LUMIPOD

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The search for a mutagenic activity of the test item L-Leucine, reaction products with 1,4:3,6- dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride (batch 200122016662) sponsored by SEPPIC SA was done by means of the Ames’ test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested both in presence and in absence of metabolic activation, in two independent assays according to OECD guideline (OECD 471, 1997), using the maximum recommended dose, i.e. 5000 µg/plate.
The acceptance criteria for the assay were considered as fulfilled. The current study was valid. Under these experimental conditions, no mutagenic activity was revealed.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity : bacterial reverse mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14/05/20 - 08/07/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Salmonella typhimurium HIS-

Deviations:

yes

Remarks:

After plating the plates are incubated with the test item for ca. 48-72h, as recommended in the guideline, knowing however that the temperature of 37 ±1°C may not be reached for the entire duration of exposure.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TEST ITEM L-Leucine, reaction products with 1,4:3,6-dianhydro-D glucitol, iso-Pr alc. and octanoyl chloride
OTHER NAME / CODE 80654L
NAME ON IDENTIFICATION TAG L-Leucine, reaction products with 1,4:3,6-dianhydr - 07/00009 - 200122016662
IPL REGISTRATION NUMBER 200503
BATCH NUMBER 200122016662
QUANTITY SUPPLIED 50 g
APPEARANCE pale yellow viscous liquid
WATER CONTENT 1.4%
PURITY / COMPOSITION 98.6%
SALT / BASE RATIO not available
CORRECTION FACTOR* 1.014
MOLECULAR WEIGHT unknown (UVCB) (Sponsor’s information) DENSITY not available
STORAGE CONDITIONS** room temperature (+15 to +28°C)
MANUFACTURING DATE 21/01/20
EXPIRY DATE 20/01/22
ANALYSIS DATE 04/03/20
STABILITY UNDER
STORAGE CONDITIONS 1 year up to 20/01/22 for the batch 200122016662 *: The correction factor was taken into account for the calculations.
**: Immediately upon receipt, the test item was registered, then stored at room temperature, in accordance with the Sponsor’s instructions. The complete description of the chemical and physical properties of the test item including stability is the responsibility of the Sponsor.

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver microsomal fraction S-9 mix

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500, 5000µg/plate


Limiting factor for maximum dose :
maximum dose according to OECD Guideline i.e. 5000 µg/plate

Vehicle / solvent:

Solvent used : DMSO

Untreated negative controls:

no

Positive controls:

yes

Remarks:

- Without S9-mix : sodium azide (Sigma, batch STBH9707) 1 µg/plate (TA1535, TA100) 9-amino-acridine (Merck, batch S7402762716) 50 µg/plate (TA1537) 2-nitro fluorene (Alfa Aesar, batch 81202641) 2 µg/plate (TA98) mitomycin C (Sigma, batch SLBD1982V)

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Evaluation criteria:

Sterility controls are presented in Appendix No. 2, Table 4.
After spreading out the test item alone under the conditions of the preliminary toxicity assay and after approximately 67-hour incubation at ca. 37°C, no colony was visible.
Historical data are shown in Appendix No. 3.
The frequencies of spontaneous revertants (solvent controls) were within the limits generally observed under our experimental conditions.
Concurrently to the main assays, tests were carried out on reference mutagenic test compounds in order to show the sensitivity of the strains tested and the efficiency of the metabolic activation system. Statistically and biologically significant increases in the numbers of revertants were observed in the presence of positive reference test substances. The values observed were greater than the lowest limit of historical controls.
At least 5 doses, with at least 2 plates per dose, were available for the assessment of mutagenicity, as required by OECD guideline (471, 1997) except in the 2nd assay in presence of metabolic activation in strain TA1537 (see Table 11 and § 14).

Statistics:

In parallel, data were analysed by means of Dunnett's method (Mahon et al, 1989) allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control. Statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response and biological relevance of the results should be considered first.
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined all the following criteria are fulfilled: - at least one of the test doses exhibits a biologically significant increase (2 or 3 fold increase in the mean number of revertants depending on the strain) compared with the concurrent negative control and,
- the increase is dose-related,
- and the mean numbers of revertants of the test doses are outside the distribution of negative control data
The test item is then considered able to induce mutations in this test system.
If, in all experimental conditions examined, none of the above criteria are fulfilled, a test item is considered clearly negative and unable to induce mutations in this test system.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The search for a mutagenic activity of the test item L-Leucine, reaction products with 1,4:3,6- dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride (batch 200122016662) sponsored by SEPPIC SA was done by means of the Ames’ test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested both in presence and in absence of metabolic activation, in two independent assays according to OECD guideline (OECD 471, 1997), using the maximum recommended dose, i.e. 5000 µg/plate.
The acceptance criteria for the assay were considered as fulfilled. The current study was valid. Under these experimental conditions, no mutagenic activity was revealed.

Executive summary:

PURPOSE  

The search for any mutagenic activity of L-Leucine, reaction products with 1,4:3,6-dianhydro-D glucitol, iso-Pr alc. and octanoyl chloride (Batch 200122016662) sponsored by SEPPIC SA was  studied by means of the Ames’ test (Salmonella his-/microsome system) in compliance with the  Commission Regulation EC 440/2008 and the OECD Guideline 471 and its anticipated deviation (see  § 12), using the maximum recommended dose, i.e. 5000 µg/plate.  

All the doses tested in this study are expressed as µg/plate of pure L-Leucine, reaction products  with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride.  

 

METHODS  

Study carried out on 5 strains both with and without metabolic activation using hepatic microsomes  from rat livers induced by Aroclor 1254 – Incubation period: ca. 48 h  

Strains used Salmonella typhimurium TA1535, TA1537, TA98, TA100, TA102  Solvent DMSO  

Stability in the solvent unknown (preparations for treatments were thus prepared  extemporaneously)  

Purity 98.6%  

Correction factor 1.014  

Expression of the doses  

used in the assays µg/plate of pure L-Leucine, reaction products with 1,4:3,6- dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride.

 

RESULTS

In 2 independent assays performed both with and without metabolic activation (the second assay was  carried out according to the pre-incubation protocol), no biologically significant increases in the  number of revertants were noted whatever the strain and the metabolic activation condition tested.  

The test item L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and  octanoyl chloride is thus considered as devoid of mutagenic hazard. 

 

CONCLUSION  

The search for a mutagenic activity of the test item L-Leucine, reaction products with 1,4:3,6- dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride (batch 200122016662) sponsored by  SEPPIC SA was done by means of the Ames’ test in the five Salmonella typhimurium strains  TA1535, TA1537, TA98, TA100 and TA102 tested both in presence and in absence of metabolic  activation, in two independent assays according to OECD guideline (OECD 471, 1997), using  the maximum recommended dose, i.e. 5000 µg/plate.  

The acceptance criteria for the assay were considered as fulfilled. The current study was valid.  Under these experimental conditions, no mutagenic activity was revealed. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)