2,5-di-tert-butylhydroquinone

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

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

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

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

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

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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/kgbody 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. 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.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- If no evidence of copulation is obtained after 14 days, the animals will be separated, re-mating of females with proven males of the same group was tried for an additional week.
- After successful mating each pregnant female was caged (how): housed individually in a plastic cage

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 samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control.
The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquot of each formulation was analyzed. The second 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 concentration 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 sacrifice 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

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

17.5 mg/kg bw/day

Remarks:

actual ingested

Dose / conc.:

35 mg/kg bw/day

Remarks:

actual ingested

Dose / conc.:

70 mg/kg bw/day

Remarks:

Actual ingested. 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:

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

Parental animals: Observations and examinations:

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 throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount 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 obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. 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 animals 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 observation from the first scheduled sacrifice of dams. The organs were collected and Relative organ weights 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 tissues 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.

Oestrous cyclicity (parental animals):

Estrous cycles was monitored during the acclimatization to evaluate its normal estrous cyclicity (4 to 5 days). Only females with normal estrous cyclicity was selected for the treatment. Vaginal smears was monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care will be taken to avoid disturbance of mucosa, which may induce pseudopregnancy. Estrous cyclicity was also be monitored on the day of sacrifice for females.

Sperm parameters (parental animals):

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.

Litter observations:

PARAMETERS EXAMINED
The day of littering was considered as lactation day 1.
The number of pups born (dead and live) in a litter, sex and external examination observations were recorded at birth.
Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded.
The ano-genital distance of each pup was measured on postnatal day 4 (lactation day 4).
The number of nipples/areolae in male pups were counted on postnatal day 13 (lactation day 13).
Fertility index for dams, sires and pup survival index and sex ratio at birth were calculated.

GROSS EXAMINATION OF DEAD PUPS:
The pups was sacrificed on lactation day 13 and the sacrificed pups and dead pups (if any) were examined for gross abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

SACRIFICE
The pups were sacrificed on lactation day 13 and the sacrificed pups and dead pups (if any) were examined for gross abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of the organs specified above

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table above were prepared for microscopic examination and weighed, respectively.

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 in the table.

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

Reproductive indices:

Mating (%): Number of females mated x 100
Number of females paired

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): Number of pregnant females x 100
Number of females mated

Gestation index (%): Number of females with living pups on Day 1 x 100
Number of pregnant females

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%): Total number of offspring born x 100
Total number of uterine implantation sites

Lactation index (%): Number of live offspring on Day 13 after littering x 100

Offspring viability indices:

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check x 100
Number of live pups at First Litter Check

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check x 100
Number of live pups at First Litter Check

Viability index (%): Number of live offspring on Day 4 before culling x 100
Number live offspring on Day 1 after littering

Lactation index (%): Number of live offspring on Day 13 after littering x 100

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 co
nducted 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 adminis
tration 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.

Dermal irritation (if dermal study):

not examined

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 cli
nical 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):

no effects observed

Description (incidence and severity):

There were no changes noted in mean feed consumption during pre-mating and post-mating treatment period at any of the tested dose group males (main group) when compared with concurrent vehicle control group. The feed consumption was not measured for main group males during cohabitation period.
There were no changes noted in mean feed consumption during pre-mating treatment period at any of the tested dose group females (main group) when compared with concurrent vehicle control group. The feed consumption was not measured for main group females during cohabitation period.
There were no changes noted in mean feed consumption during experimental period (both treatment and recovery periods) at the high dose recovery group animals of either sex when compared with concurrent vehicle control group.

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 per
iod (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 (G
4) 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):

ed 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 inhouse
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 obser
vations, assessment of grip strength and assessment of motor activity in any of the males (five ran
domly 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 concurre
nt vehicle control group.
There were no test item-related changes noted in neurological/functional examinations like home cag
e, 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

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

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

There were no irregularities observed in the oestrus cyclicity at any of the group G1, G2 and G3 females during pre-mating and mating treatment periods. The mean length of oestrus cycle per dam during pre-mating period was unaffected at all these dose groups when compared with vehicle control group.
In group G4, two surviving females and one found dead female were noted with irregular oestrus cycles during pre-mating period. The two survived females noted with normal oestrus cycles later in the study as the dose was reduced to 50 mg/kg body weight from 70 mg/kg body weight and also both the females were confirmed with mating and confirmed as fertile. The data of found dead female was excluded for mean calculation of length of oestrus cycle.
These changes can be considered as test item-related initially as the animals noted with test item-related clinical signs when dosed with 70 mg/kg body weight.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant changes (delays) observed in the copulatory interval (pre-coital interval) and no changes in mean number of days taken by the females to conceive at any of the tested dose groups when compared with vehicle control group females.
However, in group G4, slight delay in copulatory interval (pre-coital interval) was noted which can be considered as test item-related due to noted treatment related clinical signs, reduced body weights and feed consumption initially at this dose level.

Gestation Body Weight and Percent Change in Gestation Body Weight
There were no test item-related changes noted in mean body weight and percent change in mean body weight gain during gestation period in any of the tested dose group (G2, G3 and G4) females when compared with vehicle control group.

Feed Consumption during Gestation Period
There were no test item-related changes noted in mean feed consumption during gestation period in any of the tested dose group (G2, G3 and G4) females when compared with vehicle control group.
However, statistically significant reduction in mean gestation feed consumption during gestation day (GD) 14 to 20 at group G2 and during gestation day (GD) 7 to 14 at group G4 when compared with vehicle control group. This noted observation could be considered as incidental and unrelated to treatment due to lack of dose dependency and consistency throughout the gestation period and also there were no changes noted in mean body weight and percent change in body weight during this period.

Lactation Body Weight and Percent Change in Lactation Body Weight
There were no test item-related changes noted in mean body weight and percent change in mean body weight gain during lactation period in any of the tested dose group (G2, G3 and G4) females when compared with vehicle control group.
However, statistically significant increase in mean percent change in body weight gain during lactation day (LD) 7 to 13 was noted at group G3 when compared with vehicle control group. This noted observation could be considered as incidental and unrelated to treatment due to lack of consistency and also there were no changes noted in mean feed consumption during this period.

Feed Consumption during Lactation Period
There were no test item-related changes noted in mean feed consumption during lactation period in any of the tested dose group (G2, G3 and G4) females when compared with vehicle control group.
However, statistically significant reduction in mean lactation feed consumption during lactation day (LD) 1 to 4 was noted at all the tested dose groups when compared with vehicle control group. This noted observation could be considered as incidental and unrelated to treatment due to lack of dose dependency and consistency throughout the lactation period and also there were no changes noted in mean body weight and percent change in body weight during this period.

Gestation Length and Delivery Data at Birth per Litter
There were no test item-related changes observed in mean gestation length per litter at all the tested dose groups (G2, G3 and G4) when compared with vehicle control group.
The at birth parameters like, mean litter size, mean number of live / dead pups born, sex ratio (m/f) and live birth index did not reveal any statistically significant changes at any of the tested dose groups when compared with vehicle control group. However, a slight reduction in live birth index was noted at group G4 when compared with control group and other dose groups. This noted change can be considered as incidental and unrelated to treatment as there were no changes noted in pup observations and mean pup weights at birth at this dose level.

Litter observation and Pup Survival index during Lactation Period
There were no changes observed in litter observation parameters like, number of survived pups per sex per litter during lactation period, sex ratio (m/f) on LD 4/7/13, pup survival index between LD 1 to 4, 4 to 7 and 7 to 13 at all the tested dose groups (G2, G3 and G4) when compared with vehicle control group.

Uteri Observations and Pre-/Post Natal Loss per Litter
There were no statistically significant changes observed in mean number of implantations per litter and no test item related pre/post-natal losses per litter at all the tested dose groups when compared with vehicle control group.
However, slight increase in pre-natal loss (no. and %) was noted at group G4 when compared with vehicle control group. This noted change can be considered as incidental and unrelated to treatment as there were no changes noted in pup observations and mean pup weights at birth at this dose level.

1Serum Thyroxine (T4) Levels - Parental Males and Females
There were no changes observed in serum Thyroxine (T4) hormone levels at all the tested dose main group (G2, G3 and G4) males and females when compared with concurrent vehicle control group. However, statistically significant reduction in mean serum thyroxine (T4) hormone levels was noted at all the tested dose group dams when compared with vehicle control group. This noted reduction can not be considered as adverse as the the obtained values are within the in-house historical control range and also the reductions are not occurred in a dose dependent manner.

Pup observation during Postnatal Period
There were no clinical signs or external anomalies and mortalities were noted during daily observation of pups at all the tested (G2, G3 and G4) and vehicle control group litters during postnatal period. All the pups were noted with normal behaviour during daily observations.

Mean Pup Weight per Litter during Post-natal Period
There were no test item-related changes observed in mean pup [both male and female] weight per litter recorded on post-natal day (PND) 1, 4, 7 and 13 at all the tested dose (G2, G3 and G4) group litters when compared with vehicle control group.
However, an isolated statistically significant reduction in mean female pup weight on post-natal day 4 at group G4 was noted when compared with vehicle control. This change is considered incidental and unrelated to treatment as there were no changes noted in daily pup observations and the reduction was inconsistent at this dose level.

Mean Pup Ano-genital Distance per Litter on Post-natal Day (PND) 4
There were no test item-related changes observed in mean pup [both male and female] ano-genital distance measurement (mm) and ratio per litter recorded on post-natal day (PND) 4 at all the tested dose (G2, G3 and G4) group litters when compared with vehicle control group.
However, statistically significant reduction in mean male ano-genital distance ratio was noted at group G4 when compared with vehicle control. This change is considered incidental and unrelated to treatment as there were no changes noted in daily pup observations and no changes were noted in male mean pup weight on PND 4 at this dose level.

Nipple Retention in Male Pups per Litter on Postnatal Day 13
There were no occurrences of nipples in male pups examined on its postnatal day (PND) 13 in any of the tested dose group litters and vehicle control group litters.

Serum Thyroxine (T4) Levels - Postnatal Day 4/13 Pups
There were no changes observed in serum Thyroxine (T4) hormone levels of both PND 4 and 13 pups from all the tested dose group (G2, G3 and G4) litters when compared with vehicle control group. However, statistically significant reduction in mean serum thyroxine (T4) hormone levels of PND 13 pups was noted at group G4 when compared with vehicle control group. This noted reduction can not be considered as adverse as the the obtained values are within the in-house historical control range.

Key result

Dose descriptor:

NOAEL

Remarks:

(Systemic)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

(Reproduction)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at highest dose tested

Remarks on result:

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

Key result

Dose descriptor:

NOAEL

Remarks:

Development

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Remarks:

At dose of 70 mg/kg bw severe test item-related clinical signs of toxicity were observed, including reduced body weight and feed consumption and one mortality from females. Hence, the high dose was reduced to 50 mg/kg body weight from day 7 onwards.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest level tested

Remarks on result:

other: For developmental toxicity end points there ere no testitem related effects in all the tested dose groups (G2, G3 and G4).

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs or external anomalies and mortalities were noted during daily observation of pups at all the tested (G2, G3 and G4) and vehicle control group litters during postnatal period. All the pups were noted with normal behaviour during daily observations.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no clinical signs or external anomalies and mortalities were noted during daily observation of pups at all the tested (G2, G3 and G4) and vehicle control group litters during postnatal period. All the pups were noted with normal behaviour during daily observations.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in mean pup [both male and female] weight per litter recorded on post-natal day (PND) 1, 4, 7 and 13 at all the tested dose (G2, G3 and G4) group litters when compared with vehicle control group.
However, an isolated statistically significant reduction in mean female pup weight on post-natal day 4 at group G4 was noted when compared with vehicle control. This change is considered incidental and unrelated to treatment as there were no changes noted in daily pup observations and the reduction was inconsistent at this dose level.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in mean pup [both male and female] ano-genital distance measurement (mm) and ratio per litter recorded on post-natal day (PND) 4 at all the tested dose (G2, G3 and G4) group litters when compared with vehicle control group.
However, statistically significant reduction in mean male ano-genital distance ratio was noted at group G4 when compared with vehicle control. This change is considered incidental and unrelated to treatment as there were no changes noted in daily pup observations and no changes were noted in male mean pup weight on PND 4 at this dose level.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no occurrences of nipples in male pups examined on its postnatal day (PND) 13 in any of the tested dose group litters and vehicle control group litters.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Serum Thyroxine (T4) Levels - Postnatal Day 4/13 Pups
There were no changes observed in serum Thyroxine (T4) hormone levels of both PND 4 and 13 pups from all the tested dose group (G2, G3 and G4) litters when compared with vehicle control group. However, statistically significant reduction in mean serum thyroxine (T4) hormone levels of PND 13 pups was noted at group G4 when compared with vehicle control group. This noted reduction can not be considered as adverse as the the obtained values are within the in-house historical control range.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Serum Thyroxine (T4) Levels - Postnatal Day 4/13 Pups
There were no changes observed in serum Thyroxine (T4) hormone levels of both PND 4 and 13 pups from all the tested dose group (G2, G3 and G4) litters when compared with vehicle control group. However, statistically significant reduction in mean serum thyroxine (T4) hormone levels of PND 13 pups was noted at group G4 when compared with vehicle control group. This noted reduction can not be considered as adverse as the the obtained values are within the in-house historical control range.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested.

Reproductive effects observed:

no

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 item YAPOX 2245 by Oral Gavage in Sprague Dawley Rats as per OECD 422 guidelines, was to evaluatesystemic 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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The objective of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Testof test item YAPOX 2245 by Oral Gavage in Sprague Dawley Rats as per OECD 422 guidelines, was to evaluatesystemic 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.

Effects on developmental toxicity

Description of key information

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Di-tert-butyl hydroquinone is not classified for reproduction toxicity according to the CLP Regulation No. 1272/2008.