Pidolic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was considered to be non-mutagenic in a reliable in vitro genotoxicity study (Ames test) employing Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and in Escherichia colistrain WP2 uvrA in the presence and absence of a metabolic activation system.

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.

The test item, Pidolic Acid exhibited a negative response when tested for its ability to induce chromosomal aberration in cultured human peripheral blood lymphocytes up to the highest concentration of 1290 μg/mL. The data suggest that the test item is not clastogenic under the conditio ns of testing employed.

 

Link to relevant study records

Reference

Reference 1

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

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

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.

Target gene:

TK

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.

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).

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

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

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

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. 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 March 2021 to 22 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted: 29 July 2016 (OECD 2016)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

adopted: August 1998

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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 analysis of test item in sterile water (SW) as well as analytical verification of test item dosing formulations were carried out in-house using a validated analytical method as per Study No. G21146.
- Expiry Date : 05/2022

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 129000 µg/mL was prepared and further diluted to 143.33, 430 and 1290 µg/mL test concentrations

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

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- human peripheral blood lymphocytes
- Suitability of cells: Studies on exposed individuals and on cultured cells have shown that the human peripheral blood lymphocyte is an extremely sensitive indicator of both in vivo and in vitro clastogen induced chromosome structural changes and these changes in chromosome structure offer readily scored morphological evidence of damage to the genetic material.
Human peripheral blood lymphocytes are useful in in vitro cytogenetic testing because of easy availability of large numbers of human cells, their distribution throughout the body, circulation in all the tissues and a proportion of them are long-lived and they maintain a stable karyotype.

For lymphocytes:
- Blood was collected from individual male donor of age 26 years for preliminary cytotoxicity test.
- Blood was collected from individual male donor of age 30 years for chromosomal aberration test.
- Whether whole blood or separated lymphocytes were used: Whole blood cultures were established
- Whether blood from different donors were pooled or not: No
- Mitogen used for lymphocytes: A proportion of the lymphocytes can be stimulated by mitogens to undergo mitosis in culture, they are easy to culture and thus provide a ready source of dividing cells for the scoring of chromosome aberrations.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 medium supplemented with sodium bicarbonate, L-glutamine, antibiotics and 20 % fetal bovine serum (RPMI FBS20).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
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 was prepared in batches and stored in a deep freezer maintained at -68 to -86ºC. S9 homogenate was 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 typhimurium TA100 strain.
The S9 homogenate was thawed immediately before use and mixed with the co-factor solution containing 25 mg/mL NADP, 180 mg/mL Glucose-6-phosphate, 150 mM KCl and S9 fraction (at a ratio of 1:1:1:2 v/v). The co-factor solution was prepared by dissolving each of the following cofactors in 2 mL of RPMI 1640 for both preliminary cytotoxicity test and chromosomal aberration assay. These individual components are then pooled (in total 6 mL of cofactor solution) and sterilized by using a 0.2 μm disposable syringe filter: 50 mg NADP (25 mg/mL), 360 mg Glucose-6-phosphate (180 mg/mL) and 22 mg Potassium chloride (150 mM).
S9 mix was prepared by mixing 4 mL of S9 homogenate with 6 mL of the co-factor solution each for the preliminary cytotoxicity test and the chromosomal aberration assay, respectively. The S9 mix was kept in an ice bath and used within one hour of preparation.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.25 mL

Test concentrations with justification for top dose:

a) 143.33
b) 430 and
c) 1290 μg/mL
(factor of 3)

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.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE: Test item stock solution and dilutions were prepared fresh for use in the chromosomal aberration test.
For the experiment in the presence of metabolic activation, 0.5 mL S9 mix were added to the respective blood cultures to achieve a final concentration of 2% S9 (v/v) in the test medium. Similarly, for experiments in the absence of metabolic activation, 0.5 mL of 150 mM KCl were added to the respective tubes.
One hundred µL of the test item dilutions were mixed with the test medium in tubes containing the target blood cultures. 100 µL of either vehicle, or CPA or EMS solutions were added to additional blood culture tubes to serve as negative and positive controls respectively. Exposure lasted for 3 hours in experiments 1 and 2 and for 22 hours and 25 minutes in experiment 3.
After 3-hours of exposure tubes from experiments 1 and 2 were centrifuged at 1000 rpm for 5 minutes, afterwards the supernatant was discarded to stop the treatment. The cell pellet was washed twice with PBS, the tubes were re-filled with fresh RPMI FBS20 and incubated for 18 hours 50 minutes till harvest.

TREATMENT AND HARVEST SCHEDULE:
Approximately, 2 hours before harvest, Colchicine was added to cultures at a final concentration of 0.2 µg/mL to arrest dividing cells in metaphase.
Each culture from the controls and treatment groups was harvested at approximately 22 hours and 25 minutes after the beginning of the treatment and processed separately for the preparation of chromosomes. This post-treatment harvest time was selected to ensure that cells are analyzed in the first division metaphase after initiation of treatment.
Cells were harvested by centrifugation at 1000 rpm for 5 minutes. The supernatant was carefully removed and cells re-suspended in warm 0.56% KCl for 15 minutes to allow cell swelling to occur. Cells were then washed with consecutive changes of cold methanol: acetic acid fixative (3:1 v/v). This centrifugation procedure was repeated until clear white pellets were obtained. The cell suspension was kept in fixative in the refrigerator before slides were made.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Colchicine at 0.2 μg/mL was used as the spindle inhibitor. Approximately, 2 hours before harvest, Colchicine was added to cultures at a final concentration of 0.2 μg/mL to arrest dividing cells in metaphase.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Cells were pelleted and re-suspended in a minimal amount of fresh fixative so as to give a milky suspension. Several drops of suspension were transferred on to clean glass slides, flame dried and dried on a slide warmer maintained at approximately 40°C.
Five slides per replicate were made and were then marked with the study number, treatment group, activation, experiment number, replicate number and the slide number.
The slides were stained with freshly prepared 5% Giemsa stain (120 minutes)in distilled water, rinsed with tap water, air dried, immersed in xylene and mounted with DPX. The slides were then coded using random numbers before evaluation by an individual not involved in the scoring process.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A total of 300 such metaphases evenly distributed amongst the duplicate cultures were evaluated for each concentration group including controls. The three experiments were observed for Polyploidy and Endoreduplication. The total number of metaphases showing one or more aberrations both including and excluding gaps was calculated from a set of 300 metaphases for each concentration group. To calculate the mitotic index, slides were randomly observed under microscope, 1000 cells were counted in different fields and the number of metaphases per 1000 cells were recorded.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
The slides were scanned and each well spread metaphase was examined under the 100 x objective. One hundred fifty metaphases from each replicate culture were analyzed for chromosome aberrations. The number of chromosomes in each spread was counted and those containing 44 to 48 chromosomes were evaluated for aberrations. A total of 300 such metaphases evenly distributed amongst the duplicate cultures were evaluated for each concentration group including controls. The chromosome number and the microscope coordinates were recorded for the aberrant cells. Aberrations were recorded as chromatid/chromosome gaps or breaks and exchange figures.

- Determination of polyploidy: Cell containing more than the diploid number (2n) of chromosomes, in exact multiples of the haploid number (n) eg., triploid = 3n, tetraploid = 4n, etc.

- Determination of endoreplication: Chromatid alignment is maintained in a cell in which the chromosomes have duplicated but the cell has failed to cleave, a form of polyploidy.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Concurrent cytotoxicity was assessed for all treated and control cultures based on mitotic index


METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Definitions for Aberrations
The chromosome and chromatid aberrations observed have been grouped into three categories - gaps, breaks (includes deletions and displacements) and exchanges.
Gap: Chromatid gap is a non-staining region of a single chromatid in which there is a minimal misalignment of the chromatid. Chromosome gap is a non-staining region at the same locus in both chromatids of a single chromosome in which there is minimal misalignment of the chromatids.
Break: Chromatid break is discontinuity of a single chromatid in which there is clear misalignment of one of the chromatids. Chromosome break is discontinuity at the same locus in both chromatids of a single chromosome, giving rise to an acentric fragment. A chromatid deletion occurs when the bit of chromatid is missing as a result of the break. A chromosome deletion occurs when the bits of both the chromatids are missing as a result of a break. Chromatid displacement occurs when the fragment of the chromatid beyond the break point is not in alignment with the chromosome of origin. Chromosome displacement occurs when the fragments of both the chromatids beyond the break point are not in alignment with the chromosome of origin.
Exchange: Exchanges occur as a result of two or more chromatid lesions and the subsequent rearrangement of chromatid material. Ring chromosomes are formed when a chromosome undergoes two breaks and the broken ends of the chromosome reunite in a ring structure either with or without a centromere.
Pulverization: In cells with pulverization, the chromosomes are reduced to masses of fragments. This represents the degree of damage inflicted on the chromosome. This aberration indicates the cytotoxic effect of the test item.
Polyploidy: Cell containing more than the diploid number (2n) of chromosomes, in exact multiples of the haploid number (n) eg., triploid = 3n, tetraploid = 4n, etc.
Endoreduplication: Chromatid alignment is maintained in a cell in which the chromosomes have duplicated but the cell has failed to cleave, a form of polyploidy.
- Microscopic Analysis:
Metaphases of three concentrations of the test item, the vehicle control and the positive control (of Experiments 1 and 3) cultures were scored in each of the three experiments. The slides were scanned and each well spread metaphase was examined under the 100 x objective. One hundred fifty metaphases from each replicate culture were analyzed for chromosome aberrations. The number of chromosomes in each spread was counted and those containing 44 to 48 chromosomes were evaluated for aberrations. A total of 300 such metaphases evenly distributed amongst the duplicate cultures were evaluated for each concentration group including controls. The chromosome number and the microscope coordinates were recorded for the aberrant cells. Aberrations were recorded as chromatid /chromosome gaps or breaks and exchange figures. Since gaps are not considered as true aberration, the results are presented as metaphases with aberrations including gaps and excluding gaps. The three experiments were observed for Polyploidy and Endoreduplication. The total number of metaphases showing one or more aberrations both including and excluding gaps was calculated from a set of 300 metaphases for each concentration group. To calculate the mitotic index, slides were randomly observed under microscope, 1000 cells were counted in different fields and the number of metaphases per 1000 cells were recorded.

Rationale for test conditions:

A preliminary cytotoxicity test was carried out to select test concentrations for the chromosomal aberration assay concentrations of 40.3125, 80.625, 161.25, 322.5, 645, 1290 µg/mL were tested along with a SW control. The top dose of 1290 µg/mL was selected as the highest dose.
At the end of the a 3-hour exposure period, Pidolic Acid did not cause precipitation in the test solution at any of the tested concentrations, both in the presence and absence of metabolic activation. In the presence of metabolic activation, at the end of a 3-hour exposure to the test item, the pH of the test medium ranged from 7.26 to 7.28 with a pH value of 7.26 in the SW control. In the absence of metabolic activation, the pH ranged between 7.23 and 7.30 with a pH value of 7.22 in the SW control. At the end of 3-hour exposure, osmolality of the test medium at the highest soluble test item treatment condition (1290 µg/mL) was 0.317 and 0.321 OSMOL/kg in the presence and absence of metabolic activation respectively. The corresponding osmolality in the SW control was 0.311 OSMOL/kg in the presence and absence of metabolic activation.
Pidolic Acid did not exhibited the required level of cytotoxicity (reduction in the mitotic index by 45±5% of the concurrent vehicle control) at any of the tested doses both in the presence and absence of metabolic activation with 3-hour exposure or 22-hour exposure. Based on these observations, in the chromosomal aberration assay, a top concentration of 1290 µg/mL was tested in experiments 1, 2 and 3.

Evaluation criteria:

When all the validity criteria are fulfilled:
1. 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 vehicle control.
• The increase is dose-dependent when evaluated with an appropriate trend test.
• Any of the results are outside the distribution of the historical vehicle control data.
2. A test chemical is considered to be clearly negative if, in all experimental conditions examined:
• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control.
• There is no concentration-related increase when evaluated with an appropriate trend test.
• All results are inside the distribution of the historical vehicle control data.
3. The results will be considered equivocal if they do not meet the criteria specified for a positive or negative response.
4. An increase in the number of polyploidy cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the
potential to inhibit cell cycle progression.

Statistics:

The statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analysed for proportions of aberrant metaphases in each sample, excluding gaps as aberrations. Pooled data from each test concentration and the positive controls were compared with the vehicle controls using Fischer exact test. All analysis and comparisons were evaluated at 5% (p<0.05) level.

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

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

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

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

TABLE 1. Preliminary Cytotoxicity Test Results

Treatment (µg/mL)	Presence of metabolic activation (3-hour exposure)			Absence of metabolic activation (3-hour exposure)			Absence of metabolic activation (22-hour exposure)
	Mitotic Index(%)	Relative mitotic index	Per cent Inhibition	Mitotic Index (%)	Relative mitotic index	Per cent Inhibition	Mitotic Index (%)	Relative mitotic index	Per cent Inhibition
SW	8.2	100	-	8	100	0	7.6	100	0
40.3125	7.8	95	5	7.7	96	4	7.4	97	3
80.625	7.6	93	7	7.5	94	6	6.9	91	9
161.25	7.4	90	10	7.2	90	10	6.8	89	11
322.5	7	85	15	6.7	84	16	6.5	86	14
645	6.8	83	17	6.6	83	17	6.3	83	17
1290	6.4	78	22	6.4	80	20	6	79	21

 

Table 2. Summary Results of Chromosomal Aberration Test - Experiment 1

Treatment (µg/mL)	No. of metaphases scored	No.(%) of metaphases with aberrations				Total No.(%) of aberrant metaphases*		Relative Mitotic Inhibition (%)
		Gaps Cs/Ct	Breaks Cs/Ct	Exchanges Cs/Ct		Incl. Gaps	Excl. Gaps
SW (100 µL)	300	0/0	1/0	0/0		1	1	0
143.33	300	0/0	0/0	0/0		0	0	7
430	300	0/0	0/0	0/0		0	0	15
1290	300	1/0	0/0	0/0		1	0	21
CPA @14	300	2/0	20/27	23/36		95	94	43

Table 3. Summary Results of Chromosomal Aberration Test - Experiment 2

Treatment (µg/mL)	No. of metaphases scored	No.(%) of metaphases with aberrations				Total No.(%) of aberrant metaphases*		Relative Mitotic Inhibition (%)
		Gaps Cs/Ct	Breaks Cs/Ct	Exchanges Cs/Ct		Incl. Gaps	Excl. Gaps
SW (100 µL)	300	0/0	0/1	0/0		1	1	0
143.33	300	0/0	0/0	0/0		0	0	10
430	300	1/0	0/0	0/0		1	0	12
1290	300	0/0	0/0	0/0		0	0	23

Table 4. Summary Results of Chromosomal Aberration Test - Experiment 3

Treatment (µg/mL)	No. of metaphases scored	No.(%) of metaphases with aberrations				Total No.(%) of aberrant metaphases*		Relative Mitotic Inhibition (%)
		Gaps Cs/Ct	Breaks Cs/Ct	Exchanges Cs/Ct		Incl. Gaps	Excl. Gaps
SW (100 µL)	300	0/0	0/1	0/0		1	1	0
143.33	300	0/0	1/0	0/0		1	1	9
430	300	0/0	0/0	0/0		0	0	16
1290	300	0/0	0/0	0/0		0	0	22
EMS @ 600	300	0/2	40/41	23/20		94	94	37

Conclusions:

All criteria for a valid study were met as described in the study plan.
It is concluded that the test item, Pidolic Acid exhibited a negative response when tested for its ability to induce chromosomal aberration in cultured human peripheral blood lymphocytes up to the highest concentration of 1290 μg/mL. The data suggest that the test item is not clastogenic under the conditions of testing employed.

Executive summary:

The potential of the test item, Pidolic Acid to induce chromosomal aberrations in mammalian cells was evaluated using cultured human peripheral blood lymphocytes.
Human lymphocytes in whole blood culture, stimulated to divide by addition of Phytohaemagglutinin (PHA) approximately 48 hours prior to treatment, were exposed to the test item 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 chromosomal aberration assay. The chromosomal aberration assay consisted of three independent experiments: Experiments 1 and 2 with a 3-hour exposure and in the presence and absence of metabolic activation, respectively, and Experiment 3 in the absence of metabolic activation and with a 22-hour exposure.
The test item was soluble in sterile water (SW) at 129 mg/mL and was found stable in SW (Milli Q water) for 24 hours at room temperature at 500 µg/mL and 200000 µg/mL.
In a preliminary cytotoxicity test, blood cells were exposed to Pidolic Acid at the concentrations of 40.3, 80.6, 161.3, 322.5, 645 and 1290 µg/mL along with a SW control.
At the end of the 3-hour exposure period, Pidolic Acid did not cause precipitation at any of the tested doses both in the presence and absence of
metabolic activation, and did not exhibit the required level of cytotoxicity (reduction in the mitotic index by 45±5% of the concurrent vehicle control) at any of the tested doses both in the presence and absence of metabolic activation with 3-hour exposure or in the absence of metabolic activation with 22-hour exposure. Based on these observations, in the chromosomal aberration assay, a top concentration of 1290 µg/mL (~10mM) was tested in the 3-hour exposure, in the presence and absence of metabolic activation and in the absence of metabolic activation with 22-hour exposure.
In the definitive chromosomal aberration assay, blood cultures were exposed to the test item in duplicate at the concentrations of 143.3, 430 and 1290 µg/mL in Experiments 1 & 2 (presence and absence of metabolic activation with 3-hour exposure) and in Experiment 3 (absence of metabolic activation with 22-hour exposure).
In a similar way, concurrent vehicle control (SW) and the positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) were tested in duplicate cultures. In each case, the cells in the C-metaphase were harvested at approximately 22 hours after the start of the treatment from the vehicle control, test item concentrations and the positive control groups.
At the highest concentration tested, the reduction in mitotic index was 21, 23 and 22 % in Experiments 1, 2 and 3, respectively, compared to the vehicle control.
A total of 300 metaphases from duplicate cultures from each of the controls and three treatment levels were evaluated for chromosomal aberrations. The data from the treatment groups and the positive controls were statistically compared with the vehicle control.
There was no statistically significant increase in the incidence of structurally aberrant metaphases, either at 3 hours in the presence and absence of metabolic activation and at 22 hours exposure, in the absence of metabolic activation in any of the tested concentrations. Under identical conditions, the respective positive control substances produced statistically significant (p<0.05) increases in aberrant metaphases confirming the sensitivity of the test system and the activity of the S9 mix.
The results of the concentration analysis of the dose formulation samples were within the acceptable specification range and confirmed that the targeted top concentration level was achieved supporting the validity of the study conclusion.
The study indicated that, under the conditions of this study, Pidolic Acid was not clastogenic in human peripheral blood lymphocytes up to the highest tested concentration of 1290 µg/mL. 

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 August 2017 - 19 October 2017

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

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: Sponsor and 16032501
- Expiration date of the lot/batch: 24 March 2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in DMSO

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (direct plate method)
15, 50, 150, 500, 1500, 5000 μg/plate (pre-incubation method)

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

Untreated

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Evaluation criteria:

A test item will be considered mutagenic (positive) in the test system if the criteria below are met:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Bacteria

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 1535

Remarks:

Bacteria

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:

E. coli WP2 uvr A

Remarks:

Bacteria

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

Remarks:

Bacteria

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

Remarks:

Bacteria

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:

Minor statistical values were noted in Experiment 1 (TA1535 at 15 μg/plate in the presence of S9-mix and in Experiment 2 (TA100 at 50 μg/plate in the absence of S9-mix), however these responses were within the in-house historical vehicle/untreated control values and were, therefore considered of no biological relevance.

Conclusions:

The substance was considered to be non-mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and in Escherichia coli strain WP2 uvrA in the presence and absence of a metabolic activation system.

Executive summary:

In the in vitro genotoxicity study (Ames test) the substance was tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and in Escherichia coli strain WP2 uvrA in the presence and absence of a metabolic activation system. Concentrations of up to 5000 μg/plate were tested. No evidence of  mutagenic activity or cytotoxicity was seen at any concentration of the substance.
It was concluded that the substance showed no evidence of mutagenic activity in these bacterial systems under the test conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Pidolic Acid did not induce significant increase in micronucleated PCE in the bone marrow of either male or female mice at doses tested. In conclusion, Pidolic Acid was not genotoxic in the mammalian erythrocyte micronucleus test at the doses tested.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th March 2021 to 30th August 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

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
- Storage Conditions : Ambient (+15 to +25°C)
- Expiry Date : 05/2022
- The method validation and stability of the test item, Pidolic Acid in the vehicle Milli-Q water was carried under Study No.: G21489. The test item was found to be stable and resuspendable in the vehicle for 48 hours at room temperature, at the fortification levels of 1 mg/mL and 200 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding):
Required quantities of the test item were weighed in a beaker (previously calibrated to a desired volume*) for each dose level separately, then a small volume (2 to 10 mL) of vehicle (Milli-Q water) was added and stirred using a glass rod to obtain a uniform solution and the volume was made up to the mark using the vehicle to get the final desired concentration.
- Final concentration of a dissolved solid, stock liquid or gel: 50, 100, 200 mg/mL

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Dissolved in Milli-Q water

Species:

mouse

Strain:

Swiss

Details on species / strain selection:

Mouse is the most commonly used species for in-vivo mutagenicity tests and extensive data is
available on micronuclei induction by a wide variety of agents.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology (India) Pvt Ltd. 4B, M. N Park, Turkapally Village, Shameerpet Mandal, Medchal District - 500078, Telangana, India.
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: At the commencement of treatment, the weight variation of mice did not exceed ± 20 % of the mean body weight in each group and sex.
Body weights (g) Mean ± SD and body weight range on day 1 of treatment:
Dose range finding study
Males
G1 : 34.53 ± 1.16
G2 : 34.39 ± 0.81
G3 : 34.45 ± 0.29
G4 : 34.70 ± 0.57
Females
G1 : 28.43 ± 2.60
G2 : 29.17 ± 1.82
G3 : 28.86 ± 1.97
G4 : 29.29 ± 0.47
Definitive study Toxicity groups
Males
G1 : 34.83 ± 1.48
G2 : 34.96 ± 1.30
G3 : 35.04 ± 1.11
G4 : 35.02 ± 1.12
G5 : 34.90 ± 1.73
Females
G1 : 28.20 ± 0.85
G2 : 28.20 ± 0.82
G3 : 28.22 ± 0.81
G4 : 28.27 ± 0.68
G5 : 27.34 ± 1.82
PC assessment group
Males
G1PC : 38.17 ± 1.10
G2PC : 38.07 ± 0.86
G3PC : 38.07 ± 0.52
G4PC : 37.95 ± 0.07
Females
G1PC : 30.70 ± 0.80
G2PC : 30.84 ± 0.50
G3PC : 30.64 ± 0.21
G4PC : 30.66 ± 0.04
- Assigned to test groups randomly: [no/yes, under following basis: ] No. Mice were assigned to study groups by body weight stratification. Grouping was done prior to initiation of treatment.
- Fasting period before study: None
- Housing: Mice were housed individually to avoid infighting during acclimatization and treatment period in standard polysulfone cages (size: L 360 x B 205 x H 140 mm) with stainless steel top grill having provision for food and drinking water in polycarbonate bottles with stainless steel sipper tubes. Additionally, polycarbonate mouse hut was placed inside the cage as an enrichment object which was changed once during acclimatization and treatment period.
- Diet (e.g. ad libitum): Altromin (1334P) Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany was provided ad libitum to the mice.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in “Aquaguard” on-line water filter-cum-purifier (manufactured by Eureka Forbes Ltd., Mumbai - 400 001, India) was provided ad libitum to mice in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: After detailed clinical examination for good health and suitability for the study, the mice were acclimatized to the experimental room for four days for DRF study and 5 days for the definitive study before the start of the treatment. All the mice were observed once daily during acclimatization.

ENVIRONMENTAL CONDITIONS
- Mice were housed in room number A14 under standard laboratory conditions, air conditioned with adequate fresh air supply (12 - 15 air changes/hour). Environment: temperature 20-22 °C, relative humidity 65 to 66 %, and 12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 26th March 2021 To: 01 April 2021

Route of administration:

oral: gavage

Vehicle:

Vehicle: Milli-Q water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the DRF study and definitive study the dose formulations were prepared once on day 1 of treatment and used for both days of treatment. The prepared formulations were stored in experimental room (temperature 20 to 23 °C) and used within the established stability period of 48 hours.
Required quantities of the test item were weighed in a beaker (previously calibrated to a desired volume*) for each dose level separately, then a small volume (2 to 10 mL) of vehicle (Milli-Q water) was added and stirred using a glass rod to obtain a uniform solution and the volume was made up to the mark using the vehicle to get the final desired concentration.
*Pre-calibration of the beaker to desired volume: Milli-Q water was measured in a graduated measuring cylinder to the final volume of the required batch size (10 mL for DRF study and 30 mL for definitive study). The measured Milli-Q water was transferred into a clean beaker (to be precalibrated) and upper and lower meniscus of Milli-Q water was marked on the beaker using a marker. Once marking was done, the water was discarded and beaker dried. The upper meniscus mark was used to make up the volume during dose formulation preparation.

Duration of treatment / exposure:

2 days

Frequency of treatment:

once every 24 hours

Post exposure period:

Mice were sacrificed 23 to 24 hours following the final administration

Dose / conc.:

0 mg/kg bw/day

Remarks:

G1

Dose / conc.:

500 mg/kg bw/day

Remarks:

G2

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

G3

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

G4

No. of animals per sex per dose:

DRF: 2 animals/sex/dose
Definitive study toxicity group: 5 animals/sex/dose
Definitive study Plasma concentration assessment group: 2 animals/sex/dose

Control animals:

yes

Positive control(s):

cyclophosphamide monohydrate
- Justification for choice of positive control(s): Recommended in the guideline
- Route of administration: oral gavage
- Doses / concentrations: 40mg/kg bw/day

Tissues and cell types examined:

- Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A Dose Range Finding (DRF) study was carried out to select the doses for the definitive study. As per the reported information in the Safety Data Sheet provided by Sponsor, the acute oral LD50 is more than 2000 mg/kg. Hence the following dose levels of 500, 1000 and 2000 mg/kg/day, along with a concurrent vehicle control group were selected in consultation with Sponsor.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- The test item formulations and the vehicle were administered orally by gavage twice for 2 consecutive days at 24 hours interval with the variation of ± 2 hours at a dose volume of 10 mL/kg body weight. The dose volume was calculated for individual mouse according to the body weights recorded on the days of treatment.
- For vehicle control groups, vehicle (Milli-Q water) was administered.
- Cyclophosphamide monohydrate, 40 mg/kg body weight was administered to the mice belonging to the positive control group as a single oral gavage at a dose volume of 10 mL/kg body weight.

DETAILS OF SLIDE PREPARATION:
- The cell suspensions were centrifuged at 2000 rpm for 10 minutes and supernatants discarded. A sample of approximately 10 µL of the cell suspension was spread evenly on a glass slide and blow dried.
- The slide was marked with the study number, animal code number and slide number. The smears were fixed in methanol for 10 minutes. Two slides were prepared for each animal.
- Slides were stained by May-Grunwald and Giemsa stain in succession. The slides were blow-dried, immersed in xylene and cover slips mounted with DPX.

METHOD OF ANALYSIS:
A minimum of 4000 polychromatic erythrocytes (PCE) were manually scored from each mouse (slides were coded prior to scoring) for the incidence of PCE with micronuclei. The proportion of immature erythrocytes among total erythrocytes (number of PCE divided by number of total erythrocytes) was determined for each mouse by counting at least 500 erythrocytes per mouse.
From these observations, the following was derived for each mouse:
• Total erythrocytes scored
• Number of PCE differentiated
• Ratio of PCE: total erythrocytes
• Number and percentage of PCE with micronuclei
• Mean and SD of PCE with micronuclei

Evaluation criteria:

The test was considered valid as it met the following criteria:
a. The incidence of micronucleated polychromatic erythrocytes (MNPCE) distribution in the vehicle control was within 95 % control limits of the inhouse historical control data range.
b. The concurrent positive control induced responses that are compatible with the historical positive control data base and produced a statistically significant increase in the number of micronuclei compared with the concurrent vehicle control.
c. The appropriate number of doses and cells has been analyzed.
d. Appropriate selection of high dose (maximum tolerated dose).

Statistics:

Results of statistical analysis were reported in the form of Mean ± SD and sample size.
The statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package ver.12.0. All quantitative variables like change in net body weight, body temperature were tested for normality (Shapiro-Wilk test) and homogeneity (Levene’s test) of within group variance before performing ANOVA. For counts/ proportions/percentages data were normalized using suitable transformation (square root) before ANOVA. Comparison of means between the control and the treatment groups was done using Dunnett’s ‘t’ test, where ‘F’ test was significant in ANOVA.
All analyses and comparisons were evaluated at 5% (p < 0.05) level and the statistical significance was designated as given below:
* : Significantly different from the vehicle control group

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the conditions described in this report, Pidolic Acid did not induce significant increase in micronucleated PCE in the bone marrow of either male or female mice at doses tested. In conclusion, Pidolic Acid was not genotoxic in the mammalian erythrocyte micronucleus test at the doses tested.

Executive summary:

The purpose of this study was to identify the toxicity potential of Pidolic Acid as measured by its ability to induce cytogenetic damage in bone marrow cells of Swiss albino mice which results in the formation of micronuclei containing lagging chromosome fragments (clastogenicity) or whole chromosomes (aneugenicity). The concentration of the test item, Pidolic Acid in plasma was determined to assess systemic exposure of test item. This study provided a rational basis for risk assessment in humans.

Doses for the definitive study were selected based on the preliminary dose range finding (DRF) study. In the DRF study, Pidolic Acid was tested at the doses of 500 (G2), 1000 (G3) and 2000 (G4) mg/kg body weight along with a concurrent vehicle (Milli-Q water) control group (G1) at the dose volume of 10 mL/kg body weight.

Based on the results of dose range finding study (DRF), the highest dose of 2000 mg/kg was well tolerated and hence the same dose levels tested in the dose range finding study were selected for the definitive study in consultation with the Study Sponsor.

In the definitive study, Swiss albino mice (5/sex/group) were administered Pidolic Acid at dose levels of 500, 1000 and 2000 mg/kg/day for low (G2), mid (G3) and high (G4) dose group, respectively by oral gavage for 2 consecutive days. A control group (G1) of mice received the vehicle (Milli-Q water) on the same schedule. An additional group of mice (G5) was administered with positive control cyclophosphamide monohydrate (40 mg/kg) via single oral dose. Mice were sacrificed 23 to 24 hours following the final administration; both femurs were dissected from each mouse and the bone marrow was collected. The bone marrow smears were prepared, fixed, stained with May-Grunwald and Giemsa stain, and examined blinded for the treatments using oil immersion microscopy. A minimum of 4000 polychromatic erythrocytes (PCE) per mouse were examined for the presence of micronuclei indicative of chromosome damage. In addition, the proportion of PCE among total erythrocytes was also assessed for each animal as a measure of potential bone marrow toxicity.

Groups for the plasma concentration assessment consisted of 2 male and 2 female mice for control group (G1PC), low (G2PC), mid (G3PC) and high (G4PC) dose groups. Blood samples were collected on Day 2 at 1 and 3 h post dose from G1PC through G4PC groups. The concentrations of Pidolic Acid in plasma samples were analyzed to confirm the systemic exposure by oral route administration. The concentration of Pidolic Acid in mice plasma was analyzed using bioanalytical method validated prior to the sample’s analysis for specific parameters (selectivity, precision and accuracy).
The mean Pidolic Acid concentrations in the dose formulation samples were within 10% of the nominal concentrations and the relative standard deviations (% RSD) were <10% indicating that the test item was homogeneously distributed in the vehicle. Vehicle sample did not show any traces of Pidolic Acid.

The salient observations of the study are as follows:
1. There were no clinical signs and mortalities at the tested doses of 500, 1000 and 2000 mg/kg/day.
2. The body weight gains at all dose levels were comparable to vehicle control group.
3. The incidences of micronucleated PCE in the Pidolic Acid treatment groups were comparable to the vehicle control group. The ratio of PCE: total erythrocytes were also comparable between the control and treatment groups indicating that there was no bone marrow toxicity. Cyclophosphamide monohydrate, the positive control, induced a statistically significant increase (p < 0.05, Dunnett’s test) in the incidence of micronucleated PCEs and a statistically significant decrease (p < 0.05, Dunnett’s test) in the PCE: total erythrocytes ratio, as compared to  vehicle control group. The percent incidences of micronucleated PCE in the concurrent vehicle control group (0.05 to 0.07) were within the historical vehicle control range (0.00 to 0.12). These data confirm the validity of the assay.
4. Plasma samples were collected at 1 h and 3 h post dose from vehicle control group (G1PC). Plasma samples collected at 1 h and 3 h post dose from animals in the 500, 1000 and 2000 mg/kg/day dose groups (G2PC through G4PC) showed Pidolic Acid concentrations ranging from 180.08 to 1003.22 ng/mL, indicating systemic exposure to Pidolic Acid by oral administration.

Under the conditions described in this report, Pidolic Acid did not induce significant increase in micronucleated PCE in the bone marrow of either male or female mice at the doses tested. In conclusion, Pidolic Acid was not genotoxic in the mammalian erythrocyte micronucleus test at the doses tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the findings of a reliable in vitro mutagenicity study (Ames test) conducted on the substance, classification of the substance is not justified.

Based on the findings of a reliable in vitro gene mutation study in mammalian cells conducted on the substance, classification of the substance is not justified.

Based on the findings of a reliable in vitro chromosome aberration study in mammalian cells conducted on the substance, classification of the substance is not justified.

Based on the findings of a reliable in vivo mammalian somatic cell study conducted on the substance, classification of the substance is not justified.