N-(5-chloro-2-methoxyphenyl)-2-[(2-methoxy-4-nitrophenyl)azo]-3-oxobutyramide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key study: Test method OECD 422. The NOAEL of the test substance for parental and reproductive toxicity in rats by oral route was determined to be 1000 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2022 – 08 December 2022

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee, CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals). Registration No.: 1808/PO/RcBt/S/15/CPCSEA.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks (age at receipt)
- Weight at study initiation: 383-384 g (males); 265-266 g (females)
- Housing: Animals were housed in a standard polycarbonate cage (size: L 43 x B 28 x H 21 cm with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Processed corncob granules were provided as bedding material
Acclimatization: 2 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (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 were provided as a nesting material from gestation day 20 onwards.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3-23.7ºC
- Humidity (%): 43-66 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: 15 September 2022 to 08 December 2022

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

at 0.5% w/v

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed in a beaker and grind well in a mortar using pestle with a small quantity of vehicle until a homogenous suspension was formed. Thereafter the entire quantity of test formulation was transferred into a measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of contents to the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and formed homogeneous suspension in 0.5% w/v Carboxymethyl cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight). Hence, 0.5% w/v Carboxymethyl cellulose was used as vehicle for test item formulations as it is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): SLCJ7163

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 10 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- All females were confirmed as mated with the first male of same group within 10-days.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose concentration verification was determined during the first week and last week of treatment by a validated UV-visible spectrophotometer method (Study No. BIO-ANM 1837). The stability of the test item in dose formulations was established in this preliminary study. The test item formulations were stable at room temperature for 48 hours at the concentrations of 5 mg/mL and 100 mg/mL in vehicle and the results were within the acceptance criteria.

Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.

Prepared test item formulations were administered to the animals within established stability conditions.

Duration of treatment / exposure:

Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 31 days of treatment).
Main group females: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 40 to 58 days).
Recovery group animals (both sexes): until the first scheduled sacrifice of dams (total of 50 days of administration) and kept without treatment for further 15-days of observation period.

Frequency of treatment:

Once a day

Details on study schedule:

F1 animals were not mated.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1/G1R - Vehicle control / vehicle control recovery

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2 - Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3 - Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4/G4R - High dose / high dose recovery

No. of animals per sex per dose:

12 (main groups)
5 (Recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
There was no information available from repeated dose toxicity of the test substance. The estimated LD50 of the test substance is > 2000 mg/kg body weight obtained from a study conducted as per OECD test guidelines 420 (Acute Oral Toxicity) in male and female rats.
As per the ECHA Registration Dossier, the estimated NOAEL of 2-[(4-methyl-2-nitrophenyl)azo]-3-oxo-N-phenylbutyramide, a read across substance was 1000 mg/kg body weight/day obtained from a study conducted as per OECD Guideline 422. There was no indication of systemic, reproductive and developmental toxicity effects in any of the tested doses i.e. 100, 300 and 1000 mg/kg body weight/day in both the sexes.
Based on the above-mentioned information, a dose range finding study (BIO-DTX 080) was conducted with the doses of 100, 300 and 1000 mg/kg bw/day. There was no indication of systemic, reproductive and developmental toxicity effects noted in any of the tested doses and thus the same doses were selected for the present main study.

- Fasting period before blood sampling for clinical biochemistry: yes, overnight.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on day 1 before treatment and weekly thereafter during treatment. These observations were made outside the home cage and preferably at the same time. Observations included, but not be limited to, changes in the skin, fur, eyes, mucous membranes, the occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: at receipt, on the first day of dosing, weekly thereafter and at termination.
Females: at receipt, on the first day of dosing, and weekly once thereafter during pre-mating and until confirmation of mating. Pregnant animals were weighed on gestation days (GD) 0, 7, 14, and 20 and on lactation days 1, 4, 7, 13 and 14 (terminal body weight). The non-pregnant animals were weighed on the day of mating and weekly once until termination.
Recovery animals: at receipt, on the first day of dosing, weekly once thereafter and at termination.

FOOD CONSUMPTION: yes
Feed consumption (cage wise) was measured during following occasions:
Main Group Males: once in a week during premating period coinciding with body weight recording.
Main Group Females:
- once in a week during premating period coinciding with body weight recording.
- during gestation days 0 to 7, 7 to 14 and 14 to 20, during lactation days 1 to 4, 4 to 7 and
7 to 13.
- once in a week for all mated, but not littered females.
- for all non-pregnant animals once in a week coinciding with body weight recording.
Recovery group animals (both sexes): once in a week throughout the experimental period coinciding with body weight recording.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: for all animals before initiation of treatment i.e., after grouping and randomization (day 1). At the end of dosing period for all males shortly prior to scheduled sacrifice, i.e., day 30, during lactation period for littered females i.e., on lactation day 13, one day prior to scheduled sacrifice, i.e., 24th to 26th day from the day of confirmation of mating for mated but not littered/non-pregnant females and during last week i.e., day 64 for the recovery group animals.
- Dose groups that were examined: all groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for main group males on day 32; for main group females on lactation day 14 and for recovery group animals on day 66).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and from all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by an "OptiClot-4 coagulation analyzer".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for main group males on day 32; for main group females on lactation day 14 and for recovery group animals on day 66).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and from all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Na/K/Cl analyzer (Medica Corporation).

PLASMA/SERUM HORMONES/LIPIDS: Yes,
- Time of blood sample collection: in adult main group males, at termination (after completion of 31 days of treatment); in adult main group females, at termination (on lactation day 14); in pups, at termination (postnatal day 13).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: The assessment of serum T4 levels was performed in all adult males and 2 pups per litter (PND 13 pups). Further assessment of T4 levels in serum samples from the dams and PND 4 pups was not assessed as there were no treatment-related effects observed in the T4 levels of adult males and PND 13 pups.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: towards the end of the dosing period for males (shortly prior to scheduled sacrifice, i.e., on day 30) and during the lactation period for females (shortly prior to scheduled sacrifice, i.e., on lactation day 13). For recovery group animals towards the end of the recovery period (shortly prior to scheduled sacrifice, i.e.,
on day 64).
- Dose groups that were examined: 5 males and 5 females, randomly selected from each main group and all recovery group animals.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations (Hind limb foot splay), Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

Oestrous cyclicity (parental animals):

Oestrus cyclicity was monitored for two weeks after five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment.
Oestrus cyclicity was 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.
The status of oestrus cyclicity was determined on termination day.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 8 pups/litter (4/sex/litter as nearly as possible); All the sacrificed pups on the day of litter standardization were subjected for gross pathological examination.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Pup Observations: Individual pup from each litter was observed for external deformities or clinical signs once daily and for mortalities twice daily.
Pup weight: Individual live pup weight from each litter was recorded on
PND 1 (within 24 hours of parturition), 4, 7, and 13. The mean pup weight per litter (sex-wise) was reported.
Anogenital Distance: The anogenital distance of each live pup from each litter was measured on PND 4. The mean pup anogenital distance and its ratio was reported per litter (sex-wise).
Observation of male pups for nipples/areolae retention: The number of nipples/areolae in male pups was counted on PND 13 and presented litter wise.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
All the adult males were sacrificed after completion of 31 days of treatment. All pregnant females were sacrificed on lactation day 14 and the non-pregnant females were sacrificed on 25th to 27th day from the day of confirmation of mating. All the recovery group animals were sacrificed on 66th day with a total period of 50 days of treatment and 15 days of recovery period from the first scheduled sacrifice of dams.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 and 2 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on PND 4/13.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations with particular attention to the external reproductive genitals.

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 27. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 3 below.

Reproductive indices:

Mating and fertility index, cohabitation Record and Copulatory Interval (Pre-coital Interval), gestation Length, gestation index, parturation index and pregnancy index.

Offspring viability indices:

Number of male/female pups born (live/dead/cannibalized) per litter, sex ratio (m/f) and live birth index.
Number of implantation sites per dam/litter. Post implantation loss per litter was calculated based on the number of implantation sites and number of pups born. The postnatal loss per litter was calculated based on the number of pups born and number of pups survived until termination. The uterus of each female was observed for the presence of resorptions.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity in any of the animals of both sexes from all the tested dose groups throughout the experimental period. However, in group G4/G4R all animals were noted with yellow-coloured faeces from treatment day 2 and continued until termination. This coloration was due to coloured nature of the test item, but not an adverse test item related effect.
The detailed clinical examination conducted once in a week did not reveal any changes in any of the animals of both sexes from all the tested dose groups and vehicle control group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in all the tested dose groups (main and recovery) of both sexes throughout the experimental period.

There were no changes noted in mean gestational body weight and percent change in mean gestational body weight gain in all the tested dose groups when compared with the vehicle control group.

There were no changes noted in mean lactation body weight and percent change in mean lactation body weight gain in all the tested dose groups when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes noted in mean feed consumption in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control group.

There were no changes noted in mean feed consumption measured during gestation period in all the tested dose groups when compared with the vehicle control group.

There were no test item-related changes in mean lactation feed consumption in all the tested dose groups when compared with the vehicle control group.
The noted statistically significant differences across the dose groups (decrease during lactation day 1 to 4 and 4 to 7 in group G2; decrease during lactation day 4 to 7 and 7 to 13 in group G3; decrease during lactation day 4 to 7 in group G4) when compared with vehicle control group are considered as incidental and unrelated to treatment. These changes are toxicologically irrelevant and not occurred in a dose dependant manner.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in any of the animals from all the tested dose or vehicle control groups (main and recovery) of both sexes during ophthalmoscopic examination conducted shortly before scheduled sacrifice.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in obtained mean haematological values in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control group.

The noted statistically significant increase in PT (seconds) in group G3 males and decrease in APTT (seconds) in group G4R females when compared with vehicle control groups are considered as incidental and unrelated to treatment. The obtained mean values are well within lab historical control range of same species and strain and also similar changes were not occurred in other sex at the same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in mean clinical chemistry values in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control groups.

However, the following statistically significant differences were noted across the dose groups when compared with vehicle control group.
- increase in calcium level in groups G2 and G3 males.
- increase in phosphorous levels in group G4 males.
- increase in glucose levels in group G3 females.
- decrease in alkaline phosphatase levels in group G4 females.
- decrease in alkaline phosphatase levels in group G4R males.

These noted statistically significant changes are considered as incidental and unrelated to treatment as the obtained mean values are well within lab historical control range of same species and strain and also similar changes were not occurred in other sex at the same dose level.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no changes noted in mean serum T4 levels in any of the tested dose group males (main groups) when compared with the vehicle control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in mean urinalysis values in all the tested dose groups (main group males and recovery group males and females) when compared with the vehicle control group.

The noted statistically significant increase in pH from group G4R females when compared with vehicle control group is considered as incidental and unrelated to treatment, as the obtained mean value is well within lab historical control range of same species and strain and also similar change was not occurred in other sex at the same dose level.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The neurological/functional observations such as, home cage, handling, open-field, sensory, physiological observations did not reveal any test item related changes in any of the animals of both sexes from all the tested dose groups performed towards end of the dosing period for main groups (on day 30 for males and on lactation day 13 for dams) and performed towards end of recovery period for recovery groups (day 64).

There were no changes noted in mean fore/hind limb grip strengths, mean motor activity assessments and mean hind limb foot splay in all tested dose groups of both sexes when compared with vehicle control groups during conduct of neurological/functional examinations.

The noted statistically significant decrease in mean no. of movements from group G4 males when compared with vehicle control group is considered as incidental and unrelated to treatment. This noted observation is an alone occurrence of neurological assessments and also the obtained mean value is within lab historical control range of same species and strain.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related irregularities observed in oestrus cyclicity of females from any of the tested dose groups during pre-mating and mating treatment periods. However, one female from group G2 was noted with single irregular oestrus cycle (6-day cycle). This noted irregularity in duration of oestrus cyclicity in group G2 is considered as incidental and unrelated to treatment as the same female was noted with normal reproductive performance.

There were no test item-related changes in mean length of oestrus cycle per female during treatment period in all the tested dose groups when compared with the vehicle control group. The noted statistically significant increase in mean length of oestrus cycle in group G2 when compared with vehicle control group is considered as incidental and unrelated to treatment, as the obtained mean value is within lab historical control range of same species and strain and also there were no effects noted in any of the reproductive end points at this dose level.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no statistically significant differences noted in any of the tested dose groups when compared with the vehicle control group for fertility index, mean
pre-coital interval, gestation length , pregnancy index, gestation or parturation index, mean implantation sites and viable pups, and mean number and percentage of occurred post-implantation losses.

There were no changes observed in birth parameters such as, total litter size, number of live pups born, sex ratio (m/f) and live birth index (%) in any of the tested dose groups when compared with the vehicle control group. Also, the pup survival index (%) per litter during lactation period was unaffected by the test item in all tested dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects were found at any dose tested.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no changes noted in daily observation of pups at all the tested and vehicle control groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no mortalities of pups during the 13-day lactation period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean pup weight per litter in any of the tested dose groups when compared with the vehicle control group, recorded on postnatal day (PND) 1, 4, 7 and 13.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes noted in mean serum T4 hormone levels of postnatal day 13 pups (litter wise) in any of the tested dose group litters when compared with vehicle control group.
However, statistically significant increase in mean serum T4 levels in PND 13 pups of group G4 was noted when compared with vehicle control group. This noted change is considered as incidental and unrelated to treatment as the obtained mean value is within lab historical control range of same species and strain and also the change was not occurred in a dose dependent manner.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no changes in mean pup (both male and female) anogenital distance measurement (mm) and its ratio per litter in all the tested dose groups when compared with the vehicle control group, recorded on postnatal day 4.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no occurrences or evidence of retention of nipples in any of the male pups examined on the postnatal day 13 from all the tested dose groups and vehicle control group litters.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes noted in any of the pups sacrificed.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects were observed at any dose level tested.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 4. Summary of clinical signs of toxicity, detailed clinical examination and mortality record

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, M & 0	12	N (12)	NAD (12)	0 (12)
G2, M & 100	12	N (12)	NAD (12)	0 (12)
G3, M & 300	12	N (12)	NAD (12)	0 (12)
G4, M & 1000	12	N (12), Y (12)	NAD (12)	0 (12)

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, F & 0	12	N (12)	NAD (12)	0 (12)
G2, F & 100	12	N (12)	NAD (12)	0 (12)
G3, F & 300	12	N (12)	NAD (12)	0 (12)
G4, F & 1000	12	N (12), Y (12)	NAD (12)	0 (12)

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1R, M & 0	5	N (5)	NAD (5)	0 (5)
G1R, F & 0	5	N (5)	NAD (5)	0 (5)

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G4R, M & 1000	5	N (12), Y (12)	NAD (5)	0 (5)
G4R, F & 1000	5	N (12), Y (12)	NAD (5)	0 (5)

NAD: No Abnormality Detected; Y: Yellow coloured faeces.
a: observed once daily; b: observed once weekly; c: observed twice daily.

Note: All the animals were noted with yellow coloured faeces from treatment day 2 onwards.

 

Table 5. Summary of body weight (g) record

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28
G1, M & 0	Mean	384.08	417.48	437.74	447.72	455.95
	±SD	23.38	32.16	36.54	37.32	41.65
	n	12	12	12	12	12
G2, M & 100	Mean	383.21	406.66	425.52	437.09	458.53
	±SD	21.85	36.78	45.68	47.77	41.85
	n	12	12	12	12	12
G3, M & 300	Mean	383.84	416.14	431.24	441.30	449.27
	±SD	21.28	26.26	26.71	30.77	31.80
	n	12	12	12	12	12
G4, M & 1000	Mean	383.53	408.50	421.64	434.04	444.44
	±SD	23.23	36.64	44.79	40.81	38.29
	n	12	12	12	12	12

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21^#
G1, F & 0	Mean	265.48	273.08	277.18	297.46
	±SD	14.28	18.57	19.71	27.80
	n	12	12	12	3
G2, F & 100	Mean	266.41	274.84	280.12	295.80
	±SD	12.48	13.09	18.00	7.09
	n	12	12	12	2
G3, F & 300	Mean	265.88	273.13	274.86	282.09
	±SD	13.06	16.81	16.88	5.78
	n	12	12	12	3
G4, F & 1000	Mean	265.79	273.40	279.40	270.37
	±SD	13.61	15.46	16.89	18.94
	n	12	12	12	2

#: The data obtained from females in cohabitation only considered for mean calculations.

The data of day 21 body weight was not subjected to statistical analysis due to uneven number of variables.

 

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	35	42	49	56	63
G1R, M & 0	Mean	373.26	391.97	408.51	424.12	441.32	463.96	472.34	490.57	495.20	519.27
	±SD	18.27	32.44	37.69	32.32	30.60	27.00	22.20	18.58	25.89	17.65
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	376.98	406.55	419.76	432.63	442.09	465.05	486.79	506.83	512.08	535.49
	±SD	14.87	18.04	28.29	32.29	36.04	36.27	36.96	35.73	35.94	45.56
	n	5	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	35	42	49	56	63
G1R, F & 0	Mean	262.49	267.22	269.32	275.76	284.04	288.97	298.01	304.78	317.44	322.01
	±SD	10.83	11.06	14.90	13.92	11.49	12.62	17.14	16.95	14.65	16.39
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	261.74	270.89	277.16	279.41	279.22	284.29	291.74	296.80	299.69	305.70
	±SD	14.08	13.80	20.43	19.17	19.25	17.92	19.19	17.93	20.18	23.89
	n	5	5	5	5	5	5	5	5	5	5

 

 

Conclusions:

The NOAEL of the test substance for parental and reproductive toxicity in rats by oral route was determined to be 1000 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance by oral administration in rats was conducted following the OECD Test Guideline 422. A total of 116 (58 males + 58 females) Sprague Dawley rats were selected for the study and distributed to four main (G1, G2, G3 and G4) and two recovery groups (G1R and G4R). Each main group consisted of 12 males and 12 females, and each recovery group consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (0.5% w/v Carboxymethyl cellulose), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight. The test item was administered to males for a period of 31 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period) and to females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The stability of test item formulations in vehicle was established before initiation of the treatment by a validated analytical method. Prepared test item formulations were administered to the animals within established stability conditions.

For systemic toxicity assessment, all the animals (main and recovery groups) of both sexes were observed once daily for clinical signs, twice daily for mortality and morbidity and once in a week for detailed clinical examination. Body weight of all animals (main and recovery groups) of both sexes were recorded once in a week until termination. Further, the body weight of all pregnant females was recorded on gestation day (GD) 0, 7, 14 and 20 during pregnancy and on lactation day (LD) 1, 4, 7, 13 and 14 during lactation. Feed consumption for all animals (main and recovery groups) of both sexes were recorded once in a week during pre-mating. Further, feed consumption of all pregnant females was recorded during GD 0 to 7, 7 to 14 and 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13. Ophthalmological examination was carried out once before treatment and at the end of the dosing period for all males and females (main and recovery groups). Neurological/functional observations were performed for five randomly selected males and five randomly selected females from each main group towards end of the dosing period, for all animals from recovery groups towards end of the recovery period. The investigations of haematology and clinical chemistry for five randomly selected males and five randomly selected females from main group and all animals from recovery groups was conducted at termination. The investigations of urinalysis for five randomly selected males from each main group and for all recovery groups animals was conducted at termination. Serum thyroxine hormone (T4) levels were estimated for all main group males by ELISA method. The organs were collected and weighed on the day of termination for all animals (main and recovery groups) of both sexes and organ weight relative to terminal body weight was calculated. All the animals (main and recovery groups) of both sexes were observed for both external and internal gross pathological changes during conduct of necropsy. The 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 - stage aware histopathological evaluation).

For reproductive toxicity assessment, all the males (main groups) were evaluated for reproductive performances such as, mating and fertility indices. All the females (main groups) were evaluated for reproductive performances such as, mating, fertility, gestation and parturition indices. The females were also evaluated for pre-coital interval and gestation length. All females were evaluated for oestrus cyclicity during pre-mating period and the vaginal smear examination was continued until evidence of mating. At birth (delivery) parameters such as, number of live/dead pups born, litter size, sex ratio (m/f) and live birth index per litter were observed / calculated. The litter observations during lactation period such as number of survived or dead pups, sex ratio (m/f) and pup survival index per litter were observed / calculated. The total number of implantation sites was recorded for each litter during necropsy and the post-implantation and postnatal losses per litter was calculated.

For developmental toxicity assessment, all survived pups from each litter were observed once daily for external behavioural changes and twice daily for mortality until termination (PND 13). All pups from each litter were weighed individually on PND 1, 4, 7 and 13 and measured for anogenital distance on PND 4. All male pups were observed for retention of nipples on PND 13. All the pups were subjected for both external and internal gross pathological changes during conduct of necropsy. The collected serum from PND 13 pups was subjected to estimation of thyroxine hormone (T4) levels using ELISA method.

There were no clinical signs of toxicity and no mortality/morbidity noted in any of the animals from all the tested dose groups. However, in group G4/G4R all animals were noted with yellow-coloured faeces from treatment day 2 and this coloration was due to coloured nature of the test item. Detailed clinical examination did not reveal any changes in any of the tested dose group animals. No changes were noted in mean body weight, percent change in mean body weight gain and mean feed consumption of both sexes. The ophthalmological examination and neurological/functional examinations did not reveal any test item-related changes in any of the animals of both sexes. The obtained mean haematology, clinical chemistry and urinalysis values did not reveal any test item-related changes. The estimated serum T4 levels did not reveal any test item related changes. The absolute and relative organ weights of both sexes did not reveal any test item related changes. No gross pathological changes noted in any of the animals during necropsy.

There were no effects noted on reproductive performance of both sexes in all the tested dose groups. No test item-related irregularities observed in oestrus cyclicity of the females from all the tested dose groups during treatment period. No changes were noted in ‘at birth’ (delivery) and or ‘litter observations’ in all the tested dose groups. No effects were noted for live birth index and pup survival index in all tested dose groups. No test item-related post-implantation losses were noted in all the tested dose groups.

There were no test item-related developmental or external behavioural changes and no mortalities noted during postnatal period in any of the pups from all tested dose group litters. The mean pup weight, mean anogenital distance and its ratio in either sex of pups per litter were unaffected by the test item in all tested dose groups. There were no incidences of retention of nipples in male pups examined on PND 13 from all tested dose and control group litters. The estimated serum T4 levels of PND 13 (from all litters) pups did not reveal any changes in all tested dose group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested dose and control group litters.

Based on the above results, it can be concluded that the No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg bw/day as no adverse effects were found at any dose level tested.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April 2022 – 19 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

DRF study

GLP compliance:

no

Remarks:

DRF study

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks
- Weight at study initiation: 305.29-311.93 g (range of average values of each group of males at first day of dosing); 218.07-221.63 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: 2 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (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 were provided as a nesting material from gestation day 20 onwards.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4-24.0ºC
- Humidity (%): 46-65 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: 15 April 2022 to 19 June 2022

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

at 0.5% w/v

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed in a beaker or aluminum foil and grind well in a mortar using pestle with a small quantity of vehicle until a homogenous suspension was formed. Thereafter the entire quantity of test formulation was transferred into a measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of contents to the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and formed homogeneous suspension in 0.5% w/v Carboxymethyl cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight). Hence, 0.5% w/v Carboxymethyl cellulose was used as vehicle for test item formulations as it is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): SLCH1520

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- All females were confirmed as mated with the first male of same group within 14-days.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 48 hours at the concentrations of 5 mg/mL and 100 mg/mL in vehicle and the results were within the acceptance criteria (study nº BIO-ANM 1837).

Duration of treatment / exposure:

Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 34 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.

Frequency of treatment:

Once a day

Details on study schedule:

F1 animals were not mated.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 - Vehicle control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2 - Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3 - Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4 - High dose

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
There was no information available from repeated dose toxicity of the test substance. The estimated LD50 of the test substance is > 2000 mg/kg body weight obtained from a study conducted as per OECD test guidelines 420 (Acute Oral Toxicity) in male and female rats.
As per the ECHA Registration Dossier, the estimated NOAEL of 2-[(4-methyl-2-nitrophenyl)azo]-3-oxo-N-phenylbutyramide, a read across substance was 1000 mg/kg body weight/day obtained from a study conducted as per OECD Guideline 422. There was no indication of systemic, reproductive and developmental toxicity effects in any of the tested doses i.e. 100, 300 and 1000 mg/kg body weight/day in both the sexes.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The non-pregnant animals were weighed weekly once until termination.

FOOD CONSUMPTION: yes
-feed consumption was measured for all animals once a week during premating period coinciding with body weight recording. Feed consumption was measured for all pregnant animals during GD 0 to 7, 7 to 14 and 14 to 20, during LD 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for all non-pregnant animals once in a week.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before treatment for all animals (before randomization), at the end of the dosing period for all males (shortly prior to scheduled sacrifice, i.e., day 34) and females (shortly prior to scheduled sacrifice, i.e., on 26th day after confirmation of mating for mated but not littered females and on lactation day 13 for littered females).
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males on day 35, for littered females on lactation day 14 and for mated but not littered females on 26th day from the day of confirmation of mating).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all animals
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by an "OptiClot-4 coagulation analyzer".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males on day 35, for littered females on lactation day 14 and for mated but not littered females on 26th day from the day of confirmation of mating).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all animals
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Na/K/Cl analyzer (Medica Corporation).

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all animals
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: all males (shortly prior to scheduled sacrifice, i.e., on day 34) and all females (shortly prior to scheduled sacrifice, i.e., on 26th day after confirmation of mating for mated but not littered females and on lactation day 13 for littered females).
- Dose groups that were examined: all main groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations (Hind limb foot splay, Grip strength assessment and Motor activity assessment), Physiological Observation (Rectal temperature).

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored for two weeks during pre-mating treatment period to evaluate its normal oestrus cyclicity (4 to 5 days). Vaginal smears were also monitored daily from the beginning of treatment period until evidence of mating.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Individual pup from each litter was observed externally and for mortalities twice daily. Individual live pup weight from each litter was recorded on PND 1 (within 24 hours of parturition), 4, 7 and 13.
GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 35.
- Maternal animals: All surviving animals on lactation day 14

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
List of organs weighed is included in table 1 below.
No histopathology was conducted because no gross pathological changes were noted in any of the animals from all tested dose groups.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on lactation day 13.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations with particular attention to the external reproductive genitals.

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 27. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 2 below.

Reproductive indices:

Mating and fertility index, cohabitation Record and Copulatory Interval (Pre-coital Interval), gestation Length, gestation index, parturation index and pregnancy index.

Offspring viability indices:

Number of male/female pups born (live/dead/cannibalized) per litter, sex ratio (m/f) and live birth index.
Number of implantation sites per dam/litter. Post implantation loss per litter was calculated based on the number of implantation sites and number of pups born. The postnatal loss per litter was calculated based on the number of pups born and number of pups survived until termination. The uterus of each female was observed for the presence of resorptions.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity recorded in any of the animals of both sexes from all the tested dose groups throughout the experimental period.
In group G4, all the animals of both the sexes were noted with slight yellow-coloured faeces from treatment day 2 onwards, which was due to coloured nature of the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in any of the tested dose groups of both sexes when compared with the vehicle control group throughout the experimental period.

There were no test item related changes noted in mean gestational body weight and percent change in mean gestational body weight gain in any of the tested dose groups when compared with the vehicle control group.
The noted statistically significant reduction in mean percent change in gestation body weight gain during GD 7 to 14 in group G4 is considered as incidental and unrelated to test item administration, as there were no clinical signs of toxicity noted throughout the experimental period and also no changes noted in mean feed consumption during this period.

There were no changes noted in mean lactation body weight and percent change in mean lactation body weight gain in any of the tested dose groups when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes noted in mean feed consumption (including during gestation and lactation periods) in any of the tested dose groups of both sexes when compared with the vehicle control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in any of the animals of both sexes from high dose and vehicle control groups during ophthalmological examination conducted shortly before scheduled sacrifice.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in obtained mean haematological values in any of the tested dose groups of both sexes when compared with the vehicle control group.

However, statistically significant increase in mean reticulate count (% and absolute) in group G2 males, increase in mean basophils levels (%) in group G2 males, increase in mean APTT levels in group G2 males and increase in mean MCH levels in group G2 females were noted when compared with vehicle control group.
These statistically significant changes are considered as incidental and unrelated to treatment, as these changes did not occur in a dose dependant manner and also the obtained mean values are well within lab historical control range of same species and strain.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no changes noted in obtained mean clinical chemistry values in any of the tested dose groups of both sexes when compared with the vehicle control group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no changes noted in obtained mean urinalysis values in all the tested dose group males when compared with the vehicle control group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The neurological/functional observations such as, home cage, handling, open-field, sensory, physiological observations did not reveal any changes in any of the animals of both sexes from high dose and vehicle control groups performed towards end of the dosing period.
There were no changes noted in mean fore/hind limb grip strengths, mean motor activity assessments, and mean hind limb foot splay in all high dose group animals of both sexes when compared with vehicle control groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related irregularities observed in oestrus cyclicity of females in any of the tested dose groups during pre-mating and mating treatment periods. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration from all tested dose groups and comparable with the vehicle control group.
However, one female from group G4 was noted with a single irregular oestrus cycle during mating period. These observed irregular cycle was considered as incidental and not due to test item exposure, as this female revealed normal cycles during pre-mating period and was also confirmed as pregnant.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no statistically significant differences noted in any of the tested dose groups when compared with the vehicle control group for fertility index, mean
pre-coital interval, gestation length , pregnancy index, gestation or parturation index, mean implantation sites and viable pups, and mean number and percentage of occurred post-implantation losses.

There were no changes observed in birth parameters such as, total litter size, number of live pups born, sex ratio (m/f) and live birth index (%) in any of the tested dose groups when compared with the vehicle control group. Also, the pup survival index (%) per litter during lactation period was unaffected by the test item in all tested dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects were found at any dose tested.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no changes noted in daily observation of pups at all the tested and vehicle control groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no mortalities of pups during the 13-day lactation period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean pup weight per litter in any of the tested dose groups when compared with the vehicle control group, recorded on postnatal day (PND) 1, 4, 7 and 13.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes noted in any of the pups sacrificed.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects were observed at any dose level tested.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 3. Summary of delivery and litter observations record

Group & Dose (mg/kg body weight/day)		Delivery Record At birth															Sex Ratio (m/f) at Birth		Live Birth Index (%)
		Litter Size (No.)		Live Pups (No.)				Dead Pups (No.)				Cannibalized Pups (No.)
				Male	Female	Total		Male	Female	Total		Undetermined	Male	Female	Total
G1 & 0	Mean	8.00		3.80	4.20	8.00		0.00	0.00	0.00		0.00	0.00	0.00	0.00		1.53		100.00
	±SD	4.30		1.30	3.49	4.30		0.00	0.00	0.00		0.00	0.00	0.00	0.00		1.04		0.00
	n	5		5	5	5		5	5	5		5	5	5	5		5		5
G2 & 100	Mean	8.50		3.67	4.83	8.50		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.79		100.00
	±SD	2.81		1.37	1.72	2.81		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.30		0.00
	n	6		6	6	6		6	6	6		6	6	6	6		6		6
G3 & 300	Mean	10.00		4.40	5.60	10.00		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.88		100.00
	±SD	2.83		1.34	2.07	2.83		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.46		0.00
	n	5		5	5	5		5	5	5		5	5	5	5		5		5
G4 & 1000	Mean	8.00		3.80	4.20	8.00		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.92		100.00
	±SD	4.30		2.59	1.92	4.30		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.46		0.00
	n	5		5	5	5		5	5	5		5	5	5	5		5		5

Group & Dose (mg/kg body weight/day)		LD 1 to 4												Sex Ratio (m/f) on LD 4		Pup Survival Index (%) [LD 1 to 4]
		Live Pups (No.)				Dead Pups (No.)				Cannibalized Pups (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total
G1 & 0	Mean	3.80	4.20	8.00		0.00	0.00	0.00		0.00	0.00	0.00		1.53		100.00
	±SD	1.30	3.49	4.30		0.00	0.00	0.00		0.00	0.00	0.00		1.04		0.00
	n	5	5	5		5	5	5		5	5	5		5		5
G2 & 100	Mean	3.67	4.83	8.50		0.00	0.00	0.00		0.00	0.00	0.00		0.79		100.00
	±SD	1.37	1.72	2.81		0.00	0.00	0.00		0.00	0.00	0.00		0.30		0.00
	n	6	6	6		6	6	6		6	6	6		6		6
G3 & 300	Mean	4.40	5.60	10.00		0.00	0.00	0.00		0.00	0.00	0.00		0.88		100.00
	±SD	1.34	2.07	2.83		0.00	0.00	0.00		0.00	0.00	0.00		0.46		0.00
	n	5	5	5		5	5	5		5	5	5		5		5
G4 & 1000	Mean	3.80	4.20	8.00		0.00	0.00	0.00		0.00	0.00	0.00		0.92		100.00
	±SD	2.59	1.92	4.30		0.00	0.00	0.00		0.00	0.00	0.00		0.46		0.00
	n	5	5	5		5	5	5		5	5	5		5		5

Group & Dose (mg/kg body weight/day)		LD 5 to 7												Sex Ratio (m/f) on LD 7		Pup Survival Index (%) [LD 5 to 7]
		Live Pups (No.)				Dead Pups (No.)				Cannibalized (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total
G1 & 0	Mean	3.80	4.20	8.00		0.00	0.00	0.00		0.00	0.00	0.00		1.53		100.00
	±SD	1.30	3.49	4.30		0.00	0.00	0.00		0.00	0.00	0.00		1.04		0.00
	n	5	5	5		5	5	5		5	5	5		5		5
G2 & 100	Mean	3.67	4.83	8.50		0.00	0.00	0.00		0.00	0.00	0.00		0.79		100.00
	±SD	1.37	1.72	2.81		0.00	0.00	0.00		0.00	0.00	0.00		0.30		0.00
	n	6	6	6		6	6	6		6	6	6		6		6
G3 & 300	Mean	4.40	5.60	10.00		0.00	0.00	0.00		0.00	0.00	0.00		0.88		100.00
	±SD	1.34	2.07	2.83		0.00	0.00	0.00		0.00	0.00	0.00		0.46		0.00
	n	5	5	5		5	5	5		5	5	5		5		5
G4 & 1000	Mean	3.80	4.20	8.00		0.00	0.00	0.00		0.00	0.00	0.00		0.92		100.00
	±SD	2.59	1.92	4.30		0.00	0.00	0.00		0.00	0.00	0.00		0.46		0.00
	n	5	5	5		5	5	5		5	5	5		5		5

Group & Dose (mg/kg body weight/day)		LD 8 to 13												Sex Ratio (m/f) on LD 13		Pup Survival Index (%) [LD 8 to 13]
		Live Pups (No.)				Dead Pups (No.)				Cannibalized (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total
G1 & 0	Mean	3.80	4.20	8.00		0.00	0.00	0.00		0.00	0.00	0.00		1.53		100.00
	±SD	1.30	3.49	4.30		0.00	0.00	0.00		0.00	0.00	0.00		1.04		0.00
	n	5	5	5		5	5	5		5	5	5		5		5
G2 & 100	Mean	3.67	4.83	8.50		0.00	0.00	0.00		0.00	0.00	0.00		0.79		100.00
	±SD	1.37	1.72	2.81		0.00	0.00	0.00		0.00	0.00	0.00		0.30		0.00
	n	6	6	6		6	6	6		6	6	6		6		6
G3 & 300	Mean	4.40	5.60	10.00		0.00	0.00	0.00		0.00	0.00	0.00		0.88		100.00
	±SD	1.34	2.07	2.83		0.00	0.00	0.00		0.00	0.00	0.00		0.46		0.00
	n	5	5	5		5	5	5		5	5	5		5		5
G4 & 1000	Mean	3.80	4.20	8.00		0.00	0.00	0.00		0.00	0.00	0.00		0.92		100.00
	±SD	2.59	1.92	4.30		0.00	0.00	0.00		0.00	0.00	0.00		0.46		0.00
	n	5	5	5		5	5	5		5	5	5		5		5

Table 4. Summary record of reproductive performance

Group, Sex & Dose (mg/kg body weight/day)	No. of Males with Evidence of Mating (No. of Males used for Mating)	Male Mating Index (%)		No. of Males Capable of Impregnating a Female (No. of males used for Mating)	Male Fertility Index (%)
G1, M & 0	6 (6)	100.00		5 (6)	83.33
G2, M & 100	6 (6)	100.00		6 (6)	100.00
G3, M & 300	6 (6)	100.00		5 (6)	83.33
G4, M & 1000	6 (6)	100.00		5 (6)	83.33

Group, Sex & Dose (mg/kg body weight/day)		Cohabitation Record and Pre-coital Interval (Mean Time of Mating)					Gestational Length (duration of pregnancy)
		Pre-coital Interval (Days)		Conceiving Days (1 to 5)	Conceiving Days (More than 5 days)		Gestation Length (Days)
G1, F & 0	Mean	7.33	N	2	4		22.00
	±SD	3.01	%	33.33	66.67		0.00
	n	6					5
G2, F & 100	Mean	8.00	N	2	4		22.17
	±SD	3.63	%	33.33	66.67		0.41
	n	6					6
G3, F & 300	Mean	8.50	N	1	5		22.40
	±SD	2.35	%	16.67	83.33		0.55
	n	6					5
G4, F & 1000	Mean	7.83	N	3	3		22.40
	±SD	4.26	%	50.00	50.00		0.55
	n	6					5

Group, Sex & Dose (mg/kg body weight/day)	Female Mating Index (%)			Female Fertility Index (%)
	No. of Females with Evidence of Mating (No. of Females used for Mating)	Female Mating Index (%)		No. of Females Confirmed as Fertile (No. of Females used for Mating)	Female Fertility Index (%)
G1, F & 0	6 (6)	100.00		5 (6)	83.33
G2, F & 100	6 (6)	100.00		6 (6)	100.00
G3, F & 300	6 (6)	100.00		5 (6)	83.33
G4, F & 1000	6 (6)	100.00		5 (6)	83.33

Group, Sex & Dose (mg/kg body weight/day)	Female Fecundity or Pregnancy Index (%)			Gestation Index (%)				Parturition index (%)
	No. of females able to achieve pregnancy (No. of Females Confirmed with Mating)	Female Fecundity or Pregnancy Index (%)		Females with Live Born Pups at Parturition	No. of Females with Evidence of Pregnancy	Gestation Index (%)		No. of Females Littered	No. of Females with evidence of pregnancy	Parturition index (%)
G1, F & 0	5 (6)	83.33		5	5	100.00		5	5	100.00
G2, F & 100	6 (6)	100.00		6	6	100.00		6	6	100.00
G3, F & 300	5 (6)	83.33		5	5	100.00		5	5	100.00
G4, F & 1000	5 (6)	83.33		5	5	100.00		5	5	100.00

Group, Sex & Dose (mg/kg body weight/day)		Post-implantation Loss (%)					Post-natal Loss (%)
		No. of Implantations	No. of Viable (Live) Pups	Post-implantation Loss (No.)	Post-implantation Loss (%)		Total No. of Pups Found Dead/ Cannibalized during lactation period	Post-natal Loss (%)
G1, F & 0	Mean	8.60	8.00	0.60	4.29		0.00	0.00
	±SD	4.98	4.30	1.34	9.58		0.00	0.00
	n	5	5	5	5		5	5
G2, F & 100	Mean	8.50	8.50	0.00	0.00		0.00	0.00
	±SD	2.81	2.81	0.00	0.00		0.00	0.00
	n	6	6	6	6		6	6
G3, F & 300	Mean	10.40	10.00	0.40	2.67		0.00	0.00
	±SD	3.44	2.83	0.89	5.96		0.00	0.00
	n	5	5	5	5		5	5
G4, F & 1000	Mean	8.00	8.00	0.00	0.00		0.00	0.00
	±SD	4.30	4.30	0.00	0.00		0.00	0.00
	n	5	5	5	5		5	5

Table 5. Summary record of pup observations during post-natal period

Group & Dose (mg /kg body weight/day)		At Birth (PND 1)	PND 1 to 4	PND 5 to 7	PND 8 to 13
G1 & 0	No. of Dams/Litters	5	5	5	5
	No. of Live Pups	40	40	40	40
	Pup Observation/ No. of Pups observed	N/40	N/40	N/40	N/40
G2 & 100	No. of Dams	6	6	6	6
	No. of Live Pups	51	51	51	51
	Pup Observation/ No. of Pups observed	N/51	N/51	N/51	N/51
G3 & 300	No. of Dams	5	5	5	5
	No. of Live Pups	50	50	50	50
	Pup Observation/ No. of Pups observed	N/50	N/50	N/50	N/50
G4 & 1000	No. of Dams	5	5	5	5
	No. of Live Pups	40	40	40	40
	Pup Observation/ No. of Pups observed	N/40	N/40	N/40	N/40

Table 6. Summary record of mean pup weight (g) per litter

Group & Dose (mg /kg body weight/day)		PND 1			PND 4			PND 7			PND 13
		Mean Pup Weight (g)			Mean Pup Weight (g)			Mean Pup Weight (g)			Mean Pup Weight (g)
		Male	Female		Male	Female		Male	Female		Male	Female
G1 & 0	Mean	6.97	6.81		9.73	9.59		13.43	13.27		19.11	18.79
	±SD	0.29	0.41		0.42	0.53		0.21	0.41		0.31	0.77
	n	5 (19)	5 (21)		5 (19)	5 (21)		5 (19)	5 (21)		5 (19)	5 (21)
G2 & 100	Mean	7.25	6.80		9.93	9.41		13.90	13.24		19.60	18.84
	±SD	0.13	0.28		0.17	0.20		0.26	0.35		0.99	1.07
	n	6 (22)	6 (29)		6 (22)	6 (29)		6 (22)	6 (29)		6 (22)	6 (29)
G3 & 300	Mean	7.04	6.60		9.63	9.32		13.31	12.92		18.70	18.30
	±SD	0.21	0.32		0.27	0.35		0.21	0.43		0.60	0.50
	n	5 (22)	5 (28)		5 (22)	5 (28)		5 (22)	5 (28)		5 (22)	5 (28)
G4 & 1000	Mean	7.10	6.70		9.49	9.42		13.62	13.08		19.01	18.46
	±SD	0.27	0.34		0.54	0.49		0.75	0.43		0.48	0.42
	n	5 (19)	5 (21)		5 (19)	5 (21)		5 (19)	5 (21)		5 (19)	5 (21)

 

 

 

Conclusions:

The NOAEL of the test substance for parental and reproductive toxicity in rats by oral route was determined to be 1000 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance by oral administration in rats was conducted following the OECD Test Guideline 422. A total of 48 (24 males + 24 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 6 males and 6. The animals in G1 group were administered with vehicle (0.5% w/v Carboxymethyl cellulose), and the animals in G2, G3 and G4 groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight. The test item was administered to males for a period of 34 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period) and to females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The stability of test item formulations in vehicle was established before initiation of the treatment by a validated analytical method. Prepared test item formulations were administered to the animals within established stability conditions.

All animals were observed for clinical signs, mortality and morbidity, body weight, feed consumption, ophthalmological examinations, neurological/functional observations and clinical pathological (haematology, clinical chemistry and urinalysis) examinations. The gross pathology and organ weighing were performed on the day of termination for all animals. All females were observed for maternal body weight and feed consumption during gestation and lactation. Females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13 and observed for gross pathological observations at termination.

There were no test item related clinical signs of toxicity noted in any of the animals from all tested dose groups, except slight, yellow-coloured faeces from group G4 which was due to coloured nature of the test item. No mortality/morbidity noted in any of the animals from all tested dose groups. No test item related changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption in all tested dose groups of both sexes. The ophthalmological examination and neurological/functional examinations did not reveal any changes in any of the animals from high dose group of both sexes. The obtained mean haematology, clinical chemistry and urinalysis values did not reveal any test item-related changes in all tested dose groups. The absolute and relative organ weights of both sexes did not reveal any changes from all tested dose groups.

No gross pathological changes were noted in any of the animals from all tested dose groups during necropsy.

There were no effects noted on reproductive performance of both sexes in all tested dose groups. No test item-related irregularities observed in oestrus cyclicity of the females from all the tested dose groups. No changes were noted in ‘at birth’ (delivery) and or ‘litter observations’ in all tested dose groups. No effects were noted for live birth index and pup survival index in all tested dose groups. No test item-related post-implantation losses were noted in all tested dose groups.

There were no developmental/external behavioural changes noted and no test item-related mortalities noted during postnatal period in any of pups from all tested dose group litters. The mean pup weight in either sex of pups per litter were unaffected by the test item in all tested dose groups. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all tested dose and control group litters.

It can be concluded that the No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg bw/day as no adverse effects were found at any dose level tested.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A key study is available with a Klimisch score of 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

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

Based on the available information, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) no 1272/2008.