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EC number: 200-091-9 | CAS number: 51-35-4
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-05-09 to 2017-07-19
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- L-4-hydroxyproline
- EC Number:
- 200-091-9
- EC Name:
- L-4-hydroxyproline
- Cas Number:
- 51-35-4
- Molecular formula:
- C5H9NO3
- IUPAC Name:
- L-4-hydroxyproline
- Test material form:
- solid: crystalline
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Thai Hakko Biotechnologies Co., Ltd. / batch 160006
- Expiration date of the lot/batch: retest date 2018-02-22
- Purity test date: 2016-03-08
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 10 - 25 °C in a tightly closed container, stored in a dry and well ventilated place
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: highly soluble
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n.a.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Completely dissolved in highly purified water.
Method
Species / strain
- 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:
- S9 mix
- Test concentrations with justification for top dose:
- 31.6, 100, 316, 1000, 3160 and 5000 µg/plate.
Justification for top dose: High dose as positive reaction unlikely. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: higly purified water
- Justification for choice of solvent/vehicle: test item highly soluble in water
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable):
DURATION
- Preincubation period: first experment: none; second experiment: 20 min.
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 per concentration and experiment
- OTHER: - Rationale for test conditions:
- Guideline
- Evaluation criteria:
- Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by the testing laboratory.
The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20
Where concurrent negative or positive control data fall outside the range, they may be acceptable and considered for the inclusion into the historical control distribution as long as these data are not extreme outliers.
Historical background data of revertant colony numbers of the negative and positive controls without and with metabolic activation for the experiments of the years 2015 to 2017 are reported in the study report. - Statistics:
- A test item is considered to show a positive response if
- the number of revertants is significantly increased (p Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient ) may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Applicant's summary and conclusion
- Conclusions:
- Under the present test conditions, L-4- Hydroxyproline tested up to the concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
- Executive summary:
The potential of L-4-Hydroxyproline to induce gene mutations was examined in 5 Salmonella typhimuriumstrains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.
In conclusion, under the present test conditions, L-4-Hydroxyproline tested up to the concentration of 5000 µg/plate, caused no mutagenic effect in theSalmonella typhimuriumstrains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
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