Linalyl acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE; Experimental Toxicology and Ecology; Ludwigshafen; Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Linalyl acetate

EC Number:

204-116-4

EC Name:

Linalyl acetate

Cas Number:

115-95-7

Molecular formula:

C12H20O2

IUPAC Name:

1,5-dimethyl-1-vinylhex-4-en-1-yl acetate

Specific details on test material used for the study:

In the prestudy (58V0138/16A054), 11 mE peroxides /kg test substance were determined in the undiluted test substance. For the test substance formulations 5, 10, 50 % test substance in MEK, the maximum concentration of peroxides found were 5 mE/kg in the high dose group formulation. It is concluded, that formation of peroxides occured in the test substance formulations used.

In final study (58V0138/16A162), the test substance has been mainly kept under argon and has been analyzed after the last application to exclude the formation of autooxidation products. In the undiluted test substance, 0.8 mE peroxides /kg test substance were determined. For the test substance formulations 0.5, 2.5, 10% test substance in MEK, the maximum concentration of peroxides found were 0.1 mE/kg. It is concluded, that no relevant formation of peroxides occured in the test substance formulations used.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, The Netherlands
- Age at study initiation: 9 weeks
- Weight at study initiation: 18.1 – 21.6 g
- Housing: 1 animal/cage
- Diet: ad libitum; Kliba mouse/rat maintenance diet “GLP" by Provimi Kliba SA,Switzerland.
- Water: ad libitum
- Acclimation period: min. 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

0.5%, 2.5%, 10% (58V0138/16A162)
5%, 10%, 50% (58V0138/16A054)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Yes
- Irritation: Yes
- Systemic toxicity: Yes
- Ear thickness measurements: Yes
- Lymph node weight: Yes

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.

TREATMENT PREPARATION AND ADMINISTRATION:
Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal
Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
Form of application: Epicutaneous
Application volume: 25 µL per ear
Site of application: Dorsal part of both ears
Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected into a tail vein with 20 µCi of 3H-thymidine in 250 µL of sterile saline.

- Parameters assessed:
Determination of ear weight:
Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal.

Removal and weight determination of the lymph nodes:
Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.

Preparation of cell suspension and determination of cell count:
After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline (PBS) and a single cell suspension was prepared by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into phosphate-buffered physiological saline. The cell count was determined using a Casy®-Counter.

Measurement of 3H-thymidine incorporation of the lymph node cells:
The remaining cell suspensions were washed with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was measured in a β-scintillation counter.

- Criteria used to consider a positive response:
A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H thymidine at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices are considered in conjunction with the other assessed end points (i.e. cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.

Positive control substance(s):

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

Statistics:

3H-thymidine incorporation, cell count, lymph node weight and ear weight: Wilcoxon - Test

Results and discussion

In vivo (LLNA)

Results

Key result

Parameter:

EC3

Value:

1.6

Test group / Remarks:

3H-thymidine incorporation

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
SI (3H-thymidin-incorporation)
0.5%: 1.14
2.5%: 4.55**
10%: 7.62**

SI (cell counts)
0.5%: 1.04
2.5%: 1.61**
10%: 2.21**

SI (lymph node weight)
0.5%: 1.02
2.5%: 1.32**
10%: 1.71**

EAR WEIGHTS
SI:
0.5%: 1.00
2.5%: 1.00
10%: 1.09**

**p<=0.01

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were noticed in all animals during general observation. No local findings were observed during the observation period.

BODY WEIGHTS:
No relevant influence on the body weights was observed in the course of the study.

Any other information on results incl. tables

Prestudy - BASF 2016 (58V0138/16A054):

Determination of the highest non-irritant concentration in an initial pretest using 2 animals:

% Linalylacetate in MEK	lymph node weight (SI)	ear weights (SI)
10	1.35	1.06
50	2.13	1.01

Identification of skin sensitization using 5 animals:

% Linalylacetate in MEK	3H-thymidin- incorporation (SI)	cell counts (SI)	lymph node weight (SI)	ear weights (SI)
5	12.04**	2.70**	1.75**	1.02
10	19.64**	3.80**	2.14**	1.06
50	24.24**	3.61**	2.15**	1.07

**p<=0.01

Linalylacetat has been found to show a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was <5%. An increase in potency to elevate lymph node weights has been observed compared to the initial pretest. The test substance has been analyzed after completion of the test substance application and revealed a content of 11 mE peroxides/kg test substance. Therefore, the formation of peroxides in the test substance during the study and their contribution to the skin sensitization of the test substance cannot be excluded.

No clinical signs and local findings have been observed during the course of the study.

Irritation Screening -Results BASF 2017 (58V0138/16A162):

Determination of the highest non-irritant concentration in an initial pretest using 2 animals:

% Linalylacetate in MEK	lymph node weight (SI)	ear weights (SI)
5	1.51	1.06
50	2.03	1.05

At the tested concentrations the animals did not show any signs of excessive local irritation as confirmed by the ear weight measurements (compared to current vehicle values) and ear thickness measurements. However, increased lymph node weights (compared to current vehicle values) were observed in both concentrations.

The test substance has been analyzed after completion of the test substance application and revealed a content of 0.8 mE peroxides/kg test substance.

Applicant's summary and conclusion

Executive summary:

The skin sensitizing potential of Linalylacetat was assessed using the radioactive Murine Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 0.5%, 2.5% and 10% w/w preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone. The absence of relevant formation of peroxides in the test substance formulation was confirmed analytically. The study used 3 test groups and 1 control group. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation, applied to the dorsal surfaces of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. Three days after the last application the mice were injected into the tail vein with 20 μCi of 3H-thymidine in 250 μL of sterile saline and lymph node response was evaluated by measuring 3H-thymidine incorporation, cell counts and weights of each animal’s pooled lymph nodes. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

When applied as 10% and 2.5% preparations in MEK, Linalylacetate induced a statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes, which was above the cut off value for biological relevance (increase above the cut off stimulation index (SI) of 3). Concomitantly, the increase in the auricular lymph node cell counts at 10% and 2.5% was statistically significant and biologically relevant (above the cut-off value (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5). In addition there was a statistically significant increase in lymph node weights at 10% and 2.5%. The Linalylacetate preparations did not cause any excessive increase in ear weights (> 25%), demonstrating the absence of excessive ear skin irritation. The increase at 10% was statistically significant.

Thus, it is concluded that Linalylacetat exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was >0.5% <2.5%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 1.6%.