Registration Dossier

Administrative data

Description of key information

L-isoleucine did not show any dose-related effect on the stimulation index in the Local Lymph Node Assay study. As a consequence, it was concluded that L-isoleucine is not a skin sensitizing substance.

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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Only minor deviations not influencing the outcome of the study.
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
- Species: SPF-bred, CBA/Ca mice
- Source: Charles River deutschland, Sulzfeld, Germany
- Weight at study initiation: 18.5 - 21.9 g
- Housing: Individually in macrolon cages type III
- Diet (e.g. ad libitum): ad libitum; commercial diet (batch 4428) from SDS Special Diets Services, Witham, England
- Water (e.g. ad libitum): ad libitum; tap water suitable for human consumption
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-23.0°C
- Humidity (%): 30-70%, with some occasional exceeding of the upper limit, up to 77.0%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2005-07-27 To: 2005-08-08
Vehicle:
dimethylformamide
Concentration:
6.25 %, 12.5 % and 25 % L-isoleucine
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Not performed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Animal allocation by computer.
- Criteria used to consider a positive response:
3H-thymidine incorporation was compared between the different treatment groups. In evaluating changes in 3H-thymidine incorporation possible outliers were identified by means of the dose-response curve, as well as biologically relevant ranges (i.e. data within the range of background levels). Changes in 3H-thymidine incorporation in the test substance treatment groups were evaluated and expressed as stimulation index (SI). The SI was obtained by dividing the individual values of the 3H-thymidine incorporation, expressed as DPM (corrected for background), with the mean proliferation of the vehicle control group. Since also the DPM-data of the vehicle treated group are divided with its own mean DPM-data, this results in an average SI for vehicle-treated controls of 1. The decision process with regard to a positive response includes a stimulation index >= 3 together with consideration of dose response and statistical analyses.

TREATMENT PREPARATION AND ADMINISTRATION:
Once daily, during three consecutive days, the dosing formulations were applied on the dorsum of both ears (by means of pipetting 25 µl on each ear) of the test substances, the vehicles and the positive control.
On day 5 all animals received an intravenous injection with 250 µl of phosphate-buffered saline (PBS) containing 20 µl of 3H-thymidine in the tail vein.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical evaluation was performed using SAS Online Doc ®, version 8.2, 1999, SAS Institute Inc., USA. Statistical evaluation of the data (body weights and 3H-thymidine incorporation) was performed by analysis of (co)-variance followed by an appropriate test like Dunnett’s multiple comparison tests. 3H-thymidine incorporation of treatment groups were compared to vehicle-treated animals. The validity of the model was tested by comparing the positive control and the vehicle-treated group. Probability values of p<0.05 were considered significant.
Positive control results:
No effects on body weights or clinical signs were found after treatment with HCA. As expected, administration of HCA, which is a known sensitizer, resulted in a clear and statistically significant enhanced 3H-thymidine incorporation in the LLN (p<0.05). The calculated stimulation index of 16.7 supports this observation and demonstrates the sensitivity/validity of the model.
Key result
Parameter:
SI
Remarks on result:
other: Low-dose: mean SI = 2.1 Mid-dose: mean SI = 1.8 High-dose: mean SI = 1.7
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
No significant differences in 3H-thymidine incorporation were observed between the L-isoleucine treated groups and the vehicle treated group. Stimulation indices ranged from 1.7-1.8. In contrast, a significant difference (p<0.05) in 3H-thymidine incorporation was observed between the low-dose L-Isoleucine treated group and the vehicle treated group. The stimulation index of the low-dose L-Isoleucine was however only 2.1. Moreover, no dose-response relationship was observed after L-Isoleucine treatment. Taken these findings together, it was concluded that the response from L-Isoleucine treated groups is negative.

No animals treated with L-isoleucine died during the course of the experiment. Furthermore, no clinical signs were observed. No significant differences in body weights between the groups were observed. At necropsy, macroscopic signs were noted of ears and lymph nodes. In the animals from the vehicle control and test groups no aberrant signs were observed in the ears and auriculair lymphnodes. In contrast, from all animals treated with HCA, the left and right auriculair lymph nodes were enlarged. No aberrant signs were observed in the ears from HCA-treated animals.

Interpretation of results:
GHS criteria not met
Conclusions:
In a GLP guideline study according to OECD 429 L-isoleucine was not able to act as a skin sensitizer when applied at the used dose levels The maximum dose was 25 % L-isoleucine.
Executive summary:

The objective of this study was to demonstrate whether L-Isoleucine has sensitizing properties when applied on the skin in a Local Lymph Node Assay (LLNA) in mice. The LLNA is the preferred method for the use in the identification of skin sensitizing chemicals and for confirming that chemicals lack a significant potential to cause skin sensitization. This study was conducted according to the OECD guideline 429. L-Isoleucine was used in three different increasing dose levels (6.25%, 12.5% and 25%, respectively). A negative control group receiving vehicle (dimethylformamide) and a positive control group receiving hexyl cinnamic aldehyde (HCA), a known skin sensitizer were included.

No effects on body weights or clinical signs were found after treatment with L-isoleucine. Moreover, no significant differences in 3H-thymidine incorporation were observed between the L-isoleucine mid-dose and high-dose treatment groups as compared to the vehicle treated group. Although a significant difference in 3H-thymidine incorporation was observed between the low-dose L-Isoleucine and the vehicle treated group, the stimulation index from the low-dose L-isoleucine treated group was only 2.1, which is considered to be a negative response (positive response: stimulation index >= 3).

In conclusion, under the experimental conditions as described in this study, L-isoleucine is not able to act as a skin sensitizer when applied at the used dose levels.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin senitizing properties of L-isoleucine have been examined via a Local Lymph Node Assay (LLNA) in mice, which was carried out according to OECD and GLP guidelines. L-isoleucine was administered in 3 increasing dose levels (6.25%, 12.5% and 25%, respectively) on the dorsum of both ears of the test animals for 3 consecutive days. Furthermore, the study comprised a vehicle control (DMF) and positive control (HCA) group. On day 5, all animals received an intravenous injection of 3H-thymidine. Five hours later the 3H-thymidine incorporation in the draining local (auricular) lymph nodes (LLN) was determined.

No effects on body weights or clinical signs were found after treatment with L-isoleucine. Moreover, no significant differences in 3H-thymidine incorporation were observed between the L-isoleucine mid-dose and high-dose treatment groups as compared to the vehicle treated group. Although a significant difference in 3H-thymidine incorporation was observed between the low-dose L-isoleucine and the vehicle treated group, the stimulation index from the low-dose L-isoleucine treated group was only 2.1, which is considered to be a negative response (positive response: stimulation index >= 3).

In conclusion, under the experimental conditions as described in this study, L-isoleucine is not able to act as a skin sensitizer when applied at the used dose levels.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance to Directive 67/548/EEC (Dangerous Substances Directive), classification is not necessary for skin sensitisation based on the available test data for L-isoleucine.

In accordance to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for skin sensitisation based on the available test data for L-isoleucine.