2-methylvaleraldehyde

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

Study period:

2011-07-20 to 2011-08-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented guideline conform study including GLP compliance

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V.; Postbus 6174; 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 9 weeks (beginning of treatment)
- Weight at study initiation: 16.6 - 19.7 g
- Housing: single; Makrolon Type III, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimatisation: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:

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

Concentration:

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

No. of animals per dose:

5 females (nulliparous and non-pregnant) per dose

Details on study design:

RANGE FINDING TESTS: a pre-test was performed in two animals
- Treatment: 25% and 100% by topical application once daily on three consecutive days.
- Irritation: Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value >3 was observed at any observation time and/or if an increase in ear thickness of > 25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation. On day 2 and 3, the animal treated with 100% showed an erythema of the ear skin (score 1). On day 3, the animal treated with 25% showed an erythema of the ear skin (score 1) as well. Other signs of irritation or signs of systemic toxicity were not observed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:

A test item was regarded as a sensitiser in the LLNA if the following criteria were fulfilled:

- First, that exposure to at least one concentration of the test item resulted in an
incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as
indicated by the Stimulation Index.

- Second, that 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.

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice were treated by topical (epidermal) application (25 µL/ear/day) to the dorsal surface of each ear with test item concentrations once daily for three consecutive days. A further group of mice (control animals) were treated with an equivalent volume of the relevant vehicle alone. Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 19.9 µCi of 3HTdR were injected into each test and control mouse via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium. The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze. After washing two times with phosphate buffered saline the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid.The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

DETERMINATION OF LYMPH NODE WEIGHT AND CELL COUNT
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned in section 9.3.3 was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter. The values obtained were taken down manually.

DETERMINATION OF EAR WEIGHTS
After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

Positive control substance(s):

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

Statistics:

A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test and the Student Newman Keuls test. Statistical significance was set at the first per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers.

Positive control results:

alpha-Hexylcinnamaldehyde (25% in acetone:olive oil (4+1 v/v)):
Mean DPM per animal (5): 3720.5 +/- 1278.5
Stimulation Index (SI): 8.08

Parameter:

SI

Remarks on result:

other: - 0% 2-Methylpentanal: SI: 1.00 - 25% 2-Methylpentanal: SI: 0.55 - 50% 2-Methylpentanal: SI: 1.24 - 100% 2-Methylpentanal: SI: 4.03

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

- 0% 2-Methylpentanal: Mean DPM per animal: 459.1 +/- 30.6 - 25% 2-Methylpentanal: Mean DPM per animal: 252.3 +/- 112.8 - 50% 2-Methylpentanal: Mean DPM per animal: 570.5 +/- 352.0 - 100% 2-Methylpentanal: Mean DPM per animal: 1847.9 * +/- 902.5 * Statistically significant increase vs. control group (p< 0.05)

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

Under the conditions of the present skin sensitisation study the test item shows skin sensitizing properties.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In a Local Lymph Node Assay (LLNA) mice were treated by topical (epidermal) application to the dorsal surface of each ear with the test item in concentrations of 25,50 (w/v) and 100%. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone. Five days after the first topical application (day 6) 250 µL of a 3HTdR solution was injected into each test and control mouse via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised. The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal) and single cell suspensions of pooled lymph node cells were measured in a beta-scintillation counter. Furthermore, after excision the lymph nodes were weighed, lymph node cell were counted and the ear weight of the mice were determined.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant increase in ear weights was observed only in the high dose group in comparison to the vehicle control group (p<0.05). This finding is biologically not relevant since the high dose group ear weight was still within the range of historical control data and the treshold for excessive local irritation (i.e. 25% increase of ear weight in comparison to control ear weight according to OECD 429 was not exceed).

In this study a Stimulation Indices (S.I.), termed as the ratio of proliferation in test item treated groups compared to that in vehicle control, of 0.55 (25%), 1.24 (50%), and 4.03 (100%) were determined, respectively. A clear dose response was observed. Based on the S.I.s obtained with 50 and 100% test item concentration, an EC3 value of 81.5% (w/v) was calculated. A statistically significant and biologically relevant increase in DPM value was found in the high dose group in comparison to the vehicle control group (p<0.05). This was also observed for the lymph node weight. In the lymph node cell count, a statistically significant and biologically relevant increase was observed in the mid and the high dose group in comparison to the vehicle control group (p<0.05). A clear dose response was also seen in these two parameters.

In conclusion, the test item 2-Methylpentanal was a skin sensitiser under the test conditions of this study.

Migrated from Short description of key information:
2-Methylpentanal is a skin sensitizer.

Justification for selection of skin sensitisation endpoint:
Well documented and reliable guideline study.

Justification for classification or non-classification

Based on the results obtained in the skin sensitisation test, 2 -Methylpentanal is classified as follows:

R43, may cause an allergic skin reaction (according to Directive 67/548/EEC) and H317, may cause an allergic skin reaction, Cat. 1B (according to Regulation (EC) No 1272/2008).