ears in both groups is in good correlation with the ear thickness values.
-Since the ear punch weights of all animals in the study were within the acceptable range, all doses are considered to be acceptable and are evaluated.

PROLIFERATION ASSAY
-The results of the proliferation assay are summarized in Table 2. The appearance of the lymph nodes was normal in the negative control group. Slightly enlarged lymph nodes were observed in the 5 % (w/v) dose group. Larger than normal lymph nodes were observed in the 10, 25 and 50 % (w/v) dose groups and in the positive control group.
- The stimulation index values were 15.1, 13.3, 8.1 and 3.9 at concentrations of 50, 25, 10 and 5 % (w/v), respectively.

INTERPRETATION OF OBSERVATIONS
-The test material was solid flake, which was formulated in DMF. There were no confounding effects of systemic toxicity at the applied concentrations. At 50 % (w/v) there was a borderline possible increase in ear thickness, but at 25, 10 and 5 % (w/v) there were no indications of any inflammation of the ears that could interfere with the proliferation results, hence the values obtained are considered to reflect the real potential of the test material to cause lymphoproliferation in the Local Lymph Node Assay. The results followed a clear, classical dose response, confirming the positive result. The resulting stimulation indices observed above the threshold limit of 3 at all the tested concentrations under these exaggerated test conditions was considered to be good evidence that the test material is a sensitiser. The size of lymph nodes was in good correlation with this conclusion.
-The obtained data are compatible with a conventional dose response and allow the calculation of the EC3 value according to an adequate scientific method. EC3 means the effective chemical concentration required for SI=3. The calculated EC3 value of the test material is 4.3 % (w/v).

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 5.7) 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.

Any other information on results incl. tables

Table 2: 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)

3430

3397.5

8

424.7

1.0

Test Material 50 % (w/v) in DMF

51196

51163.5

8

6395.4

15.1

Test Material 25 % (w/v) in DMF

45228

45195.5

8

5649.4

13.3

Test Material 10 % (w/v) in DMF

27403

27370.5

8

3421.3

8.1

Test Material 5 % (w/v) in DMF

13372

13339.5

8

1667.4

3.9

Positive control (25 % (w/v) HCA in DMF)

19501

19468.5

8

2433.6

5.7

Applicant's summary and conclusion

Interpretation of results:
other: EU Criteria: Category 1B. May cause an allergic skin reaction (H317)
Conclusions:
Under the conditions of this study, the test material was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay. The calculated EC3 value of the test material is 4.3 % (w/v).
Executive summary:

The skin sensitisation potential of the test material following dermal exposure was investigated in accordance with the standardised guidelines OECD 429 and EU Method B.42, under GLP conditions. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.

Based on the results of the Preliminary Compatibility Test, the test material characteristics, its usage and on the recommendations of the OECD Guideline, the test material was tested for formulation compatibility in N,N-dimethylformamide (abbreviated as DMF). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test material was 50 % (w/v).

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50 and 25 % (w/v) in DMF. Based on the observations recorded in the preliminary test, the 50 % (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 50, 25, 10 and 5 % (w/v) concentrations, the negative control group received the vehicle (DMF) and the positive control group received 25 % (w/v) HCA (dissolved in DMF).

The test material 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. Alopecia or extensive alopecia was observed on the animals in the 50 % (w/v) dose group on Days 2-6 and in the 25 % (w/v) dose group on Days 3-6. Test material precipitate or minimal amount of test material precipitate was observed on the ears of the animals in the 50 and 25 % (w/v) dose groups on Days 1-6 and in the 10 % (w/v) dose group on Days 2-3. No treatment related effects were observed on animal body weights.

The stimulation index values were 15.1, 13.3, 8.1 and 3.9 at concentrations of 50, 25, 10 and 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 this study, the test material was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay. The calculated EC3 value of the test material is 4.3 % (w/v).

Categories Display