DL-alanine

Endpoint summary

Description of key information

QSAR: read-across performed with the OECD QSAR Toolbox (v4.1), Result: D,L-Alanine is predicted to be not sensitising (see also section 13 for OECD Toolbox v4.3).

QSAR: prediction for skin sensitisation (CAESAR) implemented in the VEGA tool, Result: D,L-Alanine is predicted to be a non-sensitiser by the skin sensitization model CAESAR.

Expert statement: Result: The expert statement confirms the QSAR predictions that D,L-Alanine is predicted to be not skin sensitising.

These results are corroborated by 3 in vivo studies (two LLN and one GPMT) with L-valine, L-isoleucine and L-tert-leucine, which all showed absence of skin sensitising properties.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2005-03-16 to 2005-09-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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: 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
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
According to guideline

IN-LIFE DATES: From: 2005-07-27 To: 2005-08-08

Vehicle:

dimethylformamide

Concentration:

6.25, 12.5 and 25% L-valine or 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 J3 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.

Parameter:

SI

Remarks on result:

other: Test substance L-valine: 6.25% mean SI=1.8; 12.5% mean SI = 1.7; 25% mean SI = 1.9 Test substance L-Isoleucine: 6.25% mean SI = 2.1; 12.5% mean SI = 1.8; 25% 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-valine treated groups and the vehicle treated group. Stimulation indices (3H-thymidine incorporation (dpm) divided by the average 3H-thymidine incorporation of the vehicle group) ranged from 1.7-1.9. 6.25 % dose: mean DPM = 3115.5 12.5 % dose: mean DPM = 2922.8 25 % dose: mean DPM = 3240.3 No significant differences in 3H-thymidine incorporation were observed between the L-isoleucine treated groups and the vehicle treated group. Stimulation indices (3H-thymidine incorporation (dpm) divided by the average 3H-thymidine incorporation of the vehicle group) ranged from 1.7-2.1. 6.25 % dose: mean DPM = 3622.3 12.5 % dose: mean DPM = 3101.1 25 % dose: mean DPM = 2872.3

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP guideline study according to OECD 429, L-valine and L-isoleucine did not induce skin sensitisation when applied at the used dose levels. The maximum dose was 25% L-valine or L-isoleucine.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin senitization

In a weight of evidence approach the absence of skin sensitizing properties of the test substance D,L-Alanine was predicted with two QSAR predictions. In an expert statement (Chemservice, 2018) the predictions of the applied in silico methods are in line with reported experimental evidences in human studies for alanine and other individual naturally occurring amino acids. Also two LLNA studies and one GPMT with other relevant amino acids did not show skin sensitising properties. These data give sufficient evidence that the registered substance DL-Alanine is not a skin sensitizer. Conclusively, a respective hazard classification is not warranted. Further assessments of the skin sensitizing potential of DL-Alanine are not considered necessary.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Because two LLNA studies and one GPMT with other relevant amino acids did not show skin sensitising properties (and the same is expected for DL-alanine based on QSAR analysis), it can be concluded that due to the lack of sensitizing properties, they also will not be able to sensitize the respiratory tract. This would then also be applicable to DL-alanine.

Justification for classification or non-classification

Sufficient information on the absence of skin sensitizing potential of DL-alanine has been gathered with reliable QSAR predictions, supported by an expert statement. The data give sufficient evidence that the registered substance DL-alanine is not a skin sensitizer. No structural alerts or structural analogues have been identified as potential skin sensitizer. Conclusively, a respective hazard classification according to Regulation (EC) No 1272/2008 (CLP Regulation) is not warranted.