L-4-hydroxyproline

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:

2017

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

Test material information

Constituent 1

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

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.