Fusel oil

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Erlangen, Germany

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsD

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, 33178 Borchen, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 17-24 g
- Housing: 5 animals per cage in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 0702), ad libitum
- Water: tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

25%, 50%, 100% (v/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: maximum technically applicable concentration in the vehicle was 50%
- Irritation: No signs of systemic toxicity nor signs of irritation at the application site were detected.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation (determined by beta-scintillation).
- Criteria used to consider a positive response: A substance will be regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine-incorporation into lymph node cells of the test group animals relative to that recorded for the lymph nodes of control group animals (SI ≥ 3).

TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.

Five days after the first topical application all mice were dosed with 250 µL 3H-methyl thymidine (corresponding to 20 µCi) by intravenous injection (tail vein).
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected with phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approximately 1 mL 5% TCA at 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
The 3H-methyl thymidine–incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Positive control substance(s):

other: p-phenylendiamine (CAS 106-50-3, 1% on three consecutive days; reliability check in November 2012)

Positive control results:

The positive control substance p-phenylendiamine induced an SI of 10.2 ± 2.7 (5 animals).

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Control: 2622 ± 909 25%: 2527 ± 747 50%: 2022 ± 220 100%: 1699 ± 406

Key result

Parameter:

SI

Value:

>= 1

Test group / Remarks:

25%

Remarks on result:

other:

Key result

Parameter:

SI

Value:

>= 0.8

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

>= 0.6

Test group / Remarks:

100%

Table 1: Body weights [g] and body weight gain [g] during the study period.

Concentration	Animal No.	Body weight (start)	Body weight (end)	Weight gain
25%	1	21	23	2
	2	21	23	2
	3	22	22	0
	4	20	21	1
	5	19	21	2
50%	6	20	21	1
	7	17	19	2
	8	21	21	0
	9	20	21	1
	10	22	24	2
100%	11	19	20	1
	12	21	22	1
	13	21	22	1
	14	19	20	1
	15	20	21	1
Negative control	16	24	23	-1
	17	21	22	1
	18	18	19	1
	19	22	23	1
	20	21	22	1

 

Table 2: Overview of disintegrations per minute and stimulation index.

Concentration	Animal No.	Disintegrations per minute (DPM; not corrected with background value)	Stimulation index (SI)
25%	1	3090
	2	1832
	3	3635
	4	2424
	5	1657
	Mean ± SD	2527 ± 747	1.0 ± 0.3
50%	6	1936
	7	2186
	8	2013
	9	1667
	10	2309
	Mean ± SD	2022 ± 220	0.8 ± 0.1
100%	11	1376
	12	1501
	13	1499
	14	2497
	15	1624
	Mean ± SD	1699 ± 406	0.6 ± 0.0
Negative control	16	2770
	17	1950
	18	2673
	19	1528
	20	4191
	Mean ± SD	2622 ± 909	1.0
Background (scintillation fluid and trichloroacetic acid)		60
		50
		59
		62
		53
	Mean ± SD	57 ± 5	0.0

 

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A study performed according to OECD 429 and in compliance with GLP, is available for assessment of skin sensitizing properties of Fusel oil (CAS No. 8013-75-0) (Lütkenhaus, 2013). Five CBA mice per dose were treated with 25 µl of 25%, 50%, and 100% test substance (solution in acetone/olive oil, 4:1). After 5 h the mice were sacrificed, the lymph nodes were prepared and the 3H-methyl thymidine incorporation was measured and expressed as the number of disintegrations per minute (DPM). The DPM were 2622 ± 909, 2527 ± 747, 2022 ± 220, and 1699 ± 406 for the control, 25%, 50%, and 100% dose group, respectively. The resulting stimulation indices were 1.0 ± 0.3, 0.8 ± 0.1 and 0.6 ± 0.0 for the 25%, 50%, and 100% dose group, respectively. In conclusion, as the stimulation index after treatment with Fusel oil was similar to the control values, it can be concluded that Fusel Oil possesses no skin sensitizing potential.

Migrated from Short description of key information:

Fusel oil was not sensitising in a Local Lymph Node Assay according to OECD 429.

Justification for selection of skin sensitisation endpoint:

The key study was selected for assessment.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation of Fusel oil are conclusive but not sufficient for classification according to the criteria of Directives 67/548/EEC (DSD) and Regulation 1272/2008/EC (CLP).