Pidolic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th April 2021 to 19th November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th April 2021 to 19th November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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 No. : 310DEY
- Batch Manufactured By: Glentham Life Sciences
- Supplied by: Intracrop Limited Unit 21, Raleigh, Hall Industrial Estate Eccleshall,Stafford ST21 6JL,
United Kingdom.
- Purity as per Certificate of Analysis: 99.3 %
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions : Ambient (+ 15 to +25°C)
-Expiry Date : 23-08-2029
- The stability of the test item in the vehicle was established separately under Eurofins Advinus Study
No. G21489 at 1 and 200 mg/mL. Based on the results, the test item was found to be stable in the vehicle for up to 48 hours when stored at room temperature.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): dissolved in Milli-Q Water
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- As a solution in Milli-Q water

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animals or test system and environmental conditions
TEST ANIMALS
- Source: Hylasco Biotechnology Pvt. Ltd. Plot 4B, MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12-13 weeks.
- Weight at study initiation:
Males Females
G1: 435.80 ± 27.45 266.72 ± 14.44
G2: 430.83 ± 27.46 271.35 ± 14.96
G3: 429.46 ± 29.32 268.48 ± 14.59
G4: 430.95 ± 28.77 270.84 ± 14.63
G1R: 424.79 ± 30.72 278.21 ± 6.16
G4R: 425.55 ± 19.98 267.62 ± 19.44
- Housing:
Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually. During mating, two rats (one male and one female) were housed in standard polysulfone cages with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term (Gestation Day 20).
- Diet (e.g. ad libitum): Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum to the rats. The retained sample of the diet was discarded prior to finalization of the report.
- 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 Limited., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: The rats were acclimatized for five days before the pre-treatment period. During the acclimatization and pre-treatment period, animals were observed once daily for any abnormalities.
ENVIRONMENTAL CONDITIONS
- Rats were housed in an environment-controlled room. The temperature maintained during the experiment was between 19 to 24°C and relative humidity between 58 and 68 %. The photoperiod was a 12 hour light and 12 hours dark cycle. Adequate fresh air supply of 12.5– 12.7 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.
IN-LIFE DATES: From: 29 April 2021 To: 17 July 2021

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of the normal exposure in humans.
PREPARATION OF DOSING SOLUTIONS:
Required quantities of the test item were weighed in a pre-calibrated beaker* for each dose level separately and the vehicle [ Milli-Q® water] was added up to the pre-mark and mixed using a glass rod to get the final desired concentrations of 10, 30 and 100 mg/mL for the G2, G3 and G4/G4R groups, respectively. The suspensions were mixed well by using a glass rod.
VEHICLE
- Concentration in vehicle: The test item was weighed and suspended in vehicle [Milli-Q water] and administered at the graduated dose levels of 100, 300 and 1000 mg/kg bwt/day. The dose volume administered was 10 mL/kg bw/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The estimation of the test item concentration in the dose formulations were carried out by injecting the prepared sample and standard solutions into HPLC.
For active ingredient and test item concentration analysis, prepared formulation samples were obtained in duplicate sets on Day 1 and during Week 6 (Day 37) of treatment period and was analysed in-house. For each set, composite sample was drawn from each preparation and in case of control, duplicate composite samples were drawn. The analysis was done as per the method validated under Eurofins Advinus Study No.: G21489. One set of samples (first set) was analysed for test item concentration analysis and other set (second set) of samples was stored at ambient condition for reanalysis purpose as a backup. The backup samples were discarded as analysis results of the first set of samples were within the acceptable limits.
Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±10.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 52 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 43-61 days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 10 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Frequency of treatment:

once a day

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control (G1)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose (G2)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose (G3)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose (G4)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control recovery (G1R)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose recovery (G4R)

No. of animals per sex per dose:

Main groups : 10 males and 10 females
Recovery groups : 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels of 100 (G2), 300 (G3) and 1000 (G4/G4R) mg/kg bwt/day were selected for this study
based on the results of the 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No.
N5611) and in consultation with the Sponsor.
- Rationale for animal assignment (if not random): Animals were randomly distributed to different gro
ups by body weight stratification method using ProvantisTM Software (Version 10.1.0.1, Instem LSS,
Staffordshire ST15OSD, United Kingdom). Grouping was done one day prior to the start of treatment.
Rats with extreme body weights and not selected for the treatment were excluded from the study and
killed humanely under deep isoflurane anaesthesia.
- Fasting period before blood sampling for clinical biochemistry: At sacrifice, the parental males (Day
53), parental females (LD14) and the recovery animals (Day 68) were subjected to detailed necropsy
after overnight fasting (water allowed) and the study plan specified tissues were collected.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed for morbidity and mortality twice daily. All rats were observed
for clinical signs once daily during the treatment period and recovery period
DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals the
reafter (±1 day) during the treatment period.
- During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous
membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation,
piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to h
andling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming,
repetitive circling or bizarre behaviour like self-mutilation, walking backwards).
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight of males was recorded on Day 1 and at
weekly (±1 Day) intervals thereafter. Individual body weights of females were recorded on Day 1 and
at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
- All dams were weighed on GDs 0, 7, 14 and 20 and on LDs 0, 4 and 13.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g f
ood/kg body weight/day: Yes
- Food consumption (g) was measured at weekly intervals (±1 Day) during treatment and recovery
period. Food consumption was not measured during the cohabitation period. Food consumption of pr
egnant dams was measured on GD 7, 14 and 20 and on Days 4 and 13 of the lactation periods. Food
consumption was calculated by using the food consumed at each interval per cage and dividing by
the number of rats per cage and the number of days in the intervening period to determine the food
consumption (g/rat/day).

OTHER:
Functional Observation Battery Tests (FOB): The following neurological examinations were performed
on LD 13 for randomly selected 5 main group parental females, on Day 51 for randomly selected 5
main group parental males and at the end of the recovery period (Day 66) for all recovery group an
imals.
- Home Cage Observations: Each rat was observed in the home cage for posture and for presence or
absence of abnormal vocalizations, tremors and convulsions.
- Observations during Removal of Animal from Home Cage and Handling: The objective of this phase
of neurological examination was to observe the subject’s response to handling and to conduct other
procedures of the FOB that can best be performed when the rat is being held. Each rat was observed
for the following examinations:
-- ease of removal from home cage
-- handling reactivity
-- palpebral closure
-- eye examination
-- piloerection
-- lacrimation
-- salivation
-- skin/fur examination
-- perineum wetness
-- respiration
-- muscle tone and
-- extensor thrust response
The observations were recorded using scores/ranks.
- Open Field Observation: Rat was placed (one at a time) in an open arena, on a flat surface with a
clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each
group. During this observation period, the rat was evaluated as it moved about freely/unperturbed
and the following observations were made and was recorded using score/ranks:
-- gait
-- posture
-- tremors
-- mobility score
-- arousal level
-- clonic or tonic movements
-- stereotypic behaviour
-- bizarre behaviour
-- urination
-- defecation
-- rearing
-- abnormal vocalizations
- Functional Tests: Functional testing included motor activity, sensory evaluation, landing hindlimbs f
ootsplay and measurement of grip performance.
- Motor Activity: The motor activity of rats was measured using an automated animal activity m
easuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was
individually placed in the activity cages of the
instrument. The rats were monitored for 30 minutes. During this motor activity measurement session,
parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large amb
ulatory movement) in seconds,
distance travelled in inches and resting time in seconds were monitored. The Opto-Varimex 5 motor
activity measurement system provided the data analysed at 10 minutes interval and the same was
reported.
-2 Sensory Reactivity Measurements: After the 2 minutes (approximately) observation period, while
the rat was in the open field arena, the following tests were conducted. The rat was allowed to mov
e freely in the open field box for these tests but positioned in the box by the observer in order to
administer stimulus. During sensory reactivity measurements, rats were observed for following and t
he observations were recorded using scores/ranks:
-- approach response
-- touch response
-- click response
-- tail-pinch response
-- pupil response
-- aerial righting reflex
- Landing Hindlimbs Footsplay: The landing hindlimbs footsplay was assessed by dropping the rat
on to a horizontal surface of the tabletop from a short height and measured the distance between the
hind feet upon landing. The heel portion of each hind foot of each rat was gently pressed to an ink
pad just prior to testing. The rat was suspended in a prone position and then dropped from a height
of approximately 30 cm on a SOP format, which contained the details such as study no., animal no, g
roup, sex and date. A clean recording SOP format was used for each rat. A total of 3 readings (Trial 1,
Trial 2 and Trial 3) were recorded for each rat and average of 3 footsplay values are presented in the
report along with the individual footsplay values.
- Grip Performance: Hindlimbs and forelimbs grip performance was tested using dual grip strength
meter (Model: Columbus Instruments). Three trials (Trial 1, Trial 2 and Trial 3) were conducted for
each rat i.e., three trials each for forelimbs and hindlimbs. Average of three trials for both forelimbs
and hindlimbs were calculated and presented in the report along with the individual grip strength va
lues.
- Physiological Observations: Body temperature (rectal temperature) was measured in degree Cel
sius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was
measured.
Clinical Pathology:
- At the end of the pre-mating period, 5 males and 5 females randomly selected from the main groups
and all rats at the end of recovery period, were fasted overnight (water allowed) and approximately 3
mL of blood was collected by retro-orbital plexus puncture method under isoflurane anaesthesia.
- Haematology: The following hematological parameters were determined using Advia 2120i Hae
matology system (Siemens Healthcare Diagnostics Inc., NY, USA).
1 Red Blood Corpuscles
2 Haemoglobin
3 Haematocrit
4 Mean Corpuscular Volume
5 Mean Corpuscular Haemoglobin
6 Mean Corpuscular Haemoglobin Concentration
7 Reticulocytes count
8 White Blood Corpuscles
9 Differential leukocyte count
10 Platelets
Additionally, blood smears were prepared from the haematology (K2EDTA tube) samples at terminal
blood collections and stained with Giemsa stain from all animals. The differential leukocyte counts
and/ or RBC morphology were not performed by conventional microscopy as adequate interpretation
could be made with the automated counts. The smears were discarded at the time of final report
preparation.
- Coagulation Parameters: Blood samples collected for coagulation analysis were centrifuged at 2
500 times gravity (xg) at 15°C for 10 minutes for separation of plasma and analysed for the followin
g parameters in plasma sample using STart max coagulation analyzer (Diagnostica stago, 92600
Asnieres, France):
1 Prothrombin Time
2 Activated Partial Thromboplastin Time
- Clinical Chemistry: Plasma was separated after centrifugation of the whole blood samples at 5ºC,
4000 rpm for 5 minutes and analysed using Dimension RxL MaX Clinical Chemistry System (Dade
Behring Inc. Newark, DE 19714, USA) for the following parameters:
1 Alanine Aminotransferase
2 Albumin
3 Albumin/Globulin ratio [calculated values]
4 Alkaline phosphatase
5 Aspartate Amino Transferase
6 Blood Urea Nitrogen
7 Bile acids (Total)
8 Calcium
9 Chloride
10 Creatinine
11 Creatine
12 Gamma Glutamyl Transpeptidase
13 Glucose
14 Inorganic phosphorous
15 Potassium
16 Sodium
17 Total Bilirubin
18 Total Cholesterol
19 Total protein
20 Triglycerides
- Urinalysis: Urine was collected at the end of the pre-mating period, from randomly selected 5 males
and 5 females of main groups and at the end of recovery period from all rats, in urine collection
tubes. Each rat was placed overnight in a specially fabricated cage (water allowed) and the next
morning the collected urine was sent for analysis. Urine was analyzed for:
1 Specific gravity
2 Nitrite
3 pH
4 Proteins
5 Glucose
6 Ketone bodies
7 Urobilinogen
8 Bilirubin
9 Erythrocytes
10 Leucocytes
11 Appearance (colour and clarity)
12 Volume (approximate)
Urine samples were also subjected to microscopic examination for sediments such as crystals, epith
elial cells, erythrocytes, leukocytes and casts.
- Hormone Analysis: Blood samples were collected and serum was separated as per the following
schedule for the determination of total T4 and TSH hormones:
• At least two available pups per litter on LD 4.
• All dams prior to sacrifice and two available pups per litter on LD13.
• All adult main group males, prior to sacrifice.
Pup blood was pooled by litter for thyroid hormone analyses. Pups were anesthetized with isoflurane
and the jugular vein was incised at the neck region. The collected samples were pooled together
for each litter. Blood samples were collected in plain labeled tubes and were kept on bench top till
complete clot formation, before centrifugation. Serum was separated by centrifuging the whole blood
samples at 5000 rpm for 5 minutes at 5° C. The serum samples were placed in labeled plastic tubes
and stored at ~ -70 °C until they are analysed. The left-over samples from hormone analysis were d
iscarded. Blood samples were not collected from the not littered females and dams with all pups dead/
cannibalized during lactation; they were sacrificed without blood collection.
- Thyroid Profile Hormones: The following thyroid hormones were estimated by Enzyme Linked
Immuno Sorbent Assay (ELISA) method for the samples using BIO-RAD microplate washer and BIORAD model 680.
1 Rodent Thyroid Stimulating Hormone
2 Rodent Thyroxin
The kits manufactured by Endocrine Technologies Inc., USA were used for the assay. The method de
scribed in kit insert was followed. In brief:
1. Samples and standards were loaded to the microplate wells where antibody against analyte ho
rmone was available in solid phase. Antibody-enzyme conjugate was then added to the wells.
2. The mixture was incubated (3 h for TSH and 1 h for T4) to enable the binding of hormones with an
tibody and simultaneously with the antibody in the antibody-enzyme conjugate.
3. The incubated wells were washed, chromogen/ substrate was added followed by a brief incubation
for 20 minutes.
4. Stop solution was added for stopping the colour development reaction. The colour development wa
s read at wavelength (450nm).
5. Nonlinear Curve plot was derived using ODs from standards and calculation of unknowns using
curve plot & sample OD values.

Sacrifice and pathology:

SACRIFICE
- The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated
under isoflurane anesthesia.
GROSS NECROPSY
- All adult animals and pups were subjected to detailed necropsy and findings were recorded. For app
arently non-pregnant rats, the uteri were stained with 10% aqueous ammonium sulphide (Salewski
staining method) to identify the periimplantation loss of embryos (by staining the implantation sites) fo
r confirmation of pregnancy.
The number of implantation sites was recorded for all the dams.
HISTOPATHOLOGY / ORGAN WEIGHTS
- On completion of the gross pathology examination, the following tissues/organs were collected from
all adults (parents including recovery group
animals unless otherwise specified) and preserved in 10% neutral buffered formalin and the organ
weights as percentage of body weights were determined: All gross lesions, Cervix, Epididymides,
Ovaries, Oviducts, Uterus, Vagina, Prostate, Seminal vesicles with, coagulating glands, Testes,
Thyroid with parathyroid, Levator ani bulbocavernosus, muscle complex, Cowper’s glands, Glans pen
is.
In addition, the following tissues were collected, weighed and preserved from 5 males and 5 fem
ales randomly selected from the main groups and all animals from the recovery groups and organ
weights as percentage of body weights were determined: Brain (Cerebrum, Cerebellum and pons),
Bone marrow smear, Cecum, Colon, Duodenum, Eyes, Femoral Muscle, Femur/bone, femorotibial
Joint, Glands Adrenals, Glands Mammary, Gut associated lymphoid tissue, Heart, Ileum, Jejunum,
Kidneys, Liver, Lungs, Lymph nodes mandibular, Lymph nodes mesenteric, Pituitary, Rectum, Sciatic
nerve, Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum with marrow, Stomach, Thymus,
Trachea, Urinary bladder.
Tissues/organs (including reproductive organs) collected from 5 randomly selected males and 5
randomly selected females in the control and high dose groups were examined microscopically
for histopathological changes. Histopathological examination of the testes also involved qualitative
assessment of the stages of spermatogenesis.
In the absence of test item-related effects in the high dose group (including reproductive organs and
thyroid/parathyroid), histopathological evaluation was not extended to the remaining 5 animals in the
control, recovery or treatment .
The reproductive organs (as per the list in the table) of animals failing to mate were also examined.
All gross lesions were examined in all the groups.
The tissues were processed for routine paraffin embedding and 4 – 5 micron thickness sections were
stained with Mayer’s Haematoxylin Eosin stain. In addition, testes were sectioned at 3-4 µm and
stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. U
nused tissues were archived.

Other examinations:

Oestrous cyclicity (parental animals)
Vaginal smear was examined, and the stage of oestrous cycle was recorded daily for two weeks
before start of the treatment to select females with regular 4-5 days cyclicity for the study. The
vaginal smear was also examined daily from the beginning of the treatment period until evidence of
mating to determine the Day 0 of pregnancy/treatment-related effects on mating or precoital time. T
he time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as
the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous
cycle.
Litter observations
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On LD 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield,
as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of
male or female pups prevents having four of each sex per litter. Pups were not eliminated when the lit
ter size drop below the culling target (8 pups/litter).
PARAMETERS EXAMINED
- Each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed
for any external deformities and recorded.
- The number of pups born (litter size), sex and individual pup body weight of male and female pups
on LDs 0 and 4 were recorded.
- The ano-genital distance (AGD) of each pup was measured on LD 0 and pup body weight was reco
rded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root
of body weight.
- After standardization, the individual pup body weight was measured on LD13.
- The number of nipples/areolae in male pups was counted on LD 13.
- The litters were observed daily to note the number of alive, dead and cannibalized pups.
- In addition to daily clinical observations, all pups were observed for any abnormal behavior.
- Fertility index for dams, sires as well as the pup survival index until LD 4 was calculated.
GROSS EXAMINATION OF DEAD PUPS:
Yes
- All the dead and sacrificed pups were examined for malformations and subjected to gross pathologi
cal examination.
- All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention
was paid to the external genitals, which were examined for signs of altered development.
Postmortem examinations (offspring)
SACRIFICE
- The pups were sacrificed on LD 13 after examining the external genitals for signs of altered deve
lopment.
GROSS NECROPSY
- All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention
was paid to the external genitals, which were examined for
signs of altered development.
HISTOPATHOLOGY / ORGAN WEIGTHS
On LD 13, thyroid gland was collected from one male, one female and one pup from each litter (all ra
ndomly selected), and were preserved in 10% NBF for the histopathological examination. The thyroid
weighing for pups was performed after fixation. .

Statistics:

Data was captured using the ProvantisTM laboratory information management system (LIMS).
Parameters such as body weight, body weight change, body temperature, hindlimbshind limbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry, oestrous cycle, ano-genital distance, post implantation loss (%), no. of implantations, mean litter size, sex ratio, survival index, gestation length (days), pups data and transferred (grip strength, hindlimb footsplay,motor activity, thyroid profile) data was evaluated using the Levene’s Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of nonnormal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data like pre-coital interval, mating and fertility indices were analysed using Chi-square test. When Chi-square is found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests. Data captured outside of ProvantisTM: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0. For two groups, the comparison of means between treatment and control group was done using student’s t-test.
Descriptive statistics Mean, SD, Percentages & Numbers was presented by Treatment group and Day.
All hypothesis testing was carried out at the 5% (2-sided) significance level unless otherwise specified.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

serum/plasma biochemistry

serum/plasma hormone analyses

sperm measures

urinalysis

Key result

Critical effects observed:

no

Conclusions:

As there were no treatment related effects on systemic, reproduction and fertility parameters up
to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect L
evel (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental
Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Pidolic Acid is determined to
be 1000 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental
Toxicity Screening Test in Wistar rats by oral gavage was to determine the possible health hazards
likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides
initial information on possible effects of the test item on male and female reproductive performance such
as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This
study also provides information on reversibility, persistence or delayed occurrence of systemic toxic
effects, following 14 days post treatment.
The test item was weighed and suspended in vehicle [Milli-Q water] and administered at the graduated
dose levels of 100, 300 and 1000 mg/kg bwt/day for low dose (G2), mid dose (G3) and high dose
(G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control (G1)/vehicle
control recovery (G1R) groups received vehicle alone. The dose volume administered was 10 mL/kg
bw/day. Each main group in the experiment comprised of 10 male and 10 female rats and recovery
group comprised of 5 male and 5 female rats.
The dose formulations were administered once daily to a specific group of rats for two weeks prior to
mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day
(LD) 13 for females.
The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The
authenticity of the test item was not determined at the test facility. The stability of the test item in the
vehicle was established separately under Eurofins Advinus Study No. G21489 at 1 and 200 mg/mL.
Based on the results, the test item was found to be stable in the vehicle for up to 48 hours when stored
at room temperature.
During the conduct of this study, the prepared dose formulations were analysed for test item
concentration prior to dosing on Day 1 and during Week 6 (Day 37) of the treatment period. The
results indicated that the analysed concentrations were within ± 10 % of variations from the claimed
concentrations.
All rats were observed for clinical signs once daily. Body weight was recorded prior to the start
of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at
termination. Food consumption was recorded at weekly intervals except during the cohabitation period.
After confirmation of mating by vaginal smear, the dams were weighed on presumed Gestation Days
(GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20.
The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD
0, 4 and 13.
The number, survival and mortality of pups were observed during the lactation period. The body weight
and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted
by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup.
After standardization, the individual pup body weight was recorded on LD 13. All the surviving male
pups were examined for the appearance of nipples/areolae on LD 13.
Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and
randomly selected 5 main group males on treatment day 51 and towards the end of recovery period
(Day 66) for the recovery group animals.
Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were
performed on randomly selected 5 parental males and females from each main group at the end of
two weeks pre-mating period, towards the end of recovery period for all animals after overnight fasting.
Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group
males at termination, all dams on LD 13 and from available pups on LD 4 and 13.
At sacrifice, the parental males (Day 53), parental females (LD14) and the recovery animals (Day 68)
were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified
tissues were collected. The pups were sacrificed on LD 13 after examining the external genitals for
signs of altered development. Histopathology examination was carried out on the preserved organs from randomly selected 5
males and 5 females in the control and high dose groups (including reproductive organs) and on all
gross lesions. Histopathological examination of testes included a qualitative assessment of stages of
spermatogenesis. Thyroids were examined from all adult males and females and in LD13 pups. The
reproductive organs were examined from the non-pregnant females.
As there were no treatment related effects on systemic, reproduction and fertility parameters up to and
including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL)
for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening
Test by Oral Gavage in Wistar rats for the test item Pidolic Acid is determined to be 1000 mg/kg bwt/
day under the test conditions and doses employed.

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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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 No. : 310DEY
- Batch Manufactured By: Glentham Life Sciences
- Supplied by: Intracrop Limited Unit 21, Raleigh, Hall Industrial Estate Eccleshall,Stafford ST21 6JL, United Kingdom.
- Purity as per Certificate of Analysis: 99.3 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions : Ambient (+ 15 to +25°C)
-Expiry Date : 23-08-2029
- The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G21489 at 1 and 200 mg/mL. Based on the results, the test item was found to be stable in the vehicle for up to 48 hours when stored at room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): dissolved in Milli-Q Water

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

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also
recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this
strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology Pvt. Ltd. Plot 4B, MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12-13 weeks.
- Weight at study initiation:
Males Females
G1: 435.80 ± 27.45 266.72 ± 14.44
G2: 430.83 ± 27.46 271.35 ± 14.96
G3: 429.46 ± 29.32 268.48 ± 14.59
G4: 430.95 ± 28.77 270.84 ± 14.63
G1R: 424.79 ± 30.72 278.21 ± 6.16
G4R: 425.55 ± 19.98 267.62 ± 19.44

- Housing:
Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually.

During mating, two rats (one male and one female) were housed in standard polysulfone cages with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear (Day ‘0’ pregnancy), the mated pairs were separated.
Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term (Gestation Day 20).

- Diet (e.g. ad libitum): Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum to the rats. The retained sample of the diet was discarded prior to finalization of the report.
- 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 Limited., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period:
The rats were acclimatized for five days before the pre-treatment period. During the acclimatization and pre-treatment period, animals were observed once daily for any abnormalities.

ENVIRONMENTAL CONDITIONS
- Rats were housed in an environment-controlled room. The temperature maintained during the experiment was between 19 to 24°C and relative humidity between 58 and 68 %. The photoperiod was a 12 hour light and 12 hours dark cycle. Adequate fresh air supply of 12.5– 12.7 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

IN-LIFE DATES: From: 29 April 2021 To: 17 July 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of the normal exposure in humans.

PREPARATION OF DOSING SOLUTIONS:
Required quantities of the test item were weighed in a pre-calibrated beaker* for each dose level separately and the vehicle [ Milli-Q® water] was added up to the pre-mark and mixed using a glass rod to get the final desired concentrations of 10, 30 and 100 mg/mL for the G2, G3 and G4/G4R groups, respectively. The suspensions were mixed well by using a glass rod.

VEHICLE
- Concentration in vehicle:
The test item was weighed and suspended in vehicle [Milli-Q water] and administered at the graduated dose levels of 100, 300 and 1000 mg/kg bwt/day.
The dose volume administered was 10 mL/kg bw/day.

Details on mating procedure:

- Females were placed with males from the same group in a 1:1 ratio.
- Cohabitation was continued until there was evidence of sperms in the vaginal smear. All the females copulated successfully within seven days from the day of cohabitation.
- Subsequently, pregnant females were housed individually until LD 14.
- Not-littered females were sacrificed after 25 days from the day they were found sperm positive (by vaginal smear examination).
- The day of confirmed mating was designated as GD 0. The pre-coital time(days) was calculated for each female.
- After successful mating each pregnant female was housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term (Gestation Day 20).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The estimation of the test item concentration in the dose formulations were carried out by injecting the prepared sample and standard solutions into HPLC.
For active ingredient and test item concentration analysis, prepared formulation samples were obtained in duplicate sets on Day 1 and during Week 6 (Day 37) of treatment period and was analysed in-house. For each set, composite sample was drawn from each preparation and in case of control, duplicate composite samples were drawn
The analysis was done as per the method validated under Eurofins Advinus Study No.: G21489. One set of samples (first set) was analysed for test item concentration analysis and other set (second set) of samples was stored at ambient condition for reanalysis purpose as a backup. The backup samples were discarded as analysis results of the first set of samples were within the
acceptable limits.
Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±10.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 52 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.

Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 43-61 days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.

The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).

The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.

Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 10 mL/kg bwt.

The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Frequency of treatment:

once a day

Details on study schedule:

Females were placed with males from the same group in a 1:1 ratio.
Cohabitation was continued until there was evidence of sperms in the vaginal smear. All the females copulated successfully within seven days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. Not-littered females were sacrificed after 25 days from the day they were found sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as GD 0. The pre-coital time (days) was calculated for each female.
Study initiation date : 26 April 2021
Experimental starting date : 29 April 2021
Acclimatization : Start: 29 April 2021
End: 03 May 2021
Pre-treatment period : Start: 04 May 2021
End: 17 May 2021
Treatment : Start: 18 May 2021
End: 17 July 2021
Experiment completion : 28 August 2021
Submission of draft report : 31 August 2021
Study completion : 19 November 2021

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control (G1)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low dose (G2)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Mid dose (G3)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose (G4)

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control recovery (G1R)

Doses / concentrations 6

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose recovery (G4R)

No. of animals per sex per dose:

Main groups : 10 males and 10 females
Recovery groups : 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels of 100 (G2), 300 (G3) and 1000 (G4/G4R) mg/kg bwt/day were selected for this study based on the results of the 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No. N5611) and in consultation with the Sponsor.

- Rationale for animal assignment (if not random): Animals were randomly distributed to different groups by body weight stratification method using ProvantisTM Software (Version 10.1.0.1, Instem LSS, Staffordshire ST15OSD, United Kingdom). Grouping was done one day prior to the start of treatment. Rats with extreme body weights and not selected for the treatment were excluded from the study and killed humanely under deep isoflurane anaesthesia.

- Fasting period before blood sampling for clinical biochemistry: At sacrifice, the parental males (Day 53), parental females (LD14) and the recovery animals (Day 68) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed for morbidity and mortality twice daily. All rats were observed for clinical signs once daily during the treatment period and recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter (±1 day) during the treatment period.
- During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight of males was recorded on Day 1 and at weekly (±1 Day) intervals thereafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
- All dams were weighed on GDs 0, 7, 14 and 20 and on LDs 0, 4 and 13.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption (g) was measured at weekly intervals (±1 Day) during treatment and recovery period. Food consumption was not measured during the cohabitation period. Food consumption of pregnant dams was measured on GD 7, 14 and 20 and on Days 4 and 13 of the lactation periods. Food consumption was calculated by using the food consumed at each interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food consumption (g/rat/day).

OTHER:
Functional Observation Battery Tests (FOB): The following neurological examinations were performed on LD 13 for randomly selected 5 main group parental females, on Day 51 for randomly selected 5 main group parental males and at the end of the recovery period (Day 66) for all recovery group animals.
- Home Cage Observations: Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
- Observations during Removal of Animal from Home Cage and Handling: The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
-- ease of removal from home cage
-- handling reactivity
-- palpebral closure
-- eye examination
-- piloerection
-- lacrimation
-- salivation
-- skin/fur examination
-- perineum wetness
-- respiration
-- muscle tone and
-- extensor thrust response
The observations were recorded using scores/ranks.
- Open Field Observation: Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, the rat was evaluated as it moved about freely/unperturbed and the following observations were made and was recorded using score/ranks:
-- gait
-- posture
-- tremors
-- mobility score
-- arousal level
-- clonic or tonic movements
-- stereotypic behaviour
-- bizarre behaviour
-- urination
-- defecation
-- rearing
-- abnormal vocalizations
- Functional Tests: Functional testing included motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
- Motor Activity: The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the
instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds,
distance travelled in inches and resting time in seconds were monitored. The Opto-Varimex 5 motor activity measurement system provided the data analysed at 10 minutes interval and the same was reported.
-2 Sensory Reactivity Measurements: After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks:
-- approach response
-- touch response
-- click response
-- tail-pinch response
-- pupil response
-- aerial righting reflex
- Landing Hindlimbs Footsplay: The landing hindlimbs footsplay was assessed by dropping the rat on to a horizontal surface of the tabletop from a short height and measured the distance between the hind feet upon landing. The heel portion of each hind foot of each rat was gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on a SOP format, which contained the details such as study no., animal no, group, sex and date. A clean recording SOP format was used for each rat. A total of 3 readings (Trial 1, Trial 2 and Trial 3) were recorded for each rat and average of 3 footsplay values are presented in the report along with the individual footsplay values.
- Grip Performance: Hindlimbs and forelimbs grip performance was tested using dual grip strength meter (Model: Columbus Instruments). Three trials (Trial 1, Trial 2 and Trial 3) were conducted for each rat i.e., three trials each for forelimbs and hindlimbs. Average of three trials for both forelimbs and hindlimbs were calculated and presented in the report along with the individual grip strength values.
- Physiological Observations: Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was measured.

Clinical Pathology:
- At the end of the pre-mating period, 5 males and 5 females randomly selected from the main groups and all rats at the end of recovery period, were fasted overnight (water allowed) and approximately 3 mL of blood was collected by retro-orbital plexus puncture method under isoflurane anaesthesia.
- Haematology: The following hematological parameters were determined using Advia 2120i Haematology system (Siemens Healthcare Diagnostics Inc., NY, USA).
1 Red Blood Corpuscles
2 Haemoglobin
3 Haematocrit
4 Mean Corpuscular Volume
5 Mean Corpuscular Haemoglobin
6 Mean Corpuscular Haemoglobin Concentration
7 Reticulocytes count
8 White Blood Corpuscles
9 Differential leukocyte count
10 Platelets
Additionally, blood smears were prepared from the haematology (K2EDTA tube) samples at terminal blood collections and stained with Giemsa stain from all animals. The differential leukocyte counts and/ or RBC morphology were not performed by conventional microscopy as adequate interpretation could be made with the automated counts. The smears were discarded at the time of final report preparation.
- Coagulation Parameters: Blood samples collected for coagulation analysis were centrifuged at 2500 times gravity (xg) at 15°C for 10 minutes for separation of plasma and analysed for the following parameters in plasma sample using STart max coagulation analyzer (Diagnostica stago, 92600 Asnieres, France):
1 Prothrombin Time
2 Activated Partial Thromboplastin Time
- Clinical Chemistry: Plasma was separated after centrifugation of the whole blood samples at 5ºC, 4000 rpm for 5 minutes and analysed using Dimension RxL MaX Clinical Chemistry System (Dade Behring Inc. Newark, DE 19714, USA) for the following parameters:
1 Alanine Aminotransferase
2 Albumin
3 Albumin/Globulin ratio [calculated values]
4 Alkaline phosphatase
5 Aspartate Amino Transferase
6 Blood Urea Nitrogen
7 Bile acids (Total)
8 Calcium
9 Chloride
10 Creatinine
11 Creatine
12 Gamma Glutamyl Transpeptidase
13 Glucose
14 Inorganic phosphorous
15 Potassium
16 Sodium
17 Total Bilirubin
18 Total Cholesterol
19 Total protein
20 Triglycerides
- Urinalysis: Urine was collected at the end of the pre-mating period, from randomly selected 5 males and 5 females of main groups and at the end of recovery period from all rats, in urine collection tubes. Each rat was placed overnight in a specially fabricated cage (water allowed) and the next morning the collected urine was sent for analysis. Urine was analyzed for:
1 Specific gravity
2 Nitrite
3 pH
4 Proteins
5 Glucose
6 Ketone bodies
7 Urobilinogen
8 Bilirubin
9 Erythrocytes
10 Leucocytes
11 Appearance (colour and clarity)
12 Volume (approximate)
Urine samples were also subjected to microscopic examination for sediments such as crystals, epithelial cells, erythrocytes, leukocytes and casts.
- Hormone Analysis: Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 and TSH hormones:
• At least two available pups per litter on LD 4.
• All dams prior to sacrifice and two available pups per litter on LD13.
• All adult main group males, prior to sacrifice.
Pup blood was pooled by litter for thyroid hormone analyses. Pups were anesthetized with isoflurane and the jugular vein was incised at the neck region. The collected samples were pooled together for each litter. Blood samples were collected in plain labeled tubes and were kept on bench top till complete clot formation, before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 5° C. The serum samples were placed in labeled plastic tubes and stored at ~ -70 °C until they are analysed. The left-over samples from hormone analysis were discarded. Blood samples were not collected from the not littered females and dams with all pups dead/cannibalized during lactation; they were sacrificed without blood collection.
- Thyroid Profile Hormones: The following thyroid hormones were estimated by Enzyme Linked Immuno Sorbent Assay (ELISA) method for the samples using BIO-RAD microplate washer and BIO-RAD model 680.
1 Rodent Thyroid Stimulating Hormone
2 Rodent Thyroxin
The kits manufactured by Endocrine Technologies Inc., USA were used for the assay. The method described in kit insert was followed. In brief:
1. Samples and standards were loaded to the microplate wells where antibody against analyte hormone was available in solid phase. Antibody-enzyme conjugate was then added to the wells.
2. The mixture was incubated (3 h for TSH and 1 h for T4) to enable the binding of hormones with antibody and simultaneously with the antibody in the antibody-enzyme conjugate.
3. The incubated wells were washed, chromogen/ substrate was added followed by a brief incubation for 20 minutes.
4. Stop solution was added for stopping the colour development reaction. The colour development was read at wavelength (450nm).
5. Nonlinear Curve plot was derived using ODs from standards and calculation of unknowns using curve plot & sample OD values.

Oestrous cyclicity (parental animals):

Vaginal smear was examined, and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or precoital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On LD 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter).

PARAMETERS EXAMINED
- Each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
- The number of pups born (litter size), sex and individual pup body weight of male and female pups on LDs 0 and 4 were recorded.
- The ano-genital distance (AGD) of each pup was measured on LD 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight.
- After standardization, the individual pup body weight was measured on LD13.
- The number of nipples/areolae in male pups was counted on LD 13.
- The litters were observed daily to note the number of alive, dead and cannibalized pups.
- In addition to daily clinical observations, all pups were observed for any abnormal behavior.
- Fertility index for dams, sires as well as the pup survival index until LD 4 was calculated.

GROSS EXAMINATION OF DEAD PUPS:
Yes
- All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
- All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external genitals, which were examined for signs of altered development.

Postmortem examinations (parental animals):

SACRIFICE
- The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anesthesia.

GROSS NECROPSY
- All adult animals and pups were subjected to detailed necropsy and findings were recorded. For apparently non-pregnant rats, the uteri were stained with 10% aqueous ammonium sulphide (Salewski staining method) to identify the periimplantation loss of embryos (by staining the implantation sites) for confirmation of pregnancy.
The number of implantation sites was recorded for all the dams.

HISTOPATHOLOGY / ORGAN WEIGHTS
- On completion of the gross pathology examination, the following tissues/organs were collected from all adults (parents including recovery group
animals unless otherwise specified) and preserved in 10% neutral buffered formalin and the organ weights as percentage of body weights were determined: All gross lesions, Cervix, Epididymides, Ovaries, Oviducts, Uterus, Vagina, Prostate, Seminal vesicles with, coagulating glands, Testes, Thyroid with parathyroid, Levator ani bulbocavernosus, muscle complex, Cowper’s glands, Glans penis.

In addition, the following tissues were collected, weighed and preserved from 5 males and 5 females randomly selected from the main groups and all animals from the recovery groups and organ weights as percentage of body weights were determined: Brain (Cerebrum, Cerebellum and pons), Bone marrow smear, Cecum, Colon, Duodenum, Eyes, Femoral Muscle, Femur/bone, femorotibial Joint, Glands Adrenals, Glands Mammary, Gut associated lymphoid tissue, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes mandibular, Lymph nodes mesenteric, Pituitary, Rectum, Sciatic nerve, Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum with marrow, Stomach, Thymus, Trachea, Urinary bladder.

Tissues/organs (including reproductive organs) collected from 5 randomly selected males and 5 randomly selected females in the control and high dose groups were examined microscopically for histopathological changes. Histopathological examination of the testes also involved qualitative assessment of the stages of spermatogenesis.
In the absence of test item-related effects in the high dose group (including reproductive organs and thyroid/parathyroid), histopathological evaluation was not extended to the remaining 5 animals in the control, recovery or treatment .
The reproductive organs (as per the list in the table) of animals failing to mate were also examined.
All gross lesions were examined in all the groups.
The tissues were processed for routine paraffin embedding and 4 – 5 micron thickness sections were stained with Mayer’s Haematoxylin Eosin stain. In addition, testes were sectioned at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.

Postmortem examinations (offspring):

SACRIFICE
- The pups were sacrificed on LD 13 after examining the external genitals for signs of altered development.

GROSS NECROPSY
- All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external genitals, which were examined for
signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGTHS
On LD 13, thyroid gland was collected from one male, one female and one pup from each litter (all randomly selected), and were preserved in 10% NBF for the histopathological examination. The thyroid weighing for pups was performed after fixation. .

Statistics:

Data was captured using the ProvantisTM laboratory information management system (LIMS).
Parameters such as body weight, body weight change, body temperature, hindlimbshind limbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry, oestrous cycle, ano-genital distance, post implantation loss (%), no. of implantations, mean litter size, sex ratio, survival index, gestation length (days), pups data and transferred (grip strength, hindlimb footsplay,motor activity, thyroid profile) data was evaluated using the Levene’s Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of nonnormal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data like pre-coital interval, mating and fertility indices were analysed using Chi-square test. When Chi-square is found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests.
Data captured outside of ProvantisTM: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0.
For two groups, the comparison of means between treatment and control group was done using student’s t-test.
Descriptive statistics Mean, SD, Percentages & Numbers was presented by Treatment group and Day.
All hypothesis testing was carried out at the 5% (2-sided) significance level unless otherwise specified.

Reproductive indices:

1. Male mating index (%)
= Number of males with evidence of mating x100/Number of males cohabited

2. Male fertility index (%)
= Number of males siring a litter/impregnated a female x100//Number of males with evidence of mating

3.Female mating index (%)
= Number of females mated x100//Number of females cohabited

4. Female fertility index (%)
= Number of pregnant females x100//Number of females with evidence of mating

5. Mean number of implantations/group

= Total number of implantations/Total number of pregnant animals

6. Post implantation loss (%)
= Number of implantations - Number of live pups x100//Number of implantations

Offspring viability indices:

1. Mean litter size per group
= Total Number of pups born/ Total Number of littered animals

2. Mean viable litter
= No. of viable pups/Total Number of littered animals

3. Live birth index (%)
= No. of viable pups born (at first observation) x100//Total no. of pups born (at first observation)

4. Day 4 survival index (%)

= Number of viable pups on lactation Day 4 x100//Number of viable pups born

5. Sex Ratio/ Percentage of male offspring (%)
= No. of male pups born x100//Total no. of pups born

6. Ano-genital Distance Ratio (mm/g1/3 )
= Ano-genital distance/ Cube root of body weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

neuropathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Pidolic Acid is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, following 14 days post treatment.

The test item was weighed and suspended in vehicle [Milli-Q water] and administered at the graduated dose levels of 100, 300 and 1000 mg/kg bwt/day for low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 10 mL/kg bw/day. Each main group in the experiment comprised of 10 male and 10 female rats and recovery group comprised of 5 male and 5 female rats.

The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.
The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G21489 at 1 and 200 mg/mL. Based on the results, the test item was found to be stable in the vehicle for up to 48 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during Week 6 (Day 37) of the treatment period. The results indicated that the analysed concentrations were within ± 10 % of variations from the claimed concentrations.
All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly intervals except during the cohabitation period.

After confirmation of mating by vaginal smear, the dams were weighed on presumed Gestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20.

The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 0, 4 and 13.

The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13.

Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 51 and towards the end of recovery period (Day 66) for the recovery group animals.

Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and females from each main group at the end of two weeks pre-mating period, towards the end of recovery period for all animals after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13.

At sacrifice, the parental males (Day 53), parental females (LD14) and the recovery animals (Day 68) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external genitals for signs of altered development.

Histopathology examination was carried out on the preserved organs from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. Thyroids were examined from all adult males and females and in LD13 pups. The reproductive organs were examined from the non-pregnant females.

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Pidolic Acid is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.