Octadecene, reaction products with hexadecene, hydrogenated

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 November 2019 to 12 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed with vertebrate animals as classification by use of in vitro alternatives was not possible for this test item.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No.429, Skin Sensitisation: Local Lymph Node Assay (Adopted 22 July 2010)

Deviations:

yes

Remarks:

See "Any other information" for details

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42. “Skin Sensitisation: Local Lymph Node Assay”, (Official Journal L 142, 31/05/2008)

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Oligomerisation products of alpha-alkenes C16-18 (even numbered), hydrogenated, hydroisomerised

EC Number:

832-827-5

Cas Number:

2241366-04-9

Molecular formula:

Variable - UVCB

IUPAC Name:

Oligomerisation products of alpha-alkenes C16-18 (even numbered), hydrogenated, hydroisomerised

Test material form:

liquid

Details on test material:

Name: SynNova Base Oil
CAS number: 2241366-04-9
Batch/Lot number: TS20371/TS21270
Description: Liquid - water white colorless oil.
Purity: 100%
Expiry date: 25 February 2021/31 July 2021
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaCrl mice
Source: Charles River UK Limited, Manston Road, Margate, Kent, CT9 4LT, United Kingdom
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: 21.7 – 24.3 grams (The weight variation in animals in the study did not exceed ± 20% of the mean weight.)
Acclimatisation time: 34 days

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging. / Additional enrichment (hiding tunnels) was also used during the study.
Cage type: Type II. polypropylene / polycarbonate
Bedding and nesting: Bedding and nesting materials were available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.8 – 26.0°C
Relative humidity: 26 - 76%
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were measured continuously, minimum and maximum values 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 GmbH (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, as for human consumption 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-8200 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at Charles River Laboratories Hungary Kft.

Bedding and nesting
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. SAFE 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 (SAFE 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 Charles River Laboratories Hungary Kft’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. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

Dose groups at 100% to 10% (w/v) were used to ensure any dose response for toxicity/sensitisation can be fully evaluated.

No. of animals per dose:

4 animals per dose group

Details on study design:

Dose Selection and Justification of Dose Selection
As confirmed by the main study results and all other relevant data on the test item, it is essentially non-toxic, hence maximal dose levels were used in the main study. Preliminary study data was not used in dose setting; detailed raw preliminary study data will be archived, but is not reported.
Dose groups at 100% to 10% (w/v) were used to ensure any dose response for toxicity/sensitisation can be fully evaluated.
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.

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.

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 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.

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 item is regarded as a sensitizer if both of the following criteria are fulfilled:
- 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.
- 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:
- 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 item does not cause serious systemic or local toxicity.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not specified

Results and discussion

Positive control results:

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% (w/v) in the relevant vehicle (MEK) using CBA/CaCrl mice.
No unacceptable mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study, any minor adverse effects of the OECD-specified positive control substance did not affect the clear positive outcome observed in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 8.4) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.3

Test group / Remarks:

100% (undiluted)

Results 2

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

50% (w/v) in MEK

Results 3

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

25% (w/v) in MEK

Results 4

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

10% (w/v) in MEK

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
No test item residue was observed on the ears of the animals. There were no indications of any irritancy at the site of application.

BODY WEIGHT MEASUREMENT
Marked body weight loss (>5% decrease of body weight) were observed in 1 out of 4 animals in the Negative Control and the 10% (w/v) dose group, and 3 out of 4 animals in the Positive Control group. These changes were considered to be due to individual variability, and not considered to be related to test item.

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in all test item treated animals and in the negative control group. Larger than normal lymph nodes were observed in the positive control group.

INTERPRETATION OF OBSERVATIONS
The test item was an oil, which was formulated in MEK. 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 evaluation of the stimulation index values clearly indicates that there is no sensitization effect. The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that SynNova Base Oil is a non-sensitizer. The size of lymph nodes was in good correlation with this conclusion. Based on the observed results, the test item does not need classification according to the GHS or CLP.

Any other information on results incl. tables

Individual Body Weights for all Animals with Group Means

Identity Number	Test group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change# (%)
8764	Negative (vehicle) control in MEK	22.6	22.5	-0.4
8770		23.1	21.7	-6.1
8762		22.7	22.6	-0.4
8766		23.6	23.3	-1.3
	Mean	23.0	22.5	-2.1
8773	SynNova Base Oil 100% (undiluted)	24.1	23.6	-2.1
8771		22.2	21.7	-2.3
8777		22.4	23.0	2.7
8758		22.0	21.2	-3.6
	Mean	22.7	22.4	-1.3
8765	SynNova Base Oil 50% (w/v)	22.5	21.7	-3.6
8780		22.4	23.0	2.7
8763		23.6	22.9	-3.0
8774		21.7	21.7	-1.4
	Mean	22.6	22.3	-1.3
8776	SynNova Base Oil 25% (w/v)	23.5	23.7	0.9
8775		22.9	23.3	1.7
8759		23.2	22.4	-3.4
8781		21.7	22.0	1.4
	Mean	22.8	22.9	0.1
8778	SynNova Base Oil 10% (w/v)	22.9	23.6	3.1
8761		24.3	23.3	-4.1
8767		22.2	22.0	-0.9
8779		21.7	20.5	-5.5
	Mean	22.8	22.4	-1.9
8768	Positive control 25% (w/v) HCA in MEK	22.7	21.1	-7.0
8769		23.6	22.1	-6.4
8760		22.9	21.7	-5.2
8772		22.0	21.1	-4.1
	Mean	22.8	21.5	-5.7

Notes:

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

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)	40.5	-	-	-	-
Negative control (MEK)	4252	4211.5	8	526.4	1.0
SynNova Base Oil 100% (undiluted)	9687	9646.5	8	1205.8	2.3
SynNova Base Oil 50% (w/v) in MEK	7641	7600.5	8	950.1	1.8
SynNova base Oil 25% (w/v) in MEK	2909	2868.5	8	358.6	0.7
SynNova Base Oil 10% (w/v) in MEK	3197	3156.5	8	394.6	0.7
Positive control (25% (w/v) HCA in MEK)	35380	35339.5	8	4417.4	8.4

 

Summarised Clinical Observations

Group	Animal Number	Clinical Observations
		DAY 1	DAY 2	DAY 3	DAY 4	DAY 5	DAY 6
Negative control (MEK)	8764   8770   8762   8766	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0
SynNova Base Oil 100% (undiluted)	8773   8771   8777   8758	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0
SynNova Base Oil 50% (w/v) in MEK	8765   8780   8763   8774	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0
SynNova Base Oil 25% (w/v) in MEK	8776   8775   8759   8781	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0
SynNova Base Oil 10% (w/v) in MEK	8778   8761   8767   8779	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0
Positive control (25% (w/v) HCA in MEK)	8768   8769   8760   8772	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0 BT: symptom-free; 0 AT: symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0	Symptom-free; 0   Symptom-free; 0   Symptom-free; 0   Symptom-free; 0

Note:

1. BT: Before treatment, AT: after treatment

2. 0: Erythema score 0

 

Historical Control Data

Historical Control Data of the Positive and Negative Control for CBA/CaOlaHsd mice (2014-2018)

CBA/CaCrl mice
	Vehicles
	Acetone : Olive Oil 4:1 (AOO)			Pluronic PE9200 in water (1% Plu)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	472.7	3851.3	9.0	198.7	1988.1	11.2
Range: min max	35.8 1990.1	890.3 10336.0	3.3 20.2	23.0 680.8	154.0 6755.8	3.0 33.6
Number of cases	92	88	86	234	226	218

 

	Vehicles
	N,N-Dimethylformadmide (DMF)			Dimethyl sulfoxide (DMSO)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	256.1	2738.9	11.3	466.0	3014.4	7.2
Range: min max	62.0 649.6	1201.3 5817.9	4.9 21.3	218.3 934.6	1461.3 4877.5	3.1 14.5
Number of cases	68	68	68	22	22	21

 

	Vehicles
	Propylene glycol (PG)			Methyl ethyl ketone (MEK)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	245.1	2278.6	9.4	264.5	4129.5	16.9
Range: min max	63.3 506.0	817.3 4978.0	5.8 14.4	80.5 516.2	1562.5 8682.5	8.8 36.3
Number of cases	18	18	18	18	19	19

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of the present assay, SynNova Base Oil, tested in MEK, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of SynNova Base Oil following dermal exposure in mice. The study was performed with vertebrate animals as classification by use of in vitro alternatives was not possible for this test item. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.

 

The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD 429 vehicles were assessed: Acetone: Olive oil 4:1 (v/v) mixture, N,N-dimethylformamide, Methyl ethyl ketone (MEK). The best vehicle taking into account the test item characteristics and the requirements of the relevant OECD guideline was considered to be MEK. The 100% undiluted format was the highest concentration which was suitable for the preliminary test. The formulations appeared to be solutions by visual examination.

 

Based on the known lack of toxicity with the test item (based on the information given by the Sponsor and a repeated dose toxicity study conducted at the Test Facility, 19/045-220PE), maximal dose levels were used in the main study.

 

In the main assay, twenty-four female CBA/CaCrl mice were allocated to six groups, each group comprised four animals:

- four groups of animals received SynNova Base Oil (undiluted, or formulated in MEK) at 100% (undiluted), or 50, 25, and 10% (w/v), respectively,

- a negative control group received the vehicle (MEK) only,

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

 

The test item solutions were applied to the dorsal surface of the ears of the experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3) and then maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minutes after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

 

There was no mortality or signs of systemic toxicity observed during the study.

 

Marked body weight loss (>5% decrease of body weight) were observed in 1 out of 4 animals in the Negative Control and the 10% (w/v) dose group, and 3 out of 4 animals in the Positive Control group. These changes were considered to be due to individual variability, and not considered to be related to the test item.

 

The stimulation index values were 2.3, 1.8, 0.7, and 0.7 at concentrations of 100% (undiluted), 50, 25, and 10% (w/v), respectively. The size of lymph nodes was in good correlation with this conclusion.

 

The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was 8.4, therefore demonstrating the appropriate performance of the assay.

 

In conclusion, under the conditions of the present assay, SynNova Base Oil, tested in MEK, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

 

No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017.