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Administrative data

Description of key information

Results of a local lymph node assay showed the test item to be non-sensitising (OECD 429).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 November 2016 to 08 November 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:
yes
Remarks:
broader temperature and humidity ranges than planned with no impact on results or integrity of the study (see below)
GLP compliance:
yes (incl. certificate)
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:
EXPERIMENTAL ANIMALS
- Species and strain: CBA/CaOlaHsd mice
- Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
- Hygienic level: SPF at arrival; standard housing conditions during the study
- Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
- Number of animals: 4 animals / group
- Sex: Female, nulliparous, non-pregnant
- Age of animals at starting: 10 weeks old (age-matched, within one week)
- Body weight range at starting: 20.1 – 22.1 grams (The weight variation in animals in the study did not exceed  20 % of the mean weight.)
- Acclimatisation time: 21 days

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 tunnel tubes
- 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: 18.1 - 25.7°C
- Relative humidity: 23 - 84 %
- Ventilation: 15-20 air exchanges/hour
- The temperature and relative humidity were recorded twice every day during the
acclimatisation and experimental phases.
- Room/Cabinet (preliminary experiment): 244/3
- Room/Cabinet (non-radioactive phase): 244/6
- Room/Cabinet (radioactive phase): 139 – 140

FOOD AND FEEDING
- Animals received ssniff SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” (Batch number: 278 5652, Expiry date: 30 November 2016, Batch number: 141 8884, Expiry date: 31 January 2017 and Batch number: 285 17890, Expiry date: 31 August 2017) produced by ssniff Spezialdiäten
GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), 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 (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at CiToxLAB Hungary Ltd.

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 produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Certified nest building material was also provided for animals (ARBOCEL crinklets natural 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. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file.
- The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups.
- randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
AOO
Concentration:
10 %, 5 % and 2.5 % w/v in the main assay
No. of animals per dose:
Four
Details on study design:
FORMULATION
- 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) concentration 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 AOO.
- The formulation at 10 % (w/v) using AOO (after approximately 10-minutes incubation in an ultrasonic water bath and stirring) as vehicle was the highest concentration which was suitable for the test. The formulations appeared to be in suspension by visual examination and were stirred before treatment.
- The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd.
- Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

CONTROLS
- Animals assigned to the negative control group were treated with the vehicle only concurrent to the test item treated groups.
- Based on the result of the Preliminary Compatibility Test, AOO was selected for vehicle of the study.
- Acetone (VWR; batch 15J060514; expiry date 31 October 2020; room temperature storage conditions).
- Olive oil (Sigma Aldrich; batch BCBS0084V; expiry date 31 May 2018; room temperature storage conditions protected from light).
- Animals assigned to the positive control group were treated with 25 % (w/v) α-Hexylcinnamaldehyde solution (dissolved in AOO) concurrent to the test item treated groups.

OTHER CHEMICALS USED IN THE STUDY
- Details of other chemicals used in the study are given in Table 1 (attached).

INSTRUMENT
- Name: Tri-Carb 2810 Liquid Scintillation Analyzer
- Manufacturer: PerkinElmer
- Serial Number: DG10084483
- IQ / OQ Protocol #: 1593646-1
- Date of IQ: 25 November 2008
- Date of last OQ: 16 December 2015

DOSE SELECTION AND JUSTIFICATION
- 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 10% (w/v) and 5% (w/v) in AOO. 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.
- The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 10 % (w/v).
- 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 2 (attached). 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 or signs of systemic toxicity were observed. No test item precipitate was observed on the ears of the animals. There were no indications of any irritancy at the site of application. Clinical observations are summarised in Table 8 of Appendix 4 (attached).
- No marked body weight loss (> 5% reduction of body weight) was observed on the animal body weight in the preliminary experiment (Table 6 of Appendix 4, attached).
- Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. The ear thickness values and the weights of the ear punches (2 per animal) are summarised in Table 7 of Appendix 4 (attached). The ear thickness values and ear punch weights were within the acceptable range.
- The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).
- Based on these results, 10% (w/v) dose is selected as top dose for the main test.

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
- 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).
- 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 each mouse were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The nodes of each animal were processed individually.
- A single cell suspension (SCS) of 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). 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 the lymph nodes of each individual animal.
- 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 ß-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
- The ß-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 records were maintained.

MEASUREMENT OF EAR THICKNESS
- Ear thickness of the experimental animals in the main test was determined by using a thickness gauge on Days 1, 3 and 6 and by ear punch weight determination which was performed after the animals were humanely killed.

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.

EVALUATION OF RESULTS
- The proliferative response of lymph node cells from the lymph nodes of each individual animal is expressed as radioactive disintegrations per minute (DPM) per animal. 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 disintegrations per node (DPN = DPM divided by the number of lymph nodes) for each animal following the industry standard for data presentation.
- The stimulation index (SI = mean DPN of treated group divided by mean DPN of the appropriate 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 item is regarded as a sensitiser if both of the following criteria are fulfilled:
(i) That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
(ii) 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.

ACCEPTABILITY OF THE TEST
- The Local Lymph Node Assay is considered valid if it meets the following criteria:
(i) The DPN value of the negative (vehicle) control group falls within the range of
historical laboratory control data,
(ii) The positive control substance produces a significant lymphoproliferative response
increases (SI>3),
(iii) Each treated and control group includes at least 4 animals,
(iv) The test item does not cause serious systemic or local toxicity.

USE OF RADIOACTIVE MATERIALS
- Use of radioactive materials was recorded in the appropriate register.
- Regular decontamination of the working area with a verification of decontamination was carried out.
- Radioactive waste materials were processed according to normal laboratory standards.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
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 12.2) was noted for HCA in the main experiment.
- This value was considered to confirm the appropriate performance of the assay.
Parameter:
SI
Value:
0.9
Test group / Remarks:
10 % w/v in AOO
Parameter:
SI
Value:
1.7
Test group / Remarks:
5 % w/v in AOO
Parameter:
SI
Value:
1.1
Test group / Remarks:
2.5 % w/v in AOO
Cellular proliferation data / Observations:
PROLIFERATION ASSAY
- The appearance of the lymph nodes was normal in the negative control group and in all test item treated dose groups.
- Larger than normal lymph nodes were observed in the positive control group.
- The stimulation index values were 0.9 (10 % w/v), 1.7 (5 % w/v) and 1.1 (2.5 % w/v).

CLINICAL OBSERVATIONS
- No mortality or signs of systemic toxicity were observed during the main study. No test item precipitate was observed.
- There were no indications of any irritancy at the site of application.
 
EAR THICKNESS MEASUREMENT
- Similarly to the preliminary experiment, ear thickness of the animals was measured using by a thickness gauge and by ear punch weight determination.
- Slightly increased ear thickness value was detected in one animal (only left ear) but the resultant data was below the limit of positivity.
- The ear thickness values and the ear punch weights were within the historical control range in all cases.
 
BODY WEIGHT MEASUREMENT
-No treatment related marked body weight loss (≥5%) was observed on the mean body weight changes of the groups.
- Marked body weight loss (> 5% reduction of body weight) was observed for one animal of the negative control group, for one animal of the 5% (w/v) dose group.These changes were considered as individual variability.

INTERPRETATION OF OBSERVATIONS

- The test item was solid, which was formulated in AOO. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

- The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that the substance is a non-sensitiser. The size of lymph nodes was in good correlation with this conclusion.

 

RELIABILITY OF THE RESULT

- The result of the positive control substanceα-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 (AOO) using CBA/CaOlaHsd mice.

- Furthermore, the reported mean DPN values observed for the vehicle and positive control substance were in harmony with the historical control range. Three individual vehicle control DPN values were lower than the normal historical control range, but the mean and the other available negative control DPN values and the positive control group results were in the expected historical range. The test item did not show any signs of sensitisation potential when compared with concurrent or historic controls and showed no treatment related trend. The positive control caused lymphoproliferative response in line with historic positive control data. The study was considered to be valid.

- Historical control data for the positive and negative control substances are shown in Appendix 6 (attached).

 

Interpretation of results:
GHS criteria not met
Conclusions:
The substance, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitiser) in the Local Lymph Node Assay.
Executive summary:

GUIDELINE

The study was performed in accordance with OECD Guidelines for Testing of Chemicals No. 429, Skin Sensitisation: Local Lymph Node Assay (22 July 2010).

 

METHODS

The aim of this study was to determine the skin sensitisation potential of the test material following dermal exposure. The study was performed with vertebrate animals as no full regulatoryin vitroalternative 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 item characteristics, its usage and on the recommendations of the OECD Guideline, the test item was tested for formulation compatibility in acetone:olive oil (abbreviated as AOO). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 10% (w/v).

 

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 10% (w/v) and 5% (w/v) in acetone/olive oil 4:1 v/v (AOO). Based on the observations recorded in the preliminary test, the 10% (w/v) was selected as top dose for the main test. In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received test item (formulated in AOO) at 10% (w/v), 5% (w/v) and 2.5% (w/v) concentrations,

- the negative control group received the vehicle (AOO),

- the positive control group received 25 % (w/v) HCA (dissolved in AOO).

 

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. On Day 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).

 

RESULTS

No mortality or signs of systemic toxicity were observed during the main study. No test item precipitate was observed.No treatment related marked body weight loss (≥ 5%) wasobserved on the mean body weight changes of the groups. There were no indications of any irritancy at the site of application.

 

The stimulation index values were 0.9, 1.7 and 1.1 at concentrations of 10% (w/v), 5% (w/v) and 2.5 % (w/v), respectively.

 

The result of the positive control substanceα-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle 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.

 

CONCLUSION

The substance, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitiser) in the Local Lymph Node Assay.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The key study was performed in accordance with OECD Guidelines for Testing of Chemicals No. 429, Skin Sensitisation: Local Lymph Node Assay (22 July 2010). The aim of the study was to determine the skin sensitisation potential of the test material following dermal exposure. 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 item characteristics, its usage and on the recommendations of the OECD Guideline, the test item was tested for formulation compatibility in acetone:olive oil (abbreviated as AOO). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 10% (w/v).

 

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 10% (w/v) and 5% (w/v) in acetone/olive oil 4:1 v/v (AOO). Based on the observations recorded in the preliminary test, the 10% (w/v) was selected as top dose for the main test. In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received test item (formulated in AOO) at 10% (w/v), 5% (w/v) and 2.5% (w/v) concentrations,

- the negative control group received the vehicle (AOO),

- the positive control group received 25 % (w/v) HCA (dissolved in AOO).

 

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. On Day 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 main study. No test item precipitate was observed.No treatment related marked body weight loss (≥ 5%) wasobserved on the mean body weight changes of the groups. There were no indications of any irritancy at the site of application. The stimulation index values were 0.9, 1.7 and 1.1 at concentrations of 10% (w/v), 5% (w/v) and 2.5 % (w/v), respectively.

 

The result of the positive control substanceα-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle 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.

The substance, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitiser) in the Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

LLNA results determined the test item to be non-sensitising (Stimulation Index 3) and, in accordance with ECHA Guidance on the Application of the CLP Criteria (Version 4.1; June 2015), classification as a skin sensitiser is not required under the terms of Regulation (EC) No 1272/2008.