Pidolic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th March 2021 to 29th June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted 29 July 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Pidolic acid

EC Number:

202-700-3

EC Name:

Pidolic acid

Cas Number:

98-79-3

Molecular formula:

C5H7NO3

IUPAC Name:

5-oxo-L-proline

Test material form:

solid: particulate/powder

Details on test material:

white solid crystalline powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
- Batch Manufactured by : Sigma Aldrich
- Batch Supplied by: Intracrop Limited Unit 21, Raleigh Hall, Industrial Estate Eccleshall, Stafford ST21 6JL, United Kingdom.
- Batch No. : BCCD2460
- Purity, including information on contaminants, isomers, etc.:
-Purity as per Certificate of Analysis: >99.0 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Recommended Storage Condition: Ambient (+15 to +25ºC)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability of Pidolic Acid in the vehicle as well as analytical verification of the test item dose formulations was carried out in-house as per Eurofins Advinus Study No. G21146.
- Expiry Date : 05/2022 (2 years minimum)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- dissolved in sterile water (SW) prior to testing
- Final concentration of a dissolved solid, stock liquid or gel: A stock solution of 129110 µg/mL was prepared and further diluted to 48, 143, 430 and 1291.10 µg/mL test concentrations

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- applied as dilution in sterile water.

Method

Target gene:

TK

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Mouse Lymphoma L5178Y TK+/- -3.7.2C cell line with a population doubling time of 10 to 12 hours was used.
- Suitability of cells: The autosomal and heterozygous nature of the thymidine kinase gene enables the detection of viable colonies whose cells are deficient in the enzyme thymidine kinase following mutation from TK+/- to TK-/-. This deficiency can result from genetic events affecting the TK gene including both gene mutations and chromosomal events. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Cell cycle length, doubling time or proliferation index : 10 - 12 hours

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Basic Medium: RPMI 1640 medium supplemented with L-glutamine, sodium bicarbonate, sodium pyruvate, Pluronic acid F68 and antibiotics [penicillin (105IU/L)-streptomycin (100 mg/L) and amphotericin (2 mg/L)] was the basic medium.
Complete Medium (CM10): Basic medium supplemented with 10% of heat inactivated horse serum (HS) wasthe complete medium.
Conditioned Medium (CDM): Conditioned medium was used medium (CM10) by cells for their growth, and separated by centrifugation at approximately 1000 rpm for 5 minutes. The supernatant was collected and used as conditioned medium.
Test Medium for pH & Osmolality Assessment and Exposure to Treatment: Test medium used in the assessment of initial pH & osmolality and to expose the controls and test item concentrations, was a mix of basic medium and conditioned medium at 50:50 ratio.
Cleansing Medium: Basic medium supplemented with Thymidine (0.3 mg/mL), Hypoxanthine (0.5 mg/mL) and Glycine (0.75 mg/mL) was used along with Methotrexate (1 mg/mL PBS) was used for cleansing.
Cloning Medium (CLM20): Basic medium supplemented with 20% of HS was the cloning medium. Cloning medium was also used as non-selective medium.
Selective Medium: Cloning medium supplemented with the selective agent Triflurothymidine (TFT) between 1 and 4 µg/mL was the selective medium.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Aroclor 1254 induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar rats induced with a single intraperitoneal injection of Aroclor 1254 (0.7 mL/rat ready-to-use solution), 5 days prior to sacrifice.
- method of preparation of S9 mix: The S9 homogenate is batch prepared and stored in the test facility at -68 to -86 °C. Each batch of S9 homogenate is assessed for sterility, protein content (modified Lowry Assay, Sword and Thomson, 1980) and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain.
The S9 homogenate was thawed immediately before use and mixed with the co-factor pool containing 10 mM MgCl2, 5 mM Glucose-6-phosphate, 4 mM NADP, 10 mM CaCl2 and 30 mM KCl in basic medium.
The co-factor solution was prepared by dissolving the following in 21 and 13 mL of PBS for the cytotoxicity test and the mutation assay, respectively and sterilized using a 0.2 µm disposable syringe filter.
For Preliminary Cytotoxicity Test: Mg Cl2 (10 mM) 43 mg + Glucose-6-phosphate (5 mM) 36 mg + NADP (4 mM) 66 mg + Ca Cl2 (10 mM) 31 mg + KCl (30 mM) 47 mg
For Mutation Assay: Mg Cl2 (10 mM) 26 mg + Glucose-6-phosphate (5 mM) 22 mg + NADP (4 mM) 40 mg + Ca Cl2 (10 mM) 19 mg + KCl (30 mM) 29 mg
S9 mix was prepared by mixing the following, kept on an ice bath and used within one hour:
For Preliminary Cytotoxicity: Cofactor 21 mL + PBS 16.8 mL + S9 homogenate 4.2 mL + Basic medium 42 mL
Test Mutation Assay: Cofactor 13 mL + PBS 10.4 mL + S9 homogenate 42.6 mL + Basic medium 26 mL
- concentration or volume of S9 mix and S9 in the final culture medium: 4 mL

Test concentrations with justification for top dose:

a) 48 b) 143 c) 430 and d) 1291.10 µg/mL (factor of approximately 3.0)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile water (SW)
- Justification for choice of solvent/vehicle: The test item is soluble in Sterile Water (SW). Hence SW was used as the vehicle control of choice for the cell gene mutation assay.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
For Experiments 1 and 2, 100 µL of vehicle (SW) or stock/ dilution of Pidolic Acid were transferred in to pre-labeled tubes containing the treatment stock culture (6x106 cells).
For Experiment 3, 200 µL of vehicle (SW) or stock/ dilution of Pidolic Acid were transferred in to pre-labeled tubes containing the treatment stock culture (6x106 cells).
For the test in the presence of metabolic activation with 3-hour exposure (Experiment 1), 4 mL of S9 mix was added to each tube to achieve a concentration of 2 % (v/v) of S9.
For the test in the absence of metabolic activation with 3-hour exposure (Experiment 2), 4 mL of basic medium was added to each tube.
For the test in the absence of metabolic activation with 24-hour exposure (Experiment 3), 8 mL of complete medium was added to each tube.
The tube contents were gently mixed, loosely capped and kept for incubation on a tissue culture rotator inside a CO2 incubator for the respective exposure duration.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: For Experiments 1 and 2: 3 hours. For Experiment 3: 24 hours.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): After the respective treatment period, the cell suspensions were centrifuged and the supernatant discarded.
The cell button from all the exposure conditions were washed twice with complete medium to stop the treatment and re-suspended in fresh medium and incubated for approximately 24 hours.
After this incubation period, the cells were maintained in growth for the determination of suspension growth for two days post-treatment (expression period). On each day of the expression period, cultures were maintained by counting the cells and splitting them to a density of 3x105 cells/mL.
From the cell counts, suspension growth was calculated for the first 24 hours (Day 1 SG) and the second 24 hours (Day 2 SG) to arrive at RSG.
- Selection time (if incubation with a selective agent): A sample of the cell suspension of individual replicates from the phenotypic expression phase were suspended in the selective medium and plated at 2000 cells/well in to a microtitre plate for the estimation of mutant frequency. The plates were incubated in the CO2 incubator for 12 days and then scored manually for the presence of colonies.
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used: Trifluorothymidine. between 1 and 4 µg/mL. 12 days incubation.
- Criteria for small (slow growing) and large (fast growing) colonies: Wells of the microtitre plates
containing mutant colonies were observed using an inverted microscope and the colonies were sized based on a subjective scale. Large colonies covered more than 25 % of the well and sometimes the entire well. In contrast, small colonies were visibly smaller, covering less than 25 %
of the well’s diameter.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- After the phenotypic expression period, a sample of the cell suspension from each replicate culture was plated at 1.6 cells/well in a 96 well microtitre plate for the estimation of cell survival. The plates were incubated in the CO2 incubator for 12 days and then scored manually for the presence of colonies to estimate the Cloning Efficiency (CE) and to arrive at Relative Cloning Efficiency (RCE).
From the RSG and RCE values, Relative Total Growth (RTG) was calculated.
- Any supplementary information relevant to cytotoxicity:
Cytotoxicity is defined as the Relative Total Growth (RTG) which includes Relative Suspension Growth (RSG) during the 2 day expression period and the Relative Cloning Efficiency (RCE) obtained at the end of expression period in the preliminary cytotoxicity test and at the time of mutant selection in the gene mutation test.


METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant frequency (MF) is the cloning efficiency of the mutant colonies in selective medium (CEM) adjusted by the cloning efficiency in non-selective medium at the time of mutant selection (CEV).

Rationale for test conditions:

Preliminary Cytotoxicity Test for the Selection of Test Doses
To make a rapid assessment of the toxic range of the test item, L5178Y cells were exposed to the following test concentrations 40.3, 80.7, 161.4, 322.8, 645.6 and 1291.1 µg/mL along with a Sterile Water vehicle control.
Pidolic Acid did not cause precipitation in the test media up to the highest test concentration of 1291.10 µg/mL both in the presence and absence of metabolic activation, at the beginning and end of treatment.
At the end of 3-hour exposure, pH of the test medium in the presence of metabolic activation was between 7.20 and 7.31 with 7.18 in the Sterile Water control. Similarly, the pH of the test medium in the absence of metabolic activation was between 7.20 and 7.33 with 7.20 in the Sterile Water control.
At the end of 3-hour exposure, osmolality of the test medium at the highest tested concentration (1291.10 µg/mL) was 0.301 and 0.314 OSMOL/kg in the presence and absence of metabolic activation respectively.
Osmolality of sterile water control under the same condition was 0.298 OSMOL/kg and 0.308 OSMOL/kg in the presence and absence of metabolic activation, respectively.
There was no evidence of significant growth inhibition (80 to 90% inhibition over the control) as relative total growth (RTG) at any of the tested concentrations, both in the presence of metabolic activation and in the absence of metabolic activation with 24-hour exposure.
Based on these observations, it was decided to test up to 1291.10 µg/mL in the gene mutation assay.

Evaluation criteria:

• Providing that all acceptability criteria are fulfilled, the test item is considered able to induce mutation in this test system if in any of the experimental conditions examined, the increase in MF above the concurrent vehicle control exceeds the Global Evaluation Factor (GEF) of 126 x 10-6
and the increase is concentration related.
• Providing that all acceptability criteria are fulfilled, the test item is considered unable to induce mutation in this test system if in all the experimental conditions examined, there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF of 126
x 10-6.
• There is no requirement for verification of a clearly positive or negative response.
• In cases where the response is neither clearly positive nor clearly negative, a repeat experiment may be performed using modified experimental conditions (eg. concentration spacing, other metabolic conditions etc.).
• In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results. In such cases, the test item response should be concluded as equivocal.

Statistics:

The number of negative control values, are assumed to follow Poisson Distribution. Test of significance of difference between negative control values over different dose groups and the positive control will be carried out using a method of anlaysis suggested by M.R.Thomas (Cole and Arlett, 1984), based on χ2. All analysis and significance tests will be evaluated at a 5% level of
significance (p ≤ 0.05).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

TABLE 1. In vitro Mammalian Cell Gene Mutation Test - Parallel Cytotoxicity Test Results

Treatment (µg/mL)	Experiment 1 (presence of metabolic activation with 3-hour treatment)			Experiment 2 (absence of metabolic activation With 3-hour treatment)			Experiment 3 (absence of metabolic activation With 24-hour treatment)
	RCE	RSG	RTG	RCE	RSG	RTG	RCE	RSG	RTG
Sterile water	1	1.00	100	1	1.00	100	1	1.00	100
48	0.85	0.87	83	0.97	0.90	87	0.91	0.88	80
143	0.92	0.79	73	0.90	0.81	73	0.86	0.85	73
430	0.85	0.64	54	0.85	0.74	63	0.75	0.76	57
1291.10	0.78	0.59	46	0.74	0.60	44	0.62	0.68	42
positive control	0.63	0.50	32				0.48	0.74	36

TABLE 2. In Vitro Mammalian Cell Gene Mutation Test - Small and Large colony
Mutants

	Vehicle Control		Positive Control
	Small Colony	Large Colony	Small Colony	Large Colony
Experiment 1: with S9	4	27	188	92
Experiment 2: without S9	5	30	-	-
Experiment 3: without S9	4	33	195	92

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study as described in the study plan were met. It is concluded that the test item, Pidolic Acid does not have the potential to induce gene mutation in Mouse Lymphoma L5178Y TK+/- -3.7.2C cells at the tested concentrations and under the conditions of testing employed.

Executive summary:

The genotoxic potential of the test item, Pidolic Acid, to induce gene mutation in mammalian cells was evaluated using cultured Mouse Lymphoma L5178Y
TK+/- -3.7.2C cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).
The study consisted of a preliminary cytotoxicity test and a gene mutation assay. The gene mutation assay consisted of three independent experiments:
Experiments 1 and 2 in the presence and absence of metabolic activation with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic
activation system with 24-hour exposure.
The test item was tested for its solubility in sterile water, which was found to be approximately 129110 µg/mL. The item was stable in sterile water for 24 hours
at room temperature at the concentrations of 500 and 200000 µg/mL.
In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the test item did not show evidence of significant growth
inhibition (>90 % inhibition over the control) as relative total growth (RTG) up to the highest tested concentration of 1291.10 µg/mL either in the presence or in the absence of metabolic activation. There was no precipitation of the test item in the test medium up to the highest tested concentration of 1291.10 µg/mL. The test item did not cause any appreciable change in the pH and osmolality of test medium. Based on these observations, a maximum concentration of
1291.10 µg/mL was tested in all the three experiments of the gene mutation assay.
In the cell gene mutation assay, mouse lymphoma cells were exposed to the test item in duplicate at the concentrations of 48, 143, 430 and 1291.10 µg/mL in all the three experiments, experiments 1 and 2 (presence and absence of metabolic activation with 3-hour exposure) and in experiment 3 (absence of metabolic activation with 24-hour exposure). Concurrent vehicle and positive controls Cyclophosphamide monohydrate in the presence of metabolic activation and Methyl methanesulfonate in the absence of metabolic activation were also tested in duplicate.
The reduction in cell growth as relative suspension growth (RSG) was 41, 40 and 32 % in Experiments 1, 2 and 3, respectively, compared to the vehicle
control.
The reduction in cell growth as relative total growth (RTG) was 54, 56 and 58 % in Experiments 1, 2 and 3, respectively, compared to the vehicle control.
There was no evidence of induction of gene mutation in any of these experiments either in the presence or absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequencies of mutants, under identical conditions.
The study indicated that the test item, Pidolic Acid was not mutagenic under the conditions of testing employed.