Pidolic acid

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 2

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

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
- 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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

G1

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

G2

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

G3

Doses / concentrations 4

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

Examinations

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

Results and discussion

Test results

Test results 1

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

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.