2,5-di-tert-butylhydroquinone

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 October 2019 - 22 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,5-di-tert-butylhydroquinone

EC Number:

201-841-8

EC Name:

2,5-di-tert-butylhydroquinone

Cas Number:

88-58-4

Molecular formula:

C14H22O2

IUPAC Name:

2,5-di-tert-butylhydroquinone

Test material form:

solid: crystalline

Specific details on test material used for the study:

Name of the Test Item : YAPOX 2245
Chemical Name (IUPAC) : 2,5-Di-tertiary butyl hydroquinone
CAS No. : 88-58-4
Physical Appearance
(with color) : White to light tan crystalline powder
Batch No. : 20015011219
Purity
(as per Certificate of Analysis) : 99.37%
Batch Produced by
(Name and address) : Yasho Industries Limited, Plot no. 2514/2515,
Phase IV, G.I.D.C., Vapi - 396195, Gujarat,
India
Date of Manufacture : June 2019
Date of Expiry : May 2021
Storage Conditions : Ambient (21 to 29 oC)
Test Item code by Test facility : D819-002

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: Minimum 9 to 11 weeks
- Weight at study initiation: (P) Males: 241 and 298 g; Females:180 and 222 g
- Fasting period before study: No
- Housing: in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill
i. Pre mating
Per cage two animals of the same sex and group was housed.
ii. Mating
During mating, two animals (one male and one female) of the same group was housed.
iii. Post mating
After confirming presence of sperm in the vaginal smear and/or vaginal plugs (Day 0 of pregnancy),
the mated pairs were separated. Males were housed with their former cage mates while females were
housed individually. Sterilized paper shreds will be provided as a nesting material from gestation day 20 onwards.
Maximum of three animals of same sex will be housed for recovery group animals.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum throughout the study
- Water (e.g. ad libitum): ad libitum, Deep bore-well water passed through reverse osmosis unit was provided.
- Acclimation period: 4 - 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70 %.
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item/vehicle was administered by the oral (gavage) route using a stainless steel intubation cannula attached to a disposable syringe. All the doses were administered in an equivolume of 5 mL/kg with the concentration of 3.5, 7 and 14 mg/mL for low, mid and high dose groups, respectively. Vehicle was administered to the vehicle control group at an equivolume of 5 mL/kg body weight. The actual dose volume for each animal was calculated based on the most recent body weight. The test item formulations were administered as soon as possible after preparation.

Vehicle:

corn oil

Details on oral exposure:

The test item formulations will be freshly prepared before dose administration on each treatment day.
The required quantity of test item will be weighed in aluminium foil, transferred to mortar and triturated well in a mortar using pestle. A small quantity of vehicle will be added and grind well until a homogenous suspension is formed and thereafter the entire quantity of the formulation will be transferred into measuring cylinder. A small quantity of vehicle will be added to rinse the mortar and this will be transferred into the measuring cylinder. The rinsing procedure of mortar and pestle will be repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume will be made up to required quantity with vehicle to get desired concentration of 3.5, 7 and 14 mg/mL of test item for low, mid and high dose groups respectively.
The test formulations will be maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations.
Below is an example table for dose formulation preparation;
Group Group Description Dose(mg/kg body weight/day) Concentration(mg/mL) Dose Volume (mL/kg body weight) Quantity of Test Item (mg) Volume made up with Vehicle
(mL)
G1 Vehicle Control 0 0 5 0 50
G2 Mid Dose 17.5 3.5 5 175 50
G3 Low Dose 35 7 5 350 50
G4 High Dose 70 14 5 700 50

The quantity of the test item taken and the volume prepared may vary depending on the requirement. The exact formulation preparation details will be recorded in the raw data.

VEHICLE
The corn oil will be used as vehicle for test item formulations as per in-house solubility/suspendibility test results. The test item is insoluble in distilled water and not suspended uniformly in 0.5 % w/v Carboxy Methyl Cellulose at the concentration of 20 mg/mL (considering the dose volume of 10 mL/kg body weight). The test item is uniformly suspended in corn oil at the concentration of 40 mg/mL (the highest dose concentration selected for the range finding considering the dose volume of 5 mL/kg body weight) as per in-house solubility/suspendibility test results.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis for dose concentration verification was done by Analytical
Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods
detailed in the Study Plan No. BIO-ANM 1478 and the results were presented in the report. Sampling
and analysis of formulations were performed during week 1 and week 4/5 of the treatment. The sa
mples were collected in duplicates from top, middle and bottom layers from low, mid and high dose c
oncentrations and in duplicates from single layer from vehicle control.
The collected samples were transferred to Analytical Chemistry department of Bioneeds India Privat
e Limited for dose formulation analysis. One set of aliquot of each formulation was analyzed. The s
econd aliquot was stored as a backup purpose at established stability conditions. Formulations were
considered acceptable, if mean results are within the range of 85 to 115% of the nominal concentra
tion and the relative standard deviation (% RSD) was ≤10%.
If the dose formulation analysis results failed to meet these criteria, the analysis was repeated using
second set of samples or samples of subsequent preparation during the treatment period.

Duration of treatment / exposure:

Males: The males will be treated for two weeks pre-mating, during mating and up to the day before sa crifice during the post-mating period (total of at least completion of 28 days of treatment).
Females: The females will be treated for a two week pre-mating period, during mating, pregnancy(gestation) and up to lactation day 13 after which the pups will be sacrificed on lactation day 13.
Females (dams) will be sacrificed on lactation day 14 after overnight fasting (water allowed).

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Remarks:

70 mg/kg bw/day dose from day 1 to 6. Reduced dose 50 mg/kg bw/day from day 7 till end due to severe clinical signs

No. of animals per sex per dose:

6 groups

Main Groups:
G1 - Vehicle control
G2 - Low dose
G3 - Mid dose
G4 - High dose
Recovery Groups:
G1R - Vehicle Control Recovery Group
G4R - High dose Recovery Group

Main Group: 24 (12 Males + 12 Females)
Recovery Group: 10 (5 males + 5 females)

Total number: 116 (58 Males + 58 Females)

A total of 68 females will be received and evaluated pre-exposure for estrous cyclicity and females that will fail to exhibit typical 4-5 day cycles will be excluded from the experiment and will be sacrificed after initiation of the treatment.

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection Rationale:
There was no information available on repeated dose toxicity of test item 2, 5-Di-tertiary butyl hydroquinone. However, as per ECHA registration dossier, animals treated at a dose level of 50 mg/kg showed expected gains in body weight. In necropsy, hemorrhagic non-glandular epithelium of the stomach was noted at necropsy of the animal treated at a dose level of 300 mg/kg. No abnormalities were noted at necropsy of animals treated at a dose level of 50 mg/kg. The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be in the range of 50 - 300 mg/kg bodyweight (Globally Harmonized Classification System - Category 3). Signs of systemic toxicity noted in the animal treated at a dose level of 300 mg/kg were ataxia, hunched posture, lethargy, piloerection, ptosis, occasional body tremors, tiptoe gait, emaciation, pallor of the extremities, hypothermia and elevated tail. There were no signs of systemic toxicity noted at a dose level of 50 mg/kg. The animal treated at a dose level of 300 mg/kg was killed for humane reasons, 1 day after dosing, due to the occurrence of clinical signs of toxicity that approached the severity limit.
Based on the above mentioned details, the doses of 0, 50, 100 and 200 mg/kg body weight were selected as control, low, mid and high dose groups in consultation with sponsor for 14-day range finding study with the test item 2, 5-Di-tertiary butyl hydroquinone [Bioneeds Study No. BIO-CTX 109]. The dose of 50 mg/kg body weight revealed treatment related clinical signs of toxicity, treatment related reduction in body weight and feed consumption during week 1 and were found recovered during week 2 in females only. There were no effects on other parameters like, clinical pathology, organ weight and gross pathological observations in either sex. The dose of 100 mg/kg body weight revealed treatment related clinical signs of toxicity in either sex, treatment related mortalities, treatment related reduction in body weight and feed consumption and treatment related effects in organ weights and treatment related gross pathological changes in either sex. The dose of 200 mg/kg body weight revealed treatment related clinical signs of toxicity in either sex, treatment related mortalities, treatment related reduction in body weight and feed consumption, treatment related effects in organ weights and treatment related gross pathological changes in either sex.
Based on the results obtained from the range finding study, the doses of 0, 17.5, 35 and 70 mg/kg body weight were selected as control, low, mid and high dose groups in consultation with sponsor for present Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of YAPOX 2245 by Oral Gavage in Sprague Dawley Rats.

Positive control:

Not needed

Examinations

Observations and examinations performed and frequency:

Clinical Signs of Toxicity and Mortality/Morbidity
At least once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

Detailed Clinical Examination
On day 1 before treatment and weekly thereafter (may vary by ± 1 or 2 days) during treatment. Signs
noted included, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of
secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and
unusual respiratory pattern.

Body Weight
At receipt, on the first day of dosing, at least weekly thereafter and at termination. The females were
weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during the
lactation period. The recovery group animals were weighed at receipt, on the first day of dosing, at
least weekly thereafter and at termination.

Feed Consumption
Cage wise feed consumption was measured for main group animals once a week during premating
and weekly once for main group males during the post mating period. Feed consumption was not
measured during the mating period for both males and females. Thereafter, feed consumption for
females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4,
4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals once a week throu
ghout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amo
unt of feed offered and left over in each cage and the number of rats per cage.

Ophthalmological Examination
Once before treatment for all animals, at the end of the dosing period for males (Treatment Day 34)
and during the lactation period for females (Lactation Day 13) of vehicle control and high dose main
group animals and during the last week for the recovery group animals (Day 64). The examination
was not extended to lower dose groups as there were no changes noted in high dose group animals.

Estrous Cyclicity
Estrous cycles were monitored during the acclimatization to evaluate its normal estrous cyclicity (4 to
5 days). Only females with normal oestrous cyclicity were selected for the treatment. Vaginal smears
were monitored daily from the beginning of the treatment period until evidence of mating. When obt
aining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseu
dopregnancy. Estrous cyclicity was also monitored on the day of sacrifice for females.
Recovery group females were not evaluated for oestrous cyclicity.

Neurological/Functional Examination
Performed for five males and five females, randomly selected from each group, towards the end of
the dosing period for males (day 35) and during the lactation period for females (lactation day 13).
Neurological/Functional examination was performed for both recovery group animals towards the end
of the recovery period (day 63).

Clinical Pathology
Blood samples were collected from all the main group animals for measurement of serum T4 levels
on the following schedule:
a. Two pups per litter on lactation day 4
b. All dams at termination (lactation day 14)
c. Two pups per litter (same sex) at termination (lactation day 13)
d. All adult males, at termination (after completion of at least 28 days of treatment).
The clinical pathology (haematological and clinical biochemistry) examinations was conducted in
five males and five females randomly selected from each main group and from all recovery group
animals.
Urine were collected from five randomly selected males of each main group and for all recovery ani
mals at termination.

Necropsy
The males were sacrificed after completion of at least 35 days of treatment, females were sacrificed
on lactation day 14 and recovery animals were sacrificed at least after completion of 14 days observ
ation from the first scheduled sacrifice of dams. The organs were collected and Relative organ wei
ghts were calculated against fasting body weight.

Histopathology
Histopathological examination was conducted on all the tissues collected from the vehicle control and
high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads
and histopathology of interstitial testicular cell structure) including all macroscopically abnormal tissu
es of all animals, whether died during the experiment or sacrificed at termination.
All organs and tissue samples were processed, embedded in paraffin, sectioned at a thickness of
3 to 4 micrometers and stained with hematoxylin and eosin. The testes was sectioned at 3 to 4
micrometers and stained with Hematoxylin and eosin stain and also with Periodic Acid-Schiff (PAS)
and hematoxylin stain. PAS stain aided spermatogenesis evaluation
Bone marrow smear (from one femur) was prepared at the time of necropsy. The bone marrow smear
was fixed in methanol and stained with Giemsa Stain.


Sperm parameters
The testes were sectioned at 3 to 4 micrometers and stained with Hematoxylin and eosin stain and
also with Periodic Acid-Schiff (PAS) and hematoxylin stain. PAS stain will aid spermatogenesis
evaluation.

Sacrifice and pathology:

SACRIFICE
The animals were euthanized using deep Isoflurane anaesthesia/CO2 followed by exsanguination and subjected to gross necropsy, external and internal gross pathological examination. The males were sacrificed after completion of at least 28 days of treatment. The females were sacrificed on lactation day 14 and recovery animals were sacrificed at least after completion of 14 days observation from the first scheduled sacrifice of dams. The pups were sacrificed on lactation day 13

GROSS NECROPSY
- Gross necropsy consisted of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.
The organs identified in the table below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

Organs Weighed at Necropsy for all selected animals:
Testes
Epididymides
Levator ani plus bulbocavernosus muscle complex
Cowper’s glands
Glans penis
Eye
Liver
Kidneys
Adrenals
Thymus
Spleen
Brain (cerebrum, cerebellum and pons)
Heart
Ovaries
Prostate
Seminal vesicles with coagulation glands
Organs showing macroscopic lesions
All gross lesions
Spinal cord
Stomach
Duodenum
Jejunum
Ileum with Peyer’s patches
Caecum
Colon
Rectum
Thyroid along with parathyroidW
Trachea
Lungsa
Uterus with cervix
Urinary bladder
Mandibular Lymph nodes
Mesenteric Lymph nodes
Sciatic nerve
Bone marrow smear
Skeletal muscle
Femur bone
Vagina

HISTOPATHOLOGY
Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) including all macroscopically abnormal tissues of all animals, whether died during the experiment or sacrificed at termination.

Statistics:

The data was subjected to various statistical analyses using SPSS software version 22. All analysis and comparisons will be evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests and will be designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.
The statistical analysis was followed, but not limited to the parameters, as mentioned below
Parameter Type of Analysis
• Body weight (weekly body weights and gestation/lactation body weights)
• Percent Change in body weight (weekly body weights and gestation/lactation body weights)
• Feed consumption (weekly and gestation/lactation)
• Copulatory interval
• Gestation length
• Hematology
• Clinical chemistry
• Urinalysis (whichever applicable)
• Absolute/relative organ weights
• Functional Observation parameters (whichever applicable)
• Anogenital distance ratio
• Nipple retention (if any)
• Mean pup weight
• Serum T4 values Parametric - One-way ANOVA with Dunnett’s post test

• Implantations/dam
• No. of pups/dam
• Sex ratio
• Litter size
• No. of early resorptions
• No. of late resorptions
• Pups abnormalities (if any)
• Pre implantation loss
• Pre natal loss
• Post natal loss
• Post implantation loss Non Parametric - Kruskal-Wallis followed by the Mann-Whitney test if differences were indicated

• Pregnancy rate
• No. of dams with/without live pups
• No. of dams with/without dead pups
• No. of litters with/without resorptions Cross Tabs -Chi-square test/ Fischer's Exact Test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity and no mortality/morbidity observed in any of the animals from group G1/G1R (0 mg/kg body weight/day), G2 (17.5 mg/kg body weight/day) and G3 (35 mg/kg body weight/day) of both the sex throughout the experimental period. The detailed clinical examination conducted weekly once for all the animals from group G1/G1R (0 mg/kg body weight/day), G2 (17.5 mg/kg body weight/day) and G3 (35 mg/kg body weight/day) of both the sex did not reveal any changes throughout the experimental period.
In group G4/G4R, the animals of both sex did not reveal any clinical signs on day 1 after dose administration and started to reveal test item-related clinical signs from day 2. The test item-related severe clinical signs of toxicity, reduced body weight and feed consumption initially along with one mortality from females.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female was found dead from group G4 on day 9 during evening observation. However, all the males from group G4/G4R and eleveln survived females from group G4 were completely free from clinical signs from day 21 onwards to till termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group G4/G4R, test item-related reduction in mean body weight and percent change in mean body weight gain with respect to day 1 noted initially when compared with concurrent vehicle control group, as the animals were noted with severe test item-related clinical signs when dosed with 70 mg/kg body weight/day till day 6 and later the dose was reduced to 50 mg/kg body weight/day. These noted reductions in mean body weight are statistically significant on days 7 & 14 in group G4 females, on days 14, 21 & 28 in group G4R males, on days 7, 14 & 21 in group G4R females; the noted reductions in mean percent change in body weight with respect to day 1 are statistically significant during day 1 to 7 & 1 to 14 in group G4 females, during day 1 to 7 in group G4R males and during day 1 to 7, 1 to 14 & 1 to 21 in group G4R females.
However, the group G4/G4R animals of both sex were found recover from the test item-related reduction in body weight/body weight gain later in the study and noted with normal increase when compared with concurrent vehicle control group as the dose was reduced to 50 mg/kg body weight from 70 mg/kg body weight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In group G4 animals, test item-related reduction in mean feed consumption was noted when compared with concurrent vehicle control group during pre-mating period (first 2 weeks), as the animals were noted with severe test item-related clinical signs when dosed with 70 mg/kg body weight/day till day 6 and later the dose was reduced to 50 mg/kg body weight/day. These reductions are statistically significant during week 1 and 2 in both the sex. The feed consumption was not measured during mating period for group G4 animals.
In group G4R animals, test item-related reduction in mean feed consumption was noted when compared with concurrent vehicle control group during treatment period, as the animals were noted with severe test item-related clinical signs when dosed with 70 mg/kg body weight/day till day 6 and later the dose was reduced to 50 mg/kg body weight/day. These reductions are statistically significant during week 1, 2, 3, 6 & 7 in G4R males and during week 1 & 2 in G4R females.
However, the group G4R animals of both sex were found recover from the test item-related reduction in feed consumption later in the study and noted with normal consumption when compared with concurrent vehicle control group as the dose was reduced to 50 mg/kg body weight from 70 mg/kg body weight.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in vehicle control (G1) and high dose (G4) group animals of both the sex during the ophthalmological examination conducted towards end of the dosing period (for main group males on day 35 and for main group females on lactation day 13). Therefore, the ophthalmological examination was not extended to mid and low dose group animals.
There were no ocular changes observed in vehicle control recovery (G1R) and high dose recovery (G4) group animals of both the sex during the ophthalmological examination conducted towards end of the recovery period (on day 65).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in haematology parameters at any of the tested dose group animals of both sex in either main or recovery groups when compared with concurrent vehicle control groups.

However, the following statistically significant changes were noted in haematology parameters when compared with their concurrent control groups:
- decrease in mean corpuscular hemoglobin concentration (MCHC) at group G4 males;
- increase in percent basophils at group G3 males;
- decrease in activated partial thromboplastin time (APTT) at group G2 males;
- increase in haematocrit (HCT) at group G4 females;
- decrease in absolute eosinophils at group G4R males
These observed changes are considered as incidental and unrelated to treatment, as these changes are lacking dose dependency and also the obtained values are within in-house historical control data range of same species and strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in clinical chemistry parameters at any of the tested dose group animals of both sex in either main or recovery groups when compared with concurrent vehicle control groups.

However, the following statistically significant changes were noted in clinical chemistry parameters when compared with their concurrent control groups:
- decrease in total cholesterol levels at group G2 males, G3 females and G4R males
- increase in total cholesterol levels at group G4R males;
- decrease in calcium levels at group G2, G3 and G4 males;
- increase in chloride levels at group G4 males;
- decrease in glucose levels at group G4R females;
- decrease in alanine aminotransferase levels at group G4R females
These observed changes are considered as incidental and unrelated to treatment, as these changes are lacking dose dependency and also the obtained values are within in-house historical control data range of same species and strain.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in urinalysis parameters at any of the tested dose group animals of both main or recovery groups when compared with concurrent vehicle control groups.

However, statistically significant reduction in mean urine volume at group G3 males and statistically significant reduction in mean specific gravity at group G4R females were noted when compared with their concurrent control groups. These noted changes are considered as incidental and unrelated to treatment, as these changes are lacking dose dependency and also the obtained values are within in-house historical control data range of same species and strain.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in neurological/functional examinations like home cage, handling, open field, sensory, neuromuscular (hind limb foot splay), physiological observations, assessment of grip strength and assessment of motor activity in any of the males (five randomly selected) from tested dose groups (G2, G3 and G4) performed towards end of the dosing period (day 35) and in any of the females (five randomly selected) from tested dose groups (G2, G3 and G4) performed towards end of the dosing period (lactation day 13) when compared with concurrent vehicle control group.
There were no test item-related changes noted in neurological/functional examinations like home cage, handling, open field, sensory, neuromuscular (hind limb foot splay), physiological observations, assessment of grip strength and assessment of motor activity in any of the animals of both the sex from tested dose group G4R performed towards end of the recovery period (day 65) when compared with concurrent vehicle control group.
However, there were statistically significant reductions in mean hind limb grip strength (kgf) in group G4 males; in mean fore and hind limb grip strength (kgf) in group G4R males noted. These changes are considered as incidental and unrelated to treatment as there were no changes in other functional observation battery parameters at this dose level and also the dose of 70 mg/kg body weight/day was reduced to 50 mg/kg body weight/day from day 7 and the animals were started to recover from clinical signs, found normal at the time of neurological/functional examinations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in both absolute and relative organ weights at any of the tested dose group animals of both sex in either main or recovery groups when compared with concurrent vehicle control groups.

However, the following statistically significant changes were noted in both absolute and relative organ weights when compared with their concurrent control groups:
- increase in absolute and relative spleen weight at group G3 males;
- increase in absolute epididymis weight at group G4 males;
- decrease in absolute heart weight at group G3 males;
- decrease in absolute kidneys weight at group G3 and G4 males;
- decrease in absolute brain weight at group G3;
- increase in absolute and relative thymus weight at group G4 females
These observed changes are considered as incidental and unrelated to treatment, due to lack of dose dependency and also there were no changes noted in macro or micro examination of theses organs during gross or histopathological examinations.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed during necropsy in all of the main or recovery group animals and pups except for one dead female (adult) which had external gross pathology findings such as soiled anus and black discoloration at tip of tail.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings in high dose animals of both sexes.
Few of the microscopic findings observed in this study were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings in high dose animals of both sexes.
Few of the microscopic findings observed in this study were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of YAPOX 2245 were established:
Systemic NOAEL: 50 mg/kg bw
Reproduction NOAEL: 50 mg/kg bw
Developmental NOAEL: 50 mg/kg bw

Executive summary:

The objective of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Testof test itemYAPOX 2245 by Oral Gavage in Sprague Dawley Rats as per OECD 422 guidelines, was to evaluate systemic toxicity end points, effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, parturition, and early neonatal development with repeated exposure once daily to the main group males for a period of 36 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period 50 days with a 16 days recovery.

A total of 116 (58 males + 58 females) Sprague Dawley rats were selected for the study and distributed to four main groups and two recovery groups. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle [Corn Oil], the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 17.5, 35 and 70 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively. Initially the group G4/G4R animals were treated with 70 mg/kg body weight, which was later reduced to 50 mg/kg body weight from day 7 of treatment period due to treatment related clinical signs. The vehicle and test item formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight.

The stability and homogeneity of test item in dose formulations were established before initiation of the treatment. The test item YAPOX 2245 was stable up to 6 hours at room temperature in corn oil at the concentration of 1 mg/mL and 20 mg/mL.Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1 and 5 of the dose administration period and the mean results were within the range of 85 to 115% recovery to the nominal concentration and the relative standard deviation (% RSD) was less than 10%.

All the main and recovery group animals were observed once daily for clinical signs, twice daily for mortality and morbidity, weekly once for detailed clinical examination. The body weights and feed consumption was recorded once weekly (except during cohabitation for main group animals) for all the animals. Ophthalmological examination was carried out once before treatment for all animals, towards end of the dosing period for group G1 and G4 animals and towards the end of recovery period for group G1R and G4R animals. Neurological/functional examination was performed for five randomly selected animals from each group per sex towards the end of the dosing period in case of main groups and for all the recovery group animals towards the end of the recovery period. The clinical pathological parameters such as haematology, clinical chemistry and urinalysis (only for main group males) were conducted for five randomly selected main group males and females and for all recovery group animals at termination. The serum thyroxine hormone (T4) levels were estimated for all the main group males and litters by ELISA method. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination.

All the main group males and females were evaluated for reproductive indices such as, mating and fertility index and all the main group litters were evaluated for its gestation and parturition index.

All the main group females were evaluated for oestrus cyclicity during pre-mating / cohabitation period. The pre-coital interval (copulatory interval) was calculated for each litter. The body weight and feed consumption was recorded for all the females during gestation and for all litters during lactation periods till day 14 after parturition. The gestation length per litter was calculated and at birth parameters (number of live/dead pups born, litter size, sex ratio, and live birth index per litter) were observed for all the litters of each main group. The litter observations per dam during lactation period such as, number of live/dead pups per litter during lactation period, sex ratio per litter, and pup survival index per litter were observed for all the litters of each main group. The total number of implantations per litter was recorded during necropsy and the pre-/post-natal losses per litter was calculated. All the litters were observed for presence of resorptions.

The pups were observed once daily for clinical signs/external examinations and twice daily for mortalities till termination [post-natal day (PND) 13], weighed individually on PND 1, 4, 7 and 13, measured for ano-genital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13, observed for gross pathological observations at termination and analyzed the serum collected from PND 4 and 13 pups for thyroxine hormone (T4) levels by using ELISA method.

In group G2 and G3, there were no clinical signs of toxicity or mortality/morbidity noted and no changes were noted in detailed clinical examination recorded weekly during the experimental period. There were no changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption at both these tested dose group animals. The ophthalmological examination was not conducted for these dose groups as there were no ophthalmological changes noted in high dose group (G4) animals. There were no changes noted in neurological/functional examination conducted for five randomly selected animals from each sex at both these tested dose groups. The haematology, clinical chemistry and urinalysis parameters conducted for five randomly selected animals from each sex at both these tested dose groups did not reveal any test item-related changes. There were no test item-related changes noted in serum thyroxine hormone (T4) levels estimated for all males and litters from these dose groups. There were no test item-related changes in absolute/relative organ weights and no macroscopic changes during necropsy noted in both these tested dose group animals. Histopathological examination was not conducted for these dose groups as there were no changes noted in organs/tissues from group G4 animals subjected to microscopic examination.

In group G4/G4R, the animals of both sex noted with test item-related clinical signs initially when treated with 70 mg/kg and one female was found dead on day 9. However, the dose was reduced to 50 mg/kg from day 7 onwards and the animals started to recover from the clinical signs and found normal at the end of termination. Test item-related reduction in mean body weight, percent change in mean body weight gain and mean feed consumption was noted from these dose group animals (G4/G4R) initially and found recovered later in the study. The neurological/functional examination and ophthalmological examination did not reveal any changes from both of these group animals conducted nearer to termination. The haematology, clinical chemistry and urinalysis parameters conducted for five randomly selected animals from G4 and for all animals from group G4R did not reveal any test item-related changes. There were no test item-related changes noted in serum thyroxine hormone (T4) levels estimated for all group G4 males and litters. There were no test item-related changes in absolute/relative organ weights and no macroscopic changes during necropsy noted in both G4/G4R dose group animals. There were no microscopic changes noted in organs/tissues subjected to histopathological examination from group G4 animals. Histopathological examination was not conducted for group G4R animals as there were no changes noted in organs/tissues from group G4 animals.

For reproduction toxicity end points, there were no test item related effects noted in mating and fertility index of both main group males and females in any of the tested dose groups (G2, G3 and G4) and no test item-related effects were noted in gestation and parturition index of litters from any of the tested dose groups (G2, G3 and G4) when compared with vehicle control group.

For maternal toxicity end points from group G2 and G3, there were no irregularities in oestrus cyclicity, no effects on duration of copulatory interval and no test item-related changes in mean body weight, percent change in mean body weight gain and mean feed consumption during gestation and lactation periods noted. There were no changes observed in gestation length, at birth parameters, litter observations during lactation period and number of implantations and pre-/post-natal losses per litter from both these dose groups.

For maternal toxicity end points from group G4, irregularities in oestrus cyclicity from three animals (two survived and one dead) were noted initially when dosed with 70 mg/kg and found normal later in the study as the dose was reduced to 50 mg/kg body weight and also these females were confirmed with mating and fertility. A slight test item-related delay in copulatory interval was noted at this dose group. There were no test item-related changes in mean body weight, percent change in mean body weight gain and mean feed consumption during gestation and lactation periods noted at this dose level. There were no changes observed in gestation length, at birth parameters, litter observations during lactation period and number of implantations and pre-/post-natal losses per litter from this dose group.

For developmental toxicity end points from all the tested dose groups (G2, G3 and G4), there were no clinical signs/external anomalies and no test item-related mortalities noted during post-natal period in any of the pups from tested dose group litters. There were no test item-related changes noted in mean male/female pup weight and mean male/female pup ano-genital distance ratio per litter at all the tested dose (G2, G3 and G4) groups. There were no occurrences of nipples in male pups examined on PND 13 at all the tested dose (G2, G3 and G4) group and control group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested and control group litters. There were no test item-related changes noted in serum thyroxine (T4) hormone levels estimated for PND 4 (from selected litter) and 13 (from all litters) pups at all the tested dose group litters.