Glycerides, tall-oil mono-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 March 2018 to 17 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the testing of chemicals 429. Skin Sensitisation: Local Lymph Node Assay (Adopted: 22 July 2010).

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Glycerides, tall-oil mono-

EC Number:

263-034-7

EC Name:

Glycerides, tall-oil mono-

Cas Number:

61789-12-6

Molecular formula:

C19H38O4 to C21H42O4 * (mono-glycerides) # C37H66O5 to C39H76O4 * (di-glycerides) # C55H96O6 to C57H108O4 * (tri-glycerides) # * in H2 increments (i,e, decreasing # of double bonds)

IUPAC Name:

Glycerides, tall-oil mono-

Test material form:

liquid

Details on test material:

Name: LUMULSE GMT-K
Batch no.: 385932
Appearance: opaque amber liquid
Purity: 100%
Homogeneity: homogeneous
Expiry date: 26. Oct. 2019
Storage: Room Temperature: (20 ± 5°C)

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: Mice CBA/Ca
Source: Velaz, Prague, Czech Republic
Number and Sex of Animals: 29 females
Age at First Dose: 8-11 weeks, female animals were non-pregnant and nulliparous were used
Animal Health: The health condition of animals was examined by a veterinarian before initiation of the study.
Acclimation: The animals were acclimated in identical conditions as during the experiment for 5 days prior to the start of treatment. The acclimation was according to standard operation procedures.
Housing: Condition The animals were housed in IVC polycarbonate cages (5 animals per cage) suspended on stainless steel racks, in a room equipped with central air-conditioning. The room temperature was within the range of 22 ± 3°C, relative humidity was within the range of 50 - 60 %. The light regimen was set to a 12-hour light / 12-hour dark cycle. The sanitation was performed according to standard operation procedures.
Diet: A laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was served ad libitum, each day approximately at the same time. The certificate of analysis is included in the raw data.
Water: The animals received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is periodically monitored (including microbiological control) and recorded; certificate of analysis is included in raw data.
Bedding: Lignocel S3/4, Lufa - ITL GmbH, Germany
Animals Identification: Each animal was marked with permanent pen on the tail. Each cage was affixed with a cage card containing pertinent animal and study information.
Justification for the Choice of Species: The CBA/Ca mice are the standard experimental rodent of choice and recommended by OECD Guideline No. 429.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

The doses were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5% etc. according to OECD Guideline No. 429.

No. of animals per dose:

The starting concentration was determined according to pre-screen test result.
Number of animals:
5 females – negative control (vehicle)
5 females – positive control
15 females – test item
4 females - pre-screen test

Details on study design:

Dose Preparation
The required amount of the test item (according to the concentration) was mixed with the vehicle shortly before the administration (i.e. 50% concentration was obtained by mixing of 0.5 mL of test item with 0.5 mL of vehicle). The undiluted test item was considered as 100% concentration. The dose preparation data are listed in the raw data.
The preparations were made freshly before each dosing occasion.

Dose Administration
25 μL of the test item was applied to the dorsum of each ear. The α−Hexylcinnamaldehyde (25%) as positive control and vehicle as a negative control were administrated in the same volume.

Pre-screen test
The pre-screen test was conducted under the same conditions as the main LLNA study, except the assessment of lymph node proliferation. As a 100% concentration was used undiluted test item.

Test procedure of Pre-screen test
All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any signs of irritation. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%.

Main study
Day 1:
Each animal was identified and the body weight was recorded. To the dorsum of each ear 25μL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5:
No treatment.
Day 6:
The body weight of each animal was recorded. 250μL of phosphate-buffered saline (PBS) containing 2 μCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein.
Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).

Clinical Observation
Animals were carefully observed once daily for any clinical symptoms, either of local irritation at the application site or of the systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.

Body Weight
The animal body weights were determined at the start of the test and at the scheduled day of euthanasia of the animals.

Calculation of results
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled DPM for each treatment group by the incorporation of the pooled DPM vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test when SI≥3.
EC3 value, which induce stimulation indices, is determined by linear interpolation of points on dose response curve, immediately above and below of SI value, according to the equation: EC3=c+[(3-d)/(b-d)]x(a-c)
a – higher concentration, b – SI of higher concentration, c – lower concentration, d – SI of lower concentration
If all points are below the stimulation index of three, no EC3 value can be stated.

Positive control substance(s):

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

Statistics:

The significance of difference between initial and terminal body weights was analysed by Student´s T-test; p – values less than 0.05 were considered significant.

Results and discussion

Positive control results:

The lymph node weight of control group and positive control group were 0.0375g and 0.0778g, respectively.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.91

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

1.56

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

50%

Cellular proliferation data / Observations:

Main Study
Based on the observations recorded in the preliminary test, the concentrations of 50, 25 and 10% were selected for the main test.

Clinical Observations
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.

Body Weights
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. The increase of body weight was minimal, observed at all concentrations. The increase was not statistically significant.

Lymph Node Proliferation
In comparison with the control group, an increase of the pooled lymph node weights was observed at concentrations of 50%. The pooled lymph node weights of treated groups were 0.0285g for 10% concentration, 0.0364g for 25% concentration and 0.0453g for 50% concentration of test item. The lymph node weight of control group and positive control group were 0.0375g and 0.0778g, respectively. The DPM values for the three treated groups were 1058 (10%), 1807 (25%) and 1975 (50%), respectively. The SI values for the three treated groups were 0.91 (10%), 1.56 (25%) and 1.70 (50%), respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

DISCUSSION
The skin sensitization potential of LUMULSE GMT-K was evaluated by LLNA method, which basic underlying principle is that sensitizers induce a primary proliferation of lymphocytes in the auricular lymph nodes draining the site of chemical application.
In the present study, the test item was applied to the dorsum of each ear of five female mice (CBA/Ca) per group over three consecutive days, at three concentrations. All animals survived throughout the test period without showing any clinical signs of toxicity. In comparison with the control group, the increase in lymph node weight was observed at concentration of 50%. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance as a sensitizer.
Therefore, it was not possible to calculate an EC3 value.
These results demonstrate that the test item LUMULSE GMT-K was not a skin sensitizer under the test conditions of this study.

Any other information on results incl. tables

Pre-screen test – concentration of 100%

Clinical Observations

Signs/Day	Day 1		Day 2		Day 3		Day 4		Day 5		Day 6
	Mouse 1	Mouse 2	Mouse 1	Mouse 2	Mouse 1	Mouse 2	Mouse 1	Mouse 2	Mouse 1	Mouse 2	Mouse 1	Mouse 2
Apathy	0	0	++	+	++	+	+	0	0	0	0	0
Ataxia	0	0	+	0	+	0	0	0	0	0	0	0
Intake of food	0	0	Weak	Weak	Weak	Weak	Weak	0	0	0	0	0
Intake of water			Weak	Weak	Weak	Weak	0	0	0	0	0	0
Tremor	0	0	0	0	0	0	0	0	0	0	0	0
Change in grooming activity	0	0	0	0	0	0	0	0	0	0	0	0
Piloerection	0	0	0	0	0	0	0	0	0	0	0	0

0 no effect            + weak effect       ++ moderate effect            +++ strong effect

 

Erythema score

Mouse no.	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
1	0	0	0	0	0	0
2	0	0	0	0	0	0

 

Ear thickness (mm)

Mouse no.	Day 1 (pre dose)		Day 3		Day 6
1	0.19	0.19	0.20	0.20	0.20	0.19
2	0.21	0.20	0.20	0.21	0.20	0.19
Mean	0.198		0.203		0.195
S.D.	0.010		0.005		0.006

 

Initial and terminal body weights (g)

Mouse no.	Initial body weight	Terminal body weights
1	16.60	17.49
2	17.09	17.77
Mean	16.85	17.63
S.D.	0.346	0.198

 

Pre-screen test – concentration of 50%

LUMULSE GMT-K administered at concentration of 50% did not cause any changes in monitored parameters. The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of mean body weight were observed during the test.

 

Initial and terminal body weights (g)

Mouse no.	Initial body weight	Terminal body weights
1	20.04	20.05
2	19.34	19.59
Mean	19.69	19.82
S.D.	0.495	0.325

 

No erythema was observed in both mice after test item administration.

Erythema score

Mouse no.	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
1	0	0	0	0	0	0
2	0	0	0	0	0	0

 

On day 1 (pre dose), day 3 and day 6 the ear thickness was measured.

Ear thickness (mm)

Mouse no.	Day 1 (pre dose)		Day 3		Day 6
1	0.22	0.21	0.21	0.21	0.20	0.20
2	0.19	0.19	0.20	0.20	0.20	0.19
Mean	0.203		0.205		0.197
S.D.	0.015		0.006		0.005

 

The erythema score and the increase in ear thickness did not meet the criteria which are considered as signs for excessive local skin irritation (erythema score ≥ 3; ear thickness increase ≥ 25%).

 

Main Study

Individual body weights (g)

Negative control (vehicle)

Mouse no.	Initial body weight	Terminal body weight
1	20.15	20.18
2	20.33	20.61
3	20.59	20.54
4	20.70	21.12
5	20.25	21.12
Mean	20.40	20.71
S.D.	0.232	0.405

Positive control

Mouse no.	Initial body weight	Terminal body weight
1	22.09	22.15
2	22.27	22.73
3	22.92	23.11
4	22.48	21.93
5	22.17	21.89
Mean	22.39	22.36
S.D.	0.332	0.536

LUMULSE GMT-K (10%)

Mouse no.	Initial body weight	Terminal body weight
1	20.12	20.02
2	20.45	21.19
3	20.40	20.40
4	20.93	21.82
5	20.07	20.41
Mean	20.39	20.77
S.D.	0.343	0.726

LUMULSE GMT-K (25%)

Mouse no.	Initial body weight	Terminal body weight
1	21.91	22.17
2	21.48	20.95
3	21.10	21.78
4	21.26	21.77
5	21.17	22.37
Mean	21.38	21.81
S.D.	0.327	0.544

LUMULSE GMT-K (50%)

Mouse no.	Initial body weight	Terminal body weight
1	19.61	19.94
2	19.05	20.15
3	19.79	21.50
4	19.95	19.83
5	19.95	20.26
Mean	19.67	20.34
S.D.	0.374	0.672

 

The Lymph node weight, DPM, SI. EC3 values

	Lymph node weight (g)	Number of lymph nodes	DPM	SI	EC3 (%)
Control	0.0375	10	1159	-	-
Positive Control	0.0778	10	7210	6.22
LUMULSE GMT-K 10%	0.0285	10	1058	0.91
LUMULSE GMT-K 25%	0.0364	10	1807	1.56
LUMULSE GMT-K 50%	0.0453	10	1975	1.70

 

Historical control data for the positive control

Date	Study No.	DPM – negative control (vehicle)		DPM – positive control	Stimulation Index
		Acetone/Olive oil, 4:1 (v/v)	DMSO
02/2018	600411410	612	-	3763	6.15
09/2017	600398330	1423	-	9659	6.79
06/2017	600386410	-	1575	12996	8.25
05/2017	600358670	-	172.0	1360.8	7.91
04/2017	600383610	984	-	8590	8.37
11/2016	6003584100	-	1040	6189	5.95
10/2016	600358130	-	469	3961	8.44
09/2016	600349510	777	-	4355	5.60
07/2016	600342910	1014	-	7601	7.49
04/2016	600333730	1093	-	6811	6.23

Positive control substance:

α-Hexylcinnamaldehyde (85%), Sigma Aldrich, USA

Vehicle:

Acetone/Olive oil, 4:1 (v/v) Mikrochem/Interpharm

DMSO, Sigma Aldrich

Concentration (pos. control): 25%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitizing potential of LUMULSE GMT-K was assessed using the murine local lymph node assay.
Based on the results of the study, LUMULSE GMT-K is not considered a skin sensitizer under the condition of this LLNA study.

Executive summary:

The sensitization potential of LUMULSE GMT-K was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the allergic contact sensitization potential of chemicals.

Based on the recommendations of the OECD Guideline 429, the test item was suspended in Acetone:Olive Oil 4:1 (v/v). The positive control (α-Hexylcinnamic aldehyde) (25%) was dissolved in the same vehicle.

The Pre-screen test was performed using the doses of 100 % and 50%. Based on the observations recorded in the Pre-screen tests, the concentration of 50 % was selected as top dose for the main test.

Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 10%, 25% and 50%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive¹²⁵I-iododeoxyuridine and 10^-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (10%, 25% and 50% v/v) the animals did not show visible clinical symptoms of either local irritation or systemic toxicity.

In this study the Stimulation Indices (SI) of 0.91, 1.56 and 1.70 were determined with the test item at concentrations of 10%, 25%, and 50% in Acetone:Olive Oil 4:1, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a SI greater than the threshold value of 3.

The test item LUMULSE GMT-K is not considered a skin sensitizer under the test conditions of this study.