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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

As L-4-hydroxyproline is a substance occuring in collagene and thus in human cells and body fluids there is no concern with respect to genetic toxicity. Despite of that tests were performed to confirm this fact due to Annex III considerations.

Link to relevant study records

Referenceopen allclose all

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
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
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.


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
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.
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
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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-09 to 2017-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016-07-29
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
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.


Species / strain / cell type:
mammalian cell line, other: human peripheral lymphocytes
Details on mammalian cell type (if applicable):
CELLS AND MEDIA USED
Human peripheral blood was obtained by venipuncture from young, healthy, non-smoking individuals with no known recent exposures to genotoxic chemicals or radiation, and collected in heparinised vessels. Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.

Cytokinesis block (if used):
CytoB (Cytochalasin B)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Preliminary cytotoxicity test
A preliminary cytotoxicity test was performed to narrow the range of concentrations used for the definitive test. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg L-Hydroxyproline/mL medium were employed.

Main test
At least three analysable test concentrations should be evaluated. In fact, these four concentrations were employed: 250, 500, 1000, 2000 µg/mL.

Justification for top dose
In the preliminary test no signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium in the absence and presence of metabolic activation (24-hour or 4-hour exposure).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: highly purified water as solvent, ethanol as vehicle
- Justification for choice of solvent/vehicle: L-4-hydroxyproline is higly soluble in water and very good soluble in ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
other: Colchicine as positive control for aneugenic activity
Remarks:
Negative control: highly purified water. Ethanol as solvent control
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h; then medium replaced by Ham's F10 medium with FCS (fetal calf serum)
- Exposure duration: 4 h and 24 h
- Expression time (cells in growth medium): 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): after 24 h

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B

STAIN (for cytogenetic assays): 10 % Giemsa

NUMBER OF REPLICATIONS: 2 per concentration or controls in the main study

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Each culture was harvested and processed separately. After the test item incubation, mitotic activity was arrested by the addition of CytoB to each culture at a final concentration of 5 µg/mL. After an additional incubation of 20 hours the cultures were centrifuged for 10 minutes at 800 rpm, the supernatant was discarded and the cells resuspended in KCl (0.56%). After incubation for 17 minutes at 37°C, the cell suspensions were centrifuged for 10 minutes at 800 rpm. The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added. The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm. The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide. The slides were then stained using 10% Giemsa and left to air-dry at room temperature.

NUMBER OF CELLS EVALUATED: At least 500 per replicate

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
At least 500 cells per replicate cell culture (two cultures per concentration in the main study, one culture per concentration in the preliminary test) were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity. The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
The CBPI indicates the average number of cell cycles per cell during the period of exposure to CytoB, and is used to calculate cell proliferation.
The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis:

((No. mononucleate cells)+(2×No. binucleate cells)+(3×No. multinucleate cells))
CBPI = --------------------------------------------------------------------------------------------------------------
(Total number of cells)

Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Cytostasis = 100 - RI

((No. binucleated cells)+(2×No. multinucleate cells))÷(Total number of cells)T
RI = ------------------------------------------------------------------------------------------------------------ × 100
((No. binucleated cells)+(2×No. multinucleate cells))÷(Total number of cells)C

T= treated cultures
C= control cultures


DETERMINATION OF CYTOTOXICITY
All slides, including those of the solvent controls, were independently coded before the microscopic analysis.
The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration). If substantially fewer than 1000 binucleate cells per culture are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test would be repeated using more cells, or at less toxic concentrations, whichever is appropriate. Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.


OTHER EXAMINATIONS:
- irregular shapes

Rationale for test conditions:
Guideline, results of preliminary tests
Evaluation criteria:
Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction. Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures were determined. Individual culture data were provided.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
• the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
• any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).
The test chemical is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Statistics:
See "Evaluation criteria"
Key result
Species / strain:
other: Human peripheral lymphocytes
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: highly soluble
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES: yes, preliminary study

CYTOKINESIS BLOCK (if used): cytochalasin B

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
See "Any other information on results incl. tables"

Historical Background data in vitro Micronucleus Test in cultured human peripheral lymphocytes

The micronucleus frequencies of the vehicle controls without and with metabolic activation for the last 21 studies (most recent background data of the years 2014 to 2016, not audited by the QAU-department) are given as follows:


Micronucleus frequency per 1000 cells

Negative controls

 

Without metabolic activation

With metabolic activation

 

Untreated control#1

(4- and 24-hour

exposure)

Vehicle controls

Exposure:

4-hour           24-hour

Untreated control#

Vehicle control

mean

6.6

5.9

5.6

6.4

5.3

SD

2.9

2.6

2.4

2.6

2.5

range

2.0 - 17

2.0 - 16

1.0 - 13

3.0 - 13

2 - 15

95% Confidence interval#2

5.9 - 7.3

5.2 - 6.6

4.9 - 6.3

5.7 - 7.1

4.7 - 6.0

Positive controls

 

Mitomycin C

Colchicine

Cyclophosphamide

mean

27.6

31.4

22.6

SD

10.4

21.0

8.7

range

13 - 61

12 - 125

12 - 55

SD      = Standard deviation

#1        =Data of the years 2013 to 2015 (n=21)

#2            =Poisson-based

Conclusions:
Under the present test conditions, L-4-Hydroxyproline tested up to the concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times without S9 and one exposure time with S9 revealed no indications of chromosomal damage in the in vitro micronucleus test.
Executive summary:

Test samples of L-4-Hydroxyproline were assayed in an in vitro micronucleus according to OECD 487 test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a ratliver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.

Under the present test conditions, L-4-Hydroxyprolinetested up to the concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times without S9 and one exposure time with S9 revealed no indications of chromosomal damage in the in vitro micronucleus test.

The results for the vehicle controls were within historical control range.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

L-4-hydroxyproline did not exhibit genetic toxicity.