2,2,2-trichloro-1-phenylethyl acetate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Study 1

In the OECD 407 study with Sprague Dawley rats, no treatment-related gross or histopathological findings were observed in any of the primary or secondary reproductive organs that were examined.

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, other

Remarks:

reproductive organ toxicity study in rats

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Principles of method if other than guideline:

The Repeated Dose 28-day Oral Toxicity study was designed and conducted to determine the reproductive toxicity profile of the test chemical when administered daily for 28 days in the Sprague Dawley rats.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

1) In order to meet the regulatory requirement for testing in a rodent species

2) Widely used in as a species of choice for pre-clinical toxicological studies.

3) This strain is widely used throughout the industry in the non-clinical laboratory studies.

4) This study is intended to provide information on the health hazards likely to arise from exposure to the test item.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Institute of Biosciences, Pune
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation:
Males: 132.8 g to 165.2 g
Females: 129.4 g to 156.4 g
- Fasting period before study: No Data
- Housing: The rats were housed in polycarbonate cages with paddy husk as bedding.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune was provided ad libitum from individual feeders on cage top.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. Water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature was maintained at 22 ± 3°C (actual range, 19.6 °C to 21.4 °C).
- Humidity (%): Room humidity was maintained at 30% to 70% (actual range, 54.3% to 60.6%).
- Air changes (per hr): At least 10 Air changes per hour
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was suspended in Polyethylene Glycol-400 for preparation of suspension(s). The suspension(s) of test item was made at volumes suitable for daily use for 28 days. The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight respectively were administered. The concentration of the test item was varied so as to maintain the dose volume constant at or upto 10 ml/kg body weight.

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data
- Mixing appropriate amounts with (Type of food): No Data
- Storage temperature of food: No Data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Polyethylene glycol
- Concentration in vehicle: 0 (vehicle), 250, 500 and 1000 mg/kg body weight
- Amount of vehicle (if gavage): 10 mL/Kg
- Lot/batch no. (if required): No Data
- Purity: No Data

Details on mating procedure:

No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item prepared formulation analysis for concentration, homogeneity and stability were conducted at subcontracted Laboratory.

Duration of treatment / exposure:

28 Days consecutively and 2 week recovery period.

Frequency of treatment:

Once Daily

Details on study schedule:

No data available

Dose / conc.:

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

Remarks:

Control Group (G1)

Dose / conc.:

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

Remarks:

Low Dose Group (G2)

Dose / conc.:

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

Remarks:

Mid Dose Group (G3)

Dose / conc.:

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

Remarks:

High Dose Group (G4)

No. of animals per sex per dose:

Control (0 mg/kg bw/day): 6 males and 6 females
Control Recovery (0 mg/kg bw/day): 6 males and 6 females
Low dose (250 mg/Kg/Day): 6 males and 6 females.
Mid Dose (500 mg/Kg/Day): 6 males and 6 females
High Dose (1000 mg/Kg/Day): 6 males and 6 females
High Dose Recovery(1000 mg/Kg/Day): 6 males and 6 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected based on the results of the Dose Range Finder study conducted.The study was conducted at the dose levels of 0 mg/kg, 50 mg/kg, 100 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight.

1) All the male and female animals from control and all the treated dose groups up to 1000 mg/kg survived throughout the dosing period of 14 days.

2) Male and female animals from control and all the treated dose groups exhibited normal body weight gain at the end of the dosing period of 14 days.

3) Daily clinical observations did not reveal any signs of toxicity in male and female animals from different dose groups during the dosing period of 14 days.

4) Gross pathological examination did not reveal any abnormality attributable to the treatment.

Based on these results, the 28 day study dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight and animals were exposed to the treatment, every day, for a period of 28 days.

- Rationale for animal assignment (if not random): Animals were randomized on the basis of sex and body weights. A total of 48 animals (24 males + 24 females) were selected and randomly distributed into four groups with 6 animals/sex/group and 3/sex/cage.

- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No Data

Positive control:

Not included

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily for mortality. Rats were examined once daily for clinical signs.

- Cage side observations checked in table [No.?] were included. Viability, clinical signs such as skin and fur changes, eye and mucous membrane changes, respiratory, circulatory and general changes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed once before the start of dose administration and once a week thereafter until scheduled sacrifice.
a) Home Cage Observations:
In home cage, rats were observed for behavior, alterations, vocalizations, respiration and palpebral closer
1) Behavior in Home cage:
The behavior of the rat was observed in home cage upon initial approach by the observer and description of behavior in home cage was recorded as:
1 = apparently sleeping.
2 = Awake, but immobile; apparently normal posture.
3 = Engaged in apparently normal movement such as rearing, drinking, or grooming.
4 = Immobile, with unusual posture or tonic convulsion (lying on side with legs extended, flattened body or arched back. (opisthotonos or emprosthonos).
5 = Unusual behaviour or muscular patterns (stereotyped behaviour such as head bobbing or weaving, circling, repetitive licking or grooming, bizarre behaviour such as self mutilation, writhing or retropulsion, unusual muscular patterns such as tremors, spasms or clonic convulsion).
2) Alterations Home cage:
The alterations of the rat were observed in home cage upon initial approach by the observer and description of alterations in home cage was recorded as:
1 = No alterations in behaviour or posture (Normal).
2 = Stereotyped behaviour pattern (head bobbing or weaving, circling, repetitive licking or grooming).
3 = Bizarre behaviours such as self-mutilation, writhing or retropulsion.
4 = Twitches or tremors in the limbs or repetitive movements of the mouth or jaws.
5 = Whole body tremors or spasms.
6 = Unusual posture (opisthotonos, emprosthotonos, tonic extension, head tilt, straub tail).
7 = Tonic-clonic seizure.
3) Vocalizations:
The occurrence of spontaneous or unprovoked vocalization was recorded as:
1= No vocalization/ Normal
2= Vocalization noted
If vocalization observed the actual number was recorded.
4) Respiration:
The observations for respiration were recorded as:
1 = Normal
2 = Abnormal
The type of abnormal respiration e.g. bradypnea, hyperpnea, dyspnea, rals was recorded in the clinical signs.
5) Palpebral closer:
The degree of closure of the eyelids was recorded as:
1 = Eyelids wide open (Normal).
2 = Eyelids slightly closed.
3 = Eyelids dropping, approximately half-closed.
4 = Eyelids completely shut.
b) Handling Observations:
After completing home cage observations, the rat was observed for reaction to removal, reaction to handling, urination, defecation, prominence of eye, lacrimation, salivation, piloerection, examination of mucous membrane, examination of skin / fur, examination of natural orifices and animal appearance.
1) Reaction to Removal:
The reaction of the rat removed from home cage was recorded as:
1 = Sits quietly and is easily removed.
2 = Vocalization without resistance to being picked up.
3 = Runs around cage with or without vocalization, or freezes or rears, following the investigator’s hand.
4 = Tail and throat rattles, may attack.
2) Reaction to handling:

The reaction of the rat to handling from home cage was recorded as:
1 = Quiet with no resistance.
2 = Vocalization without resistance.
3 = Tense or rigid.
4 = Squirming and twisting, may attempt to bite.

3) Urination:
The frequency of urination was recorded as:
0 = No urination during the observation period.
1 = Urine present; quantity is not excessive.
2 = the amount of urination is excessive.

4) Defecation:

The frequency of defecation was recorded as:
0 = No defecation during the observation period.
1 = Fecal boluses have normal consistency.
2 = Soft or liquid feces.

5) Prominence of Eye:

The eyes were examined for prominence of eye and observation was recorded as:
1 = Normal
2 = Exophthalmos
3 = Enophthalmos

6) Lacrimation:

The degree of lacrimation was recorded as:
1 = No excess lacrimation (normal).
2 = Excess moisture at the margin of the eyelid.
3 = Persistent dampness at the margin of the eyelid
4 = Dampness extends beyond the margin of the eyelid.

7) Salivation:

The degree of salivation was recorded as:
1 = No excess salivation (normal).
2 = Margin of mouth wet.
3 = Wet zone ¼ to ½ of submandibular area.
4 = Wet zone extends to the entire submandibular area.

8) Piloerection:
Piloerection was differentiated from a scruffy or ungroomed coat by patting the back of the animal in a rostral to caudal direction. Piloerection was considered in case the animal hairs were erect after patting. The observation for piloerection was recorded as:
0 = Absent
1 = Present
9) Examination of Mucous Membrane:
The observation for visual mucous membrane was recorded as:
¬¬¬1 = Normal
2 = Abnormal (discolouration)
10) Examination of Skin / Fur:
The observation for skin / fur examination was recorded as:
1 = Normal
2 = Abnormal
11) Examination of Natural Orifices:
The observation for natural orifices examination was recorded as:
1 = Normal
2 = Abnormal
12) Animal Appearance:
The observation for appearance of animal was recorded as:
1 = Clean and groomed.
2 = Unkempt (with scruffy and ungroomed coat)
3 = Stained by urine and/or feces.
c) Open field observation:
The animal was placed in the open field and its appearance and behavior were observed. The following observations were made and recorded:
1) Stereotype Behaviour:
The stereotype behaviour can be defined as the pronounced repetition of specific gesture or movements i.e. presence of excessive or repetitive behaviour that appears purposeless to the observer. The observation was recorded as:
0 = Absent
1 = Excessive grooming / licking / head bobbing or weaving
2 = Circling movements
2) Bizarre Behaviour:
The bizarre behaviour includes any unusual behaviour that will not be normally observed in the test species. The observation was recorded as:
0 = Absent
1 = Retropulsion
2 = Biting of cage
3 = Biting to other animal(s)
4 = Self destructive biting or mutilation
3) Rearing (Rears):
The number of times the rat raises both forelimbs off the surfaces is considered as rearing. The number of these actions was counted and total number of rearing was recorded.
4) Movements:
In the open field, each animal was observed for presence of clonic and tonic movements:
4.1) Clonic Movements:
The observation for clonic movement was recorded as:
0 = None /Normal
1 = repetitive mouth/jaw motion, such as, Chewing, clones of jaw
2 = Mild clonic tremors of whole body
3 = Repetitive clonic tremors/ seizure of whole body
4.2) Tonic Movements:
The observation for tonic movement was recorded as:
0 = None /Normal
1 = Contractions of limbs
2 =Unusual posture (Opisthotonos, Emprosthotonous, tonic extension, head tilt, straub tail).
3= seizure

5) Gait Pattern:
The gait pattern was evaluated by observing the movement of the rat in the open field and the observation was recorded as:
1 = Normal
2 = Ataxic
3 = Hind limbs or forelimbs show exaggerated or overcompensated movements, drag or appear splayed.
4 = Spastic
5 = Duck walk
6 = Scissor
7 = Hunched back
6) Mobility Score:
A measure of the ability of the animal to locomote despite gait abnormalities was recorded. The ranking of the degree of impairment of locomotion was recorded as:
1 = Normal
2 = Slightly impaired
3 = Totally impaired

7) Severity of Gait:
The severity of the gait abnormalities is graded and documented as follows:
1 = Slight gait abnormality
2 = Moderate gait abnormality
3 = Extreme gait abnormality

8) Pupillary response:
The animal eyes were briefly covered for 30 seconds with hand/cloth and then the penlight was pointed and the response to penlight was recorded as:

1 = Response
2 = No response

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on the day of randomization, day of first dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29 and day 43.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, The quantity of feed consumed by control and different treatment groups was recorded on commencement of treatment and weekly thereafter until scheduled sacrifice and the feed consumption per animal was calculated for each group.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No Data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all the animals were examined prior to the initiation of the dosing and at scheduled sacrifice.
- Dose groups that were examined: All animals were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at terminations.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, Food was withheld overnight from all rats prior to sampling.
- How many animals: Blood samples were collected from all rats from each group.
- Parameters checked in table [No.?] were examined. Hb, RBC, HCT, MCV, MCH, MCHC, WBC, N, L, E, M, B, Pt

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at respective terminations.
- Animals fasted: Yes, Food was withheld overnight from all rats prior to sampling.
- How many animals: Blood samples were collected from all rats from each group.
- Parameters checked in table [No.?] were examined. Total Protein , Blood Urea Nitrogen , Urea Nitrogen, ALT, AST, ALP, GGT, Glucose, Ca, P, Albumin, Total bilirubin, creatinine, total cholesterol, triglycerides, globulin, Na, K, Cl

URINANALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the exposure period of 28 days and towards the end of the recovery period on day 42.
- Dose groups that were examined: All animals from all groups.
- Battery of functions tested:
Sensory activity: Towards the end of the exposure period, sensory reactivity to stimuli of different types (e.g., auditory, visual and proprioceptive stimuli) was conducted for all the animals.

1) Arousal level:
The activity/arousal level of the animal was described during the observation period and is documented as follows:
1 = Very low (stuporous, comatose)
2 = Low (sluggish, some exploratory movement possible).
3 = Apparently normal (alert with exploratory movement).
4 = High (sudden startle, darting or freezing without apparent stimuli).
5 = Very high (sudden bouts of running, freezing with spontaneous vocalization).

2) Sensory Activity:
Sensory activity was assessed by following methods:
2.1) Assessment of Visual Response: A blunt probe was held approximately 4 cm away from front of the face / eye and moved away steadily and reaction was documented.
2.2) Touch Response: Avoiding the animal’s field of vision, the rump was gently touched with blunt probe. The animals reaction to this stimulus was observed and was documented.
2.3) Auditory Response: Using a clicker approximately 5 cm above the back of the animal sudden sound was made. The animals reaction to this stimulus was observed and was documented.
2.4) Tail Pinch Response: The reaction to a tail pinch was rated (The tail pinch was applied by the blunt forceps at approximately 3 cm from the tip of tail. The reaction to tail pinch was observed and was documented.
All above four responses were graded as follows:
1 = No reaction.
2 = orientating response: Slowly turns towards the stimulus or walks away.
3 = Startle response or freezing reaction.
4 = More energetic response than “2” or “3”.
5 = Jumps at or away, attack or bites.
3) Visual Placing Response:
The animal was removed from its cage and held at the base of the tail (holding the tail more distally can strip off the skin) then slowly lowered forward towards the edge of the observation area or another raised edge (such as the rim of an overturned cage). The visual placing response is graded and documented:
1 = Early extension of forelimbs to reach for the screen.
2 = Extends limbs only after contact with the vibrissae or nose.
3 = No extension even after contact with nose.
4) Air righting response:
Holding the animal in a supine position, it was dropped from approximately 30cm and the righting response was rated:
1 = Lands with all feet on the ground.
2 = Uncoordinated landing or lands on side.
3 = Lands on back.

Grip strength: Grip strength of fore limbs was measured with a digital grip strength meter (Columbus Instruments International Corporation, Ohio, USA) to determine the ability of the rat to grasp and hold on the mesh platform. The grip strength of each rat was measured in Kilogram (Kg) for 3 consecutive times and average of the three grip strength values was calculated.

Motor activity: Motor activity of each animal was monitored using an automated animal activity measuring system (Columbus Instruments, OPTO-M3, Ohio, USA). Animals were monitored for three consecutive 10 minutes intervals allowing for examination of both exploratory and acclimation activity levels. During this period, total and ambulatory activity of the animal was recorded. Stereotypic activity was calculated by subtracting ambulatory activity from total activity.
Other: No Data

Postmortem examinations (parental animals):

SACRIFICE: The rats were sacrificed by CO2 asphyxiation, after 28 consecutive days of oral administration, all surviving study rats were sacrificed on day 29 (Group I, III, IV, V).

GROSS PATHOLOGY: Yes, All the rats survived through the dosing period of 28 days and were sacrificed and gross lesions were noted.

ORGAN WEIGHTS: Liver, Kidneys, Adrenals, Epididymides, Prostate + Seminal Vesicle with Coagulation gland as whole, Thymus, Spleen, Brain, Heart, Lungs, Uterus, Testes/Ovaries were dissected free of fat and weighed. The paired organs were weighed together.

HISTOPATHOLOGY: Yes, From each rat, samples or the whole of the tissues listed below were preserved. All tissues were fixed in 10% neutral buffered formalin except, eyes and testes of all animals were preserved in Davidson’s solution for 24 hours and transferred to 10% neutral buffered formalin. Procedure for preparation of slides of tissues of various organs from the rats of various dose groups were performed.

Following tissue samples of organs from control and animals treated at different dose groups were preserved and those from control and treated at the highest dose level of 1000 mg/kg were subjected to histopathological examination:
Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Oesophagus, Ovaries, Pancreas, Pituitary gland, Pharyngeal Lymphnodes, Prostate, Rectum, Skeletal Muscles, Skin with Mammary Gland, Spleen, Sternum with bone marrow, Sciatic Nerve, Spinal Cord (Cervical, mid thoracic and lumbar), Stomach, Seminal Vesicles with Coagulation Gland, Testes, Thymus, Thyroid, Trachea, Vagina, Urinary Bladder, Uterus.

Statistics:

Raw data was processed and analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using in-house developed and validated MS-Excel 2003 based statistical software. All the parameters characterized by continuous data such as body weight, per cent body weight change, feed consumption, organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analyses of Variance (ANOVA) and Dunnett’s t-test. Where the data did not meet the homogeneity of variance, Student’s t-test was performed to calculate significance.

Significance was calculated at 1% as well as 5% level and indicated in the summary tables as follows:

* = Significant than control at 95% level of confidence (p≤ 0.05).
** = Significant than control at 99% level of confidence (p≤ 0.01).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All the animals from control and all the treated dose groups survived throughout the dosing period of 28 days.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Animals from control and all treated dose groups exhibited normal feed consumption at the end of the dosing period of 28 days.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological investigations conducted at the end of dosing period on day 29, revealed following significant changes in the values of different parameters studied when compared with that of respective controls.

Male :
MCV (p≤0.01), MCH (p≤0.01) and Total WBC (P≤0.05) : Increased values were obtained for animals from 250 mg/kg dose group,
Platelets : Increased values were obtained for animals from 250 mg/kg and 1000 mg/kg dose groups (p≤0.05),
Hb and HCT : Decreased values were obtained for animals from 250 mg/kg (p≤0.05), 500 mg/kg (p≤0.01) and 1000 mg/kg (p≤0.01) dose groups,
Total RBC : Decreased values were obtained for animals from 250 mg/kg and 500 mg/kg dose groups (p≤0.01) and
MCV and MCH : Decreased values were obtained for animals from 1000 mg/kg dose group (p≤0.01).

Female :
Platelets : Increased values were obtained for animals from 500 mg/kg dose group (p≤0.01),
Hb : Decreased values were obtained for animals from 250 mg/kg (p≤0.05) and 500 mg/kg (p≤0.01) dose groups,
Total RBC (p≤0.01), HCT (p≤0.05) and Total WBC (P≤0.05) : Decreased values were obtained for animals from 500 mg/kg dose group,
MCV : Decreased values were obtained for animals from 1000 mg/kg dose group (p≤0.01) and
MCH and MCHC : Decreased values were obtained for animals from 250 mg/kg (p≤0.05) and 1000 mg/kg (p≤0.01) dose groups.

The observed changes in hematology either lacked dose-dependency, consistency between sexes, or gross or histopathological correlations and were therefore considered incidental or non-adverse.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Biochemical investigations conducted at the end of dosing period on day 29, revealed following significant changes in the values of different parameters studied when compared with that of respective controls.

Male :
Total Protein : Elevated levels were observed in animals from 500 mg/kg dose group (p≤0.05),
Creatinine : Elevated levels were observed in animals from 1000 mg/kg dose group (p≤0.05),
Cholesterol : Elevated levels were observed in animals from 500 mg/kg and 1000 mg/kg dose groups (p≤0.05),
Bilirubin : Decreased levels were observed in animals from 250 mg/kg (p≤0.05) and 500 mg/kg (p≤0.01) dose groups and
Chloride : Decreased levels were observed in animals from 500 mg/kg dose group (p≤0.01).

Female :
Sodium : Elevated levels were observed in animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups (p≤0.01),
Alkaline Phosphatase : Decreased levels were observed in animals from 500 mg/kg dose group (p≤0.01) and
Chloride : Decreased levels were observed in animals from 1000 mg/kg dose group (p≤0.05).

The observed changes in clinical chemistry either lacked dose-dependency, consistency between sexes, or gross or histopathological correlations and were therefore considered incidental or non-adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Sensory Reactivity Observations:
All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4.

Grip Strength:
Grip strength values observed in male and female animals for control and different dose groups were comparable.

Motor Activity:
Higher values were observed in male animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups for second interval (p≤0.05). Lower values were observed in female animals from 1000 mg/kg dose group for first interval (p≤0.01) and for second interval (p≤0.05).

These changes were within laboratory range and were considered to be of no toxicological importance

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):

No treatment related histopathological changes were evident in male and female rats from control and high dose groups.
Histopathological examination revealed minimal focal to multifocal periportal mononuclear cell infiltration in the liver; minimal interstitial haemorrhages and/or tubular dilatation in the kidneys; minimal alveolar haemorrhages and/or alveolar histiocytosis in the lungs; minimal multifocal haemosiderosis and/or diffused congestion in spleen; minimal eosinophilic cells infiltration and/or luminal dilatation and/or endometrial gland dilatation in uterus; minimal luminal seminal coagulum in the urinary bladder; minimal dilatation of zona reticularis and/or vacuolation in zona fasiculata in adrenals; minimal multifocal haemorrhages in thymus; presence of ultimobranchial cysts in thyroid; in male or female animals from control and high dose group. All the changes observed in the control and high dose treatment group animals were similar, incidental and mode of death related, physiological and are covered in the facility historical data of the histopathology findings.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Critical effects observed:

not specified

Organ:

not specified

Remarks on result:

not measured/tested

Critical effects observed:

no

Reproductive effects observed:

not specified

Treatment related:

not specified

VIABILITY

	Dose		Mortality
Group	mg/kg		Males		Females
Number	Male	Female	Absolute	Relative %	Absolute	Relative %
I	0	0	0/6	0	0/6	0
II	250	250	0/6	0	0/6	0
III	500	500	0/6	0	0/6	0
IV	1000	1000	0/6	0	0/6	0

GROUP MEAN BODY WEIGHT (g)

 

Sex - Male

Group	Dose		Day
Number	mg/kg bw		0	1	8	15	22	28
I	0	Mean	154.23	159.17	214.55	266.05	299.30	323.63
		±SD	11.21	11.28	14.09	19.19	20.96	19.45
II	250	Mean	152.37	157.73	207.88	261.47	292.90	316.53
		±SD	12.54	12.53	12.50	15.07	14.41	14.22
III	500	Mean	153.25	159.88	201.80	248.33	285.63	306.90
		±SD	11.25	11.82	11.18	11.82	13.66	13.97
IV	1000	Mean	155.10	161.08	205.98	260.42	291.13	311.83
		±SD	9.84	9.88	7.48	16.30	12.05	10.81

Sex - Female

Group	Dose		Day
Number	mg/kg bw		0	1	8	15	22	28
I	0	Mean	138.18	141.05	179.03	204.52	220.50	234.18
		±SD	9.51	9.69	8.67	10.74	11.85	11.00
II	250	Mean	138.95	142.18	169.42	192.18	210.32	224.70
		±SD	8.80	8.94	4.88	7.42	15.75	15.72
III	500	Mean	139.28	142.22	169.40	189.75	206.50	221.45
		±SD	9.47	9.97	5.81	5.78	12.09	12.83
IV	1000	Mean	140.22	143.20	169.85	187.55	202.20	218.48
		±SD	10.30	10.14	7.09	6.25	10.41	8.98

GROUP MEAN FEED CONSUMPTION (g/animal/day)

Sex - Male

Group	Dose		WEEK
Number	mg/kg		0 Day	1	2	3	4
I	0	Mean	15.73	17.73	19.65	21.50	24.18
II	250	Mean	15.46	17.63	19.43	21.82	23.62
III	500	Mean	15.60	17.96	18.58	20.97	23.38
IV	1000	Mean	15.55	17.68	19.32	21.02	23.32

Sex - Female

Group	Dose		WEEK
Number	mg/kg		0 Day	1	2	3	4
I	0	Mean	11.76	13.38	15.53	16.85	19.62
II	250	Mean	11.88	13.55	15.38	16.70	19.21
III	500	Mean	11.62	13.22	15.03	16.48	18.62
IV	1000	Mean	12.03	13.58	15.22	16.22	18.58

SUMMARY OF CLINICAL OBSERVATIONS

AND GENERAL APPEARANCE

Sex : Male

 

Group Number	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days from - to	Mortality
I	0	Nil	6	1 - 6	1 - 28	0/6
II	250	Nil	6	13 - 18	1 - 28	0/6
III	500	Nil	6	25 - 30	1 - 28	0/6
IV	1000	Nil	6	37 - 42	1 - 28	0/6

Sex : Female

 

Group Number	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days from - to	Mortality
I	0	Nil	6	7 - 12	1 - 28	0/6
II	250	Nil	6	19 - 24	1 - 28	0/6
III	500	Nil	6	31 - 36	1 - 28	0/6
IV	1000	Nil	6	43 - 48	1 - 28	0/6

GROUP MEAN HAEMATOLOGY

Sex : Male

Day : 29

 

Group	Dose		Hb	Total RBC	HCT	MCV	MCH	MCHC
Number	mg/kg		(g/dL)	(x 10^6/µL)	(%)	(fL)	(pg)	(g/dL)
I	0	Mean	17.62	8.05	43.97	54.63	21.92	40.07
		±SD	0.31	0.08	0.55	0.45	0.38	0.42
II	250	Mean	16.75*	7.40**	41.75*	56.48**	22.68**	40.12
		±SD	0.48	0.28	1.31	0.79	0.18	0.52
III	500	Mean	15.62**	7.21**	39.40**	54.77	21.72	39.68
		±SD	0.74	0.51	2.13	2.84	1.10	0.28
IV	1000	Mean	16.17**	7.91	40.68**	51.48**	20.43**	39.70
		±SD	0.47	0.33	1.27	0.89	0.46	0.40

 

 

Group	Dose		Platelets	Total WBC	Differential %					Pt.
Number	mg/kg		(x 10^3/ µL)	(x 10^3/µL)	N	L	E	M	B	(Sec.)
I	0	Mean	305.33	8.08	19.33	78.00	1.67	1.00	0.00	14.67
		±SD	7.15	1.19	2.73	3.63	0.82	0.89	0.00	3.56
II	250	Mean	321.00*	13.20*	18.67	79.00	1.17	1.17	0.00	15.83
		±SD	15.58	4.02	2.34	1.90	1.17	0.75	0.00	2.86
III	500	Mean	341.50	7.92	18.67	79.00	1.33	1.00	0.00	15.67
		±SD	45.31	3.91	2.34	2.97	1.21	0.89	0.00	3.14
IV	1000	Mean	338.17*	9.07	18.67	79.17	1.17	1.00	0.00	13.50
		±SD	32.50	1.77	2.16	2.79	0.75	0.89	0.00	1.87

 

Hb         : Hemoglobin                    RBC     : Red Blood Corpuscules                         

HCT      : Hematocrit                       MCV    : Mean Corpuscular Volume

MCH    : Mean Corpuscular Hemoglobin        MCHC  : Mean Corpuscular Hemoglobin Concentration

WBC    : White Blood Corpuscles                  Pt.        : Prothrombin time

N          : Neutrophils                      L          : Lymphocytes

E          : Eosinophils                      M         : Monocytes

B          : Basophils

*  = Significant at 95% level of confidence (p≤0.05)

** = Significant at 99% level of confidence (p≤0.01)

Sex : Female

Day : 29

 

Group	Dose		Hb	Total RBC	HCT	MCV	MCH	MCHC
Number	mg/kg		(g/dL)	(x 10^6/µL)	(%)	(fL)	(pg)	(g/dL)
I	0	Mean	17.43	8.00	43.10	53.85	21.80	40.47
		±SD	0.31	0.30	1.14	0.72	0.49	0.41
II	250	Mean	16.40*	8.07	41.30	51.32	20.40*	39.78*
		±SD	0.79	0.39	2.03	3.53	1.45	0.22
III	500	Mean	15.18**	6.94**	37.90*	54.58	21.90	40.10
		±SD	0.54	0.25	1.43	0.37	0.17	0.54
IV	1000	Mean	16.63	8.12	42.25	52.07**	20.48**	39.38**
		±SD	0.85	0.52	2.32	0.96	0.37	0.35

 

 

Group	Dose		Platelets	Total WBC	Differential %					Pt.
Number	mg/kg		(x 10^3/ µL)	(x 10^3/µL)	N	L	E	M	B	(Sec.)
I	0	Mean	295.83	8.57	17.83	80.33	1.17	0.67	0.00	15.33
		±SD	30.90	2.43	2.32	2.34	0.75	0.52	0.00	3.08
II	250	Mean	308.17	11.80	17.83	79.50	1.67	1.00	0.00	16.33
		±SD	59.19	3.96	2.99	3.33	0.82	0.63	0.00	2.80
III	500	Mean	372.33**	5.48*	17.33	80.83	1.17	0.67	0.00	16.17
		±SD	18.48	0.17	2.88	2.48	0.75	0.52	0.00	2.79
IV	1000	Mean	318.00	8.33	17.67	79.83	1.00	1.50	0.00	15.33
		±SD	39.65	2.37	3.01	2.93	0.63	0.55	0.00	2.16

 

Hb         : Hemoglobin                    RBC     : Red Blood Corpuscules                         

HCT      : Hematocrit                       MCV    : Mean Corpuscular Volume

MCH    : Mean Corpuscular Hemoglobin        MCHC  : Mean Corpuscular Hemoglobin Concentration

WBC    : White Blood Corpuscles                  Pt.        : Prothrombin time

N          : Neutrophils                      L          : Lymphocytes

E          : Eosinophils                      M         : Monocytes

B          : Basophils

*  = Significant at 95% level of confidence (p≤0.05)

** = Significant at 99% level of confidence (p≤0.01)

GROUP MEAN CLINICAL BIOCHEMISTRY

 Sex : Male

Day : 29

 

Group Number	Dose      mg/kg		Total Protein (g/dL)	BUN (mg/dL)	Urea Nitrogen (mg/dL)	ALT (U/L)	AST (U/L)	ALP (U/L)	Glucose (mg/dL)
I	0	Mean	6.57	14.17	30.88	48.17	101.50	171.50	74.33
		±SD	0.29	1.72	3.75	3.25	17.95	37.13	6.53
II	250	Mean	6.77	16.00	34.88	46.67	92.17	172.67	76.50
		±SD	0.29	1.26	2.76	7.45	13.12	34.20	8.92
III	500	Mean	7.04*	15.17	33.06	48.17	104.33	185.50	78.50
		±SD	0.19	1.47	3.21	9.87	24.20	33.12	5.21
IV	1000	Mean	6.77	14.83	32.34	48.83	104.00	193.67	65.33
		±SD	0.28	2.48	5.41	4.79	8.46	22.26	5.43

 

Group Number	Dose mg/kg		Calcium (mmol/L)	Phospho-rous (mg/dL)	GGT (U/L)	Total Bilirubin (mg/dL)	Albumin (g/dL)	Globulin (g/dL)	Creatinine (mg/dL)
I	0	Mean	3.59	7.62	6.50	0.31	1.17	5.40	0.45
		±SD	0.11	0.83	1.22	0.04	0.13	0.39	0.03
II	250	Mean	3.69	7.48	6.33	0.20*	1.28	5.48	0.46
		±SD	0.12	0.88	0.52	0.09	0.09	0.29	0.05
III	500	Mean	3.76	8.03	5.17	0.13**	1.21	5.82	0.47
		±SD	0.10	0.89	0.75	0.02	0.09	0.17	0.04
IV	1000	Mean	3.68	8.27	5.67	0.27	1.27	5.48	0.52*
		±SD	0.10	0.82	0.82	0.02	0.10	0.25	0.04

 

Group Number	Dose mg/kg		Sodium (mmol/L)	Potassium (mmol/L)	Chloride (mmol/L)	Total Cholesterol (mg/dL)	Triglycerides (mg/dL)
I	0	Mean	146.34	4.22	109.07	31.33	74.17
		±SD	1.30	0.19	1.26	8.31	20.54
II	250	Mean	145.43	4.54	107.77	41.00	66.67
		±SD	0.80	0.26	1.67	7.16	14.46
III	500	Mean	146.76	4.67	105.66**	43.33*	81.17
		±SD	1.46	0.43	1.90	6.38	23.66
IV	1000	Mean	146.40	4.23	108.67	42.83*	77.33
		±SD	1.91	0.32	1.01	8.01	30.87

 

BUN : Blood Urea Nitrogen            ALT : Alanine Aminotransferase        AST : Aspartate Transaminase ALP : Alkaline Phosphatase

*  = Significant at 95% level of confidence (p≤0.05)

** = Significant at 99% level of confidence (p≤0.01)

GROUP MEAN CLINICAL BIOCHEMISTRY

 

Laboratory Test Item Code : TAS/122/001

Test System : Sprague Dawley Rat

Sex : Female

Day : 29

 

Group Number	Dose      mg/kg		Total Protein (g/dL)	BUN (mg/dL)	Urea Nitrogen (mg/dL)	ALT (U/L)	AST (U/L)	ALP (U/L)	Glucose (mg/dL)
I	0	Mean	6.53	15.17	33.06	44.83	107.33	164.00	72.83
		±SD	0.31	2.23	4.86	4.31	15.58	20.07	4.58
II	250	Mean	6.48	15.17	33.06	45.50	113.33	132.00	64.00
		±SD	0.39	1.60	3.49	9.03	23.59	30.57	8.92
III	500	Mean	6.53	13.67	29.79	42.50	90.83	99.00**	70.50
		±SD	0.43	0.82	1.78	6.60	8.98	20.42	6.41
IV	1000	Mean	6.42	15.33	33.43	41.50	95.67	125.83	71.67
		±SD	0.44	3.72	8.12	5.86	9.07	33.47	7.45

 

Group Number	Dose mg/kg		Calcium (mmol/L)	Phospho-rous (mg/dL)	GGT (U/L)	Total Bilirubin (mg/dL)	Albumin (g/dL)	Globulin (g/dL)	Creatinine (mg/dL)
I	0	Mean	3.71	6.57	6.50	0.11	1.29	5.25	0.55
		±SD	0.11	0.64	1.05	0.02	0.15	0.29	0.05
II	250	Mean	3.71	7.48	6.67	0.13	1.19	5.32	0.53
		±SD	0.11	0.75	1.37	0.06	0.17	0.33	0.04
III	500	Mean	3.68	7.17	5.17	0.12	1.29	5.23	0.51
		±SD	0.04	0.84	0.75	0.02	0.10	0.31	0.05
IV	1000	Mean	3.71	6.77	6.33	0.11	1.22	5.20	0.55
		±SD	0.04	0.94	1.75	0.02	0.13	0.40	0.06

 

Group Number	Dose mg/kg		Sodium (mmol/L)	Potassium (mmol/L)	Chloride (mmol/L)	Total Cholesterol (mg/dL)	Triglycerides (mg/dL)
I	0	Mean	144.61	4.34	108.89	52.33	76.00
		±SD	0.66	0.40	1.06	5.09	15.49
II	250	Mean	146.38**	4.43	107.33	48.17	63.17
		±SD	0.77	0.22	2.43	8.91	19.01
III	500	Mean	146.32**	4.25	108.89	47.83	83.33
		±SD	0.54	0.14	1.53	7.88	25.73
IV	1000	Mean	146.45**	4.21	106.32*	50.17	88.00
		±SD	1.42	0.27	1.21	5.85	28.71

 

BUN : Blood Urea Nitrogen            ALT : Alanine Aminotransferase        AST : Aspartate Transaminase ALP : Alkaline Phosphatase

*  = Significant at 95% level of confidence (p≤0.05)

** = Significant at 99% level of confidence (p≤0.01)

 

GROUP MEAN ABSOLUTE ORGAN WEIGHTS (g)

 Sex : Male

Day : 29

 

GroupNumber	Dose mg/kg		Body Weight (g)	Brain	Liver	Kidneys	Adrenals	Testes	Prostate + Seminal Vesicle with Coagulation gland
I	0	Mean	304.27	2.033	13.192	2.429	0.0607	3.182	1.491
		±SD	19.75	0.144	1.775	0.195	0.0133	0.139	0.284
II	250	Mean	298.33	1.936	12.352	2.244	0.0545	3.077	1.258
		±SD	15.62	0.154	1.338	0.202	0.0028	0.318	0.154
III	500	Mean	288.80	1.975	12.340	2.162	0.0523	2.594	1.133
		±SD	13.01	0.078	0.903	0.409	0.0067	0.276	0.246
IV	1000	Mean	294.33	1.999	12.163	2.347	0.0474	2.964	1.213
		±SD	10.28	0.086	1.233	0.214	0.0070	0.249	0.122

 

GroupNumber	Dose mg/kg		Heart	Spleen	Lungs	Thymus	Epididymides
I	0	Mean	1.364	1.404	1.760	0.430	0.764
		±SD	0.162	0.247	0.238	0.100	0.058
II	250	Mean	1.231	1.349	1.623	0.372	0.671
		±SD	0.045	0.413	0.188	0.068	0.048
III	500	Mean	1.233	1.411	1.817	0.363	0.658
		±SD	0.072	0.227	0.441	0.066	0.032
IV	1000	Mean	1.133	1.488	1.531	0.343	0.673
		±SD	0.144	0.513	0.315	0.081	0.076

 

 

GROUP MEAN ABSOLUTE ORGAN WEIGHTS (g)

Sex : Female

Day : 29

 

GroupNumber	Dose mg/kg		Body Weight (g)	Brain	Liver	Kidneys	Adrenals	Ovaries
I	0	Mean	217.20	1.908	8.040	1.646	0.0559	0.0822
		±SD	11.85	0.098	0.889	0.136	0.0087	0.0105
II	250	Mean	207.97	1.849	7.651	1.475	0.0556	0.0669
		±SD	16.31	0.111	1.027	0.176	0.0078	0.0095
III	500	Mean	206.17	1.813	7.788	1.523	0.0558	0.0757
		±SD	11.88	0.124	1.110	0.261	0.0044	0.0192
IV	1000	Mean	201.28	1.828	7.225	1.478	0.0512	0.0703
		±SD	8.75	0.069	0.878	0.150	0.0082	0.0076

 

Group No.	Dose mg/kg		Heart	Spleen	Lungs	Thymus	Uterus
I	0	Mean	0.955	1.130	1.394	0.435	0.346
		±SD	0.085	0.271	0.361	0.097	0.077
II	250	Mean	0.881	1.396	1.462	0.386	0.313
		±SD	0.088	0.605	0.204	0.147	0.106
III	500	Mean	0.848	1.028	1.632	0.363	0.293
		±SD	0.113	0.198	0.181	0.085	0.079
IV	1000	Mean	0.815	0.834	1.322	0.345	0.293
		±SD	0.048	0.149	0.217	0.050	0.150

GROUP MEAN RELATIVE ORGAN WEIGHTS (%)

Sex : Male

Day : 29

 

GroupNumber	Dose mg/kg		Body Weight (g)	Brain	Liver	Kidneys	Adrenals	Testes	Prostate + Seminal Vesicle with Coagulation gland
I	0	Mean	304.27	0.668	4.326	0.798	0.0199	1.048	0.490
		±SD	19.75	0.029	0.404	0.034	0.0039	0.043	0.082
II	250	Mean	298.33	0.650	4.138	0.753	0.0183	1.033	0.423
		±SD	15.62	0.055	0.368	0.066	0.0012	0.120	0.057
III	500	Mean	288.80	0.685	4.280	0.752	0.0182	0.899*	0.393
		±SD	13.01	0.036	0.381	0.153	0.0025	0.099	0.085
IV	1000	Mean	294.33	0.680	4.131	0.798	0.0161	1.008	0.412
		±SD	10.28	0.031	0.379	0.072	0.0026	0.083	0.041

 

GroupNumber	Dose mg/kg		Heart	Spleen	Lungs	Thymus	Epididymides
I	0	Mean	0.448	0.461	0.578	0.141	0.251
		±SD	0.042	0.068	0.063	0.031	0.015
II	250	Mean	0.413	0.448	0.546	0.125	0.225
		±SD	0.014	0.115	0.080	0.022	0.018
III	500	Mean	0.428	0.491	0.631	0.126	0.228
		±SD	0.041	0.095	0.162	0.025	0.014
IV	1000	Mean	0.385	0.505	0.521	0.116	0.228
		±SD	0.048	0.173	0.110	0.026	0.022

*= Significant at 95% level of confidence

 

GROUP MEAN RELATIVE ORGAN WEIGHTS (%)

Sex : Female

Day : 29

 

GroupNumber	Dose mg/kg		Body Weight (g)	Brain	Liver	Kidneys	Adrenals	Ovaries
I	0	Mean	217.20	0.880	3.695	0.758	0.0257	0.0378
		±SD	11.85	0.047	0.262	0.038	0.0031	0.0047
II	250	Mean	207.97	0.892	3.672	0.709	0.0270	0.0324
		±SD	16.31	0.060	0.323	0.055	0.0049	0.0055
III	500	Mean	206.17	0.879	3.766	0.736	0.0272	0.0368
		±SD	11.88	0.032	0.372	0.095	0.0029	0.0094
IV	1000	Mean	201.28	0.910	3.588	0.734	0.0253	0.0349
		±SD	8.75	0.053	0.396	0.066	0.0032	0.0030

 

Group No.	Dose mg/kg		Heart	Spleen	Lungs	Thymus	Uterus
I	0	Mean	0.439	0.519	0.641	0.201	0.160
		±SD	0.022	0.118	0.159	0.047	0.041
II	250	Mean	0.423	0.673	0.709	0.183	0.149
		±SD	0.024	0.304	0.131	0.057	0.041
III	500	Mean	0.410	0.496	0.792	0.177	0.142
		±SD	0.041	0.081	0.089	0.045	0.038
IV	1000	Mean	0.405	0.413	0.654	0.171	0.146
		±SD	0.015	0.060	0.084	0.021	0.076

 

 SUM

MARY OF GROSS PATHOLOGY FINDINGS

Sex : Male

 

Site and lesion observed	Group	I	II	III	IV
	Dose (mg/kg)	0	250	500	1000
No Abnormality Detected		1 - 6	13 - 18	25 - 30	37 - 42

 

SUMMARY OF GROSS PATHOLOGY FINDINGS

Sex : Female

 

Site and lesion observed	Group	I	II	III	IV
	Dose (mg/kg)	0	250	500	1000
No Abnormality Detected		7 - 12	19 - 24	31 - 36	43 - 48

 

 

                                         

 

 

 

 

 

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for the test chemical in Sprague Dawley Rats, via oral gavage, for 28 days was considered to be 1000 mg/kg bw/day.

Executive summary:

The Reproductive organ toxicity study for 28 -days by oral route was designed and conducted to determine the reproductive toxicity profile of the test chemical when administered daily for 28 days in the Sprague Dawley rats. The test chemical was dissolved in Polyethylene glycol and used at dose level of 0, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight. During the study period, the treated animals were observed or mortality, cllinical signs, body weight and food consumption changes, hematology and clinical chemistry, neurobehaviour, urinalysis, ophthalmology and were subjected to gross and histopathology. All the male and female animals from control and all the treated dose groups up to 1000 mg/kg survived throughout the dosing period of 28 days. Male and female animals from control and all the treated dose groups exhibited normal body weight gain and food intake at the end of the dosing period of 28 days. Daily and detailed (weekly) clinical observations did not reveal any signs of toxicity in male and female animals from different dose groups during the dosing period of 28 days. Towards the end of the exposure period in week 4, functional observation battery such as sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) revealed no abnormalities attributable to the treatment. Grip strength values observed in male and female animals for control and different dose groups were comparable. Higher values for motor activity were observed in male animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups for second interval. Lower values for motor activity were observed in female animals from 1000 mg/kg dose group for first and second interval. These changes were within laboratory range and were considered to be of no toxicological importance. Haematological analysis performed on 29^thday revealed statistically significant increase in the values of MCV, MCH and Total WBC of male rats dosed at 250 mg/kg, increase values of Platelets of male rats dosed at 250 mg/kg and 1000 mg/kg and increased values of Platelets of female rats dosed at 500 mg/kg. In addition, statistically significant decrease was observed in the values of Hb and HCT of male rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg, decreased values of Total RBC of male rats dosed at 250 mg/kg and 500 mg/kg, decreased values of MCV and MCH of male rats dosed at 1000 mg/kg, decreased values of Hb of female rats dosed at 250 mg/kg and 500 mg/kg, decreased values of Total RBC, HCT and Total WBC of female rats dosed at 500 mg/kg, decreased values of MCV of female rats dosed at 1000 mg/kg, decreased values of MCH and MCHC of female rats dosed at 250 mg/kg and 1000 mg/kg, the increase / decrease in the values of different parameters were marginal and within the normal laboratory limits. Statistically significant increase of Total Protein (in male rats at 500 mg/Kg/day), Creatinine (in male rats at 1000 mg/Kg/day), Cholesterol (in male rats at 500 and 1000 mg/Kg/day), Sodium (in female rats at 250, 500 and 1000 mg/Kg/day) and statistically significant decrease in Bilirubin (in male rats at 250 and 500 mg/Kg/day), Chloride and Alkaline Phosphatase (in female rats at 500 mg/Kg/day) was observed. Although there was an increase/decrease in the values of various biochemical parameters, the deviations were marginal and within the range of normal laboratory limits. Organ weight data of male animals sacrificed on day 29, revealed decreased relative weights of testes of animals from 500 mg/kg dose group. Although significant changes in organ weights were observed in male animals from intermediate dose group, no related gross pathological or histological changes were seen and hence considered to be of no toxicological importance. Organ weight data of female animals sacrificed on day 29, was found to be comparable with that of controls. Gross pathological examination conducted in rats of all dose groups during necropsy did not reveal any abnormality attributable to the treatment. Histopathological examination conducted in rats of control and high dose groups did not reveal any abnormality attributable to the treatment. Hence based on the observations made, the No Observed Adverse Effect Level (NOAEL) for the test chemical in Sprague Dawley Rats, via oral gavage, for 28 days was considered to be 1000 mg/kg bw/day.

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:

Klimisch 1

Effects on developmental toxicity

Description of key information

Study 1

In the OECD 414 conducted with Wistar rats, the NOAELs for maternal developmental toxicity and foetal developmental toxicity were established at 1000 and 500 mg/kg bw/day, respectively. NOAEL for maternal general toxicity could not be established due to minor but statistically significant decreases in body weight and body weight gain at 250 mg/kg bw/day. The foetal effects observed at 1000 mg/kg bw/day included significant decreases in body weight (sex-combined [i.e. 11.0% decrease vs controls] and of females [i.e. 11.0% decrease vs controls]) that were minimally or slightly below the historical control range of the test facillity. These foetal effects were considered secondary to maternal general toxicity, which at 1000 mg/kg bw/day included significant decreases in body weight (on GD 8, by 5.3%; on GD 14, by 5.5%; on GD 17, by 6.1%; and on GD 20, by 7.3%) and significant decreases in body weight gain (on GD 0-8, by 53.5%; on GD 0-11, by 32.4%; on GD 0-14, by 28.6%; on GD 0-17, by 24.0%; and on GD 0-20, by 22.0%). A significant positive correlation was found between test item dose and the incidence of unossified sternebrae (skeletal variation) at 500 and 1000 mg/kg bw/day. The increased incidence of this skeletal variation was considered treatment-related but non-adverse.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

The health status of each animal used in this study was examined on receipt (initial examination) by a veterinarian. All received animals were found to be in good health and were acclimatised to the laboratory conditions. Before mating, a veterinary examination was carried out by the study veterinarian and it was ascertained that all animals were still in good condition. Prior to receipt of animals for the study, the study room, racks and cages were cleaned and disinfected. During the study, the floor of the experimental room and work tops were swept and mopped with disinfectant solution at least once every day and more frequently when required. Cages were cleaned regularly. One to three rats were housed in each polycarbonate cage (length 37 cm X breadth 21 cm X height 20 cm). During mating, one male and two female rats were housed in a single cage. Pregnant females were housed individually. Sterilized corn-cob produced from pure corn, dried and free from dust, procured from an approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean. Bedding material of batch no. 720 (Krishna Corncob Industries, Aurangabad) was used and a copy of the analyses for microbial and chemical contaminants of bedding material provided by the manufacturer has been incorporated in the raw data.
Environmental conditions: The room temperature was maintained between 21.00 to 23.30 °C and the relative humidity achieved was in the range of 50.10 to 63.80%. Artificial light was set to give a cycle of 12 hours light and 12 hours dark. Adequately filtered air with at least 12 changes per hour was provided. A conventional laboratory pellet diet (Batch no. 040820) from approved supplier (Nutrivet Life Sciences, Pune) was available ad libitum. Copies of the composition, microbial and chemical contaminant reports analysed by the manufacturer have been incorporated in the raw data.Aquaguard™ filtered drinking water was available ad libitum in regularly cleaned bottles. Samples of the drinking water at sa-FORD are periodically subjected to tests for bacteriological and chemical contaminants. The latest test results are included in the raw data.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methyl cellulose

Details on exposure:

The dose volume for each animal was calculated based on the most recently recorded body weight. Each formulation was administered in a single dose through oral gavage at a constant volume of 10 ml per kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulation samples [of doses viz; G1 (Control), G2 (Low dose), G3 (Mid dose), and G4 (High dose)] were analysed by HPLC method. Two replicates of approximately 2 ml samples from each upper, middle, and lower layer were sent to Chemistry Department for homogeneity and active ingredient analysis. The concentrations were calculated and reported. The dose formulation analysis was carried out during the first and the last week of treatment using a validated analytical method

Details on mating procedure:

For mating, two females were kept with a single male (2:1 pairing) until evidence of copulation was observed. The presence of sperm in vaginal smear was considered as day 0 of pregnancy/gestation. Females showing sperm positive smear were separated and housed individually.

Duration of treatment / exposure:

15 days (i.e. on GD 5 up to and including 19).

Frequency of treatment:

Once daily.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

G2

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

G3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Maternal examinations:

All animals were observed twice daily (once before and once after the day’s activities) for any morbidity and/or mortality during acclimatization and treatment. All animals were observed daily for clinical signs and symptoms after dose administration. These observations were regularly performed approximately 1 hour after dose administration sincethe onset of clinical signs after oral gavage can be anticipated in that time frame. Animals were weighed at the time of receipt, on GD 0, on the first day of dosing (GD 5), and on gestation days 8, 11, 14, 17 and 20. The weight of feed provided, and that leftover was recorded for each cage on the same day as animal body weight. This data was used to calculate mean feed consumption (g/day/rat) on gestation days 5, 8, 11, 14, 17, and 20. Blood samples were collected from all dams on the day of termination through retro-orbital plexus. Serum from all blood samples were separated, stored under appropriate conditions, and measured for serum levels of thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) using commercially available Rat ELISA kits (KinesisDx USA).

Ovaries and uterine content:

Immediately after the termination, uteri were removed, and the pregnancy status of each animal was evaluated. Uteri that appear non-gravid were further examined using ammonium sulphide staining to confirm non-pregnant status.The total weight of the gravid or non-gravid uteri, including the cervix, was recorded for each animal. The number of corpora lutea and the ovarian and placental weights were recorded for each pregnant animal. Uterine contents were examined for the number of implantation sites, number of live/viable foetuses, number of dead foetuses and number of resorptions (early and late resorptions). Using the above information, percent pre-implantation and post-implantation losses were calculated as follows: pre-implantation loss = [(no. of corpora lutea – no. of implantations)/ no. of corpora lutea] x 100 and post-implantation loss = [(no. of implantations – no. of viable foetuses/ no. of implantations] x 100. All animals were subjected to complete gross necropsy. From all dams at termination, thyroid glands were collected, weighed (after fixation) and preserved for histopathological examination.

Fetal examinations:

From every individual litter, each foetus was weighed, sexed and examined for external abnormalities and crown to rump length. For each live foetus, anogenital distances (AGD) was measured. One-half of the foetuses from each litter were prepared and examined for skeletal abnormalities (growth retardation, delayed ossification, etc.) using Alcian blue and Alizarin red double-staining technique. The remaining foetuses from each litter were prepared for and examined for soft tissue alterations (visceral and head razor sectioning) with special emphasis on thereproductive tract. Each male foetus was also evaluated for incomplete testicular descent/cryptorchidism.

Statistics:

Raw data were processed using statistical software “Sigma Plot 14.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviations were calculated using the software and all data were summarized in tabular form. All continuous data (body weight, feed consumption, hormone estimation, absolute and relative organ weights, maternal and pup parameters) were checked for normality using Shapiro-Wilk test. All homogenous data were analysed using ANOVA and data showing significance in their variance was subjected to Dunnett’s t-test. All heterogeneous data was analysed using Kruskal-Wallis, ANOVA on ranks. Gross, skeletal, and visceral abnormalities were represented as both the total number of foetuses and litters affected and the percentage of foetuses and litters affected. Further, the data of percent incidences of abnormalities were analysed for dose dependency by Pearson’s correlation. P values of ≤ 0.05 were deemed to be statistically significant.

Historical control data:

Inhouse.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs or symptoms were observed in G1, G2 or G3 during the study period. Clinical signs in the G4 animals included hypothermia and incoordination (all animals on the first day of treatment) and numerous cases of chromodacryorrhea from the 2nd to 10th day of treatment

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All animals survived to planned death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases in body weight in treated pregnant dams vs. controls were observed at 250 mg/kg (on GD 20, by 4.4%), at 500 mg/kg (on GD 17, by 5.5%; on GD 20, by 6.3%) and at 1000 mg/kg (on GD 8, by 5.3%; on GD 14, by 5.5%; on GD 17, by 6.1%; and on GD 20, by 7.3%).
Significant decreases in body weight gain in treated pregnant dams vs controls were observed at 250 mg/kg (on GD 0-20, by 11.2%), at 500 mg/kg (on GD 0-8, by 25.9%; on GD 0-14, by 14.5%; on GD 0-17, by 16.5%; and on GD 0-20, by 15.3%) and at 1000 mg/kg (on GD 0-8, by 53.5%; on GD 0-11, by 32.4%; on GD 0-14, by 28.6%; on GD 0-17, by 24.0%; and on GD 0-20, by 22.0%).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No significant decreases in food intake were observed in pregnant dams treated at 250 mg/kg. At 500 mg/kg, significant decreaes in food intake were observed on GD 8 (mean, 13.92 g. vs mean 17.89 g. at 0 mg/kg), GD 11 (mean, 17.41 g. vs. mean, 19.46 g at 0 mg/kg) and GD 14 (mean, 20.95 g. vs. mean, 22.74 g. at 0 mg/kg). At 1000 mg/kg, significant decreaes in food intake were observed on GD 8 (mean, 9.53 g. vs. mean 17.89 g. at 0 mg/kg) and GD 14 (mean, 20.05 g. vs. mean, 22.74 g. at 0 mg/kg).

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):

No significant differences were observed in Triiodothryronine (T3) and Thyroxin (T4) levels in pregnant females of any dose group as compared to the G1 control group. Thyroid stimulating hormone (TSH) level was slighly but statistically decreased in the G4 group (mean, 0.16 microlitre IU/ml) as compared to control group (mean 0.21 microlitre IU/ml)). Since no associated changes were observed in Triiodothryronine (T3) or Thyroxin (T4) levels and no associated gross or histopathological alterations of the thyroid were noted; the observed small decrease in TSH in G4 animals was considered incidental/non-adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and organ weight relative weight to body weight of ovary and thyroid-parathyroid remained statistically comparable between all the groups. A significant reduction in absolute uterus weight was observed in G4 pregnant dams (mean 55.68 g.) as compared to the control group (mean, 67.15 g). No other significant changes in uterus weight (either absolute or relative to body weight) were observed in the study. Since also no gross findings were made in the uteri from G4, the significant decrease in absolute uterus weight was considered incidental/non-adverse. A significant increase in placenta weight was observed in G3 (mean, 0.47 g) and G4 (mean, 0.48 g) as compared to G1 (mean, 0.44g) group. The observed placenta weights in G3 and G4 group were well within the historical control range of the facility (0.40-0.52 g); hence the significant changes in placenta weights were considered incidental/non-adverse.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological alteration was observed in any of the dams.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Thyroid and parathyroid gland from G1 and G4 did not exhibit any microscopic lesion of toxicologic significance. Focal ultimobranchial cysts were found in 2 animals of G1 and in one animal from G4 group. The cyst formations were considered congenital as they were noticed in both control and treated groups, and therefore it was considered a spontaneous and non-test item-related effect. Dilatations of follicles of the thyroid gland were also noticed in G1 and G4 groups (viz., Dam No. 1, 5, 10, 13, for G1 and Dam No.76, 82, 89, 93 for G4). These dilated follicles were considered to be incidental changes as they appeared in both control and treated groups at comparable incidences.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

No cases of abortion were observed in any of the groups.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No significant changes were observed.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of total litter resorption were 1 of 22 dams with confirmed pregnancy at necropsy at 500 mg/kg and 2 of 24 dams with confirmed pregnancy at necropsy at 1000 mg/kg. All three cases were attributed to early resorptions. This effect was considered to be incidental/non-adverse since no significant changes in pregnancy rate with live foetuses were observed. The pregnancy rate with live foetuses were 96, 92, 84, and 88% at G1, G2, G3, and G4, respectively, and all of these values were within the historical control range of the test facility (i.e. of 72-96%).

Early or late resorptions:

no effects observed

Description (incidence and severity):

No significant changes were observed.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetus was observed in any of the groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Pregnancy rates were 92, 84, 88, and 96% in G1, G2, G3, and G4, respectively.

Other effects:

no effects observed

Description (incidence and severity):

No significant differences in the number of corpora lutea were observed.

Dose descriptor:

NOAEL

Remarks:

maternal general toxicity

Based on:

test mat.

Remarks on result:

not determinable because of methodological limitations

Remarks:

Mild but statistically significant changes in body weight were observed the lowest dose level

Dose descriptor:

LOAEL

Remarks:

maternal general toxicity

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

changes in number of pregnant

dead fetuses

early or late resorptions

maternal abnormalities

number of abortions

pre and post implantation loss

total litter losses by resorption

other: Number of corpora lutea

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases in the mean body weight of foetuses (both sex-combined) were observed in G3 (mean, 3.18 g, i.e. 5.9% decrease vs. controls) and G4 group (mean, 3.01 g, i.e. 11.0% decrease vs controls) as compared to G1 (mean, 3.38 g). Significant decreases in mean body weight of male foetuses were observed in G3 (mean, 3.29 g, i.e. 5.7% decrease vs controls) and G4 groups (mean, 3.14 g, i.e. 10.5% decrease vs controls) as compared to control group (mean, 3.49 g). Furthermore, significant decreases in the mean body weight of female foetuses were observed in G3 (mean, 3.03 g, i.e. 7.6% decrease vs controls) and G4 groups (mean, 2.92 g, i.e. 11.0% decrease vs controls) as compared to G1 (mean, 3.28 g).
The mean body weights of foetuses from the G3 group were well within the historical control range (male & female combined weight: 3.02-3.9 g; male: 3.11-4.03 g, female: 2.99-3.75 g). The significant decreases in foetal weight in G4 for sex-combined and females were minimally/slightly below the historical control range and were therefore considered treatment-related and adverse.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Mean numbers of live foetuses per dam were 12.44, 10.24, 9.56, and 10.00 in G1, G2, G3, and G4, respectively. No significant differences were observed.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

M/F sex ratios were 1.02, 1.26, 1.48, and 0.83 in G1, G2, G3 and G4, respectively. No significant differences were observed.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Mean litter sizes were 12.96, 11.13, 11.38, and 11.36 in G1, G2, G3, and G4, respectively. No significant differences were observed.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Gross external observation of foetuses revealed that 3.22 (29.17), 1.56 (17.39), 3.35 (28.57) and 3.20 (36.36) percentages of foetuses (litters) from G1, G2, G3 and G4, respectively demonstrated variations whereas 0.96 (12.50), 0.78 (4.35), 0.42 (4.76) and 2.80 (31.82) percentages of foetuses (litters) from G1, G2, G3 and G4, respectively showed malformations. The variation included haemorrhage observed in 3.22 (29.17), 1.56 (17.39), 3.35 (28.57) and 3.20 (36.36) percent of foetuses (litters) from G1, G2, G3 and G4, respectively. The malformations included: retarded growth (Runt): 0.64 (8.33) in G1, 0.78 (4.35) in G2, 0.42 (4.76) in G3 and 2.80 (31.82) in G4 percent of foetuses (litters); dome-shaped head: 0.32 (4.17) (G1) and 0.40 (4.55) (G4) percent of foetuses (litters); Short neck and opened eye each observed in 0.40 (4.55) (G4) percent of foetuses (litters). No significant difference were observed when individual or total malformations or variations were compared between the groups.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal malformation included unossified supraoccipital observed in skull of 0.76 (4.55) percentage of foetuses (litters) from G4. No skeletal malformation was found in foetuses of G1, G2 and G3 groups. The total percentage of foetuses (litters) with skeletal variations were 6.25 (25.00), 3.01 (17.39), 4.03 (14.29) and 12.88 (40.91) in G1, G2, G3 and G4 group, respectively. Skeletal variations observed in rib included: rudimentary rib in 0.76 (4.55) percentage of foetuses (litters) from G4; misaligned ribs found in 5.00 (20.83), 2.26 (13.04), 3.23 (9.52) and 4.55 (18.18) percentage of foetuses (litters) from G1, G2, G3 and G4, respectively. Skeletal variations observed in sternebrae included: unossified/incompletely ossified sternebrae observed in 4.38 (4.17), 0.75 (4.35), 3.23 (14.29) and 9.85 (31.82) percentage of foetuses (litters) of G1, G2, G3 and G4, respectively; misaligned sternebrae were seen in 3.75 (20.83), 2.26 (13.04), 3.23 (9.52) and 4.55 (18.18) percentage of foetuses (litters) from G1, G2, G3 and G4.
Most skeletal abnormalities noted in treatment groups were concurrently seen in the control group. Moreover, the occurrence and incidences of these malformations/variations are common in developing foetuses. However, a positive correlation was observed in the doses of the test item and % litters with the incidences of unossified sternebrae (P≤0.05 and r = 0.970). The increased incidence of this skeletal variation, which was notable at 500 and 1000 mg/kg bw/day, was considered treatment-related but non-adverse

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral variations observed included: hematoma found in 1 foetus each from G2 and G3 group; and congestion on left kidney and liver found in 2 foetuses of 1 dam (dam no. 65) from G3 group. These observations were considered spontaneous in nature and not treatment-related due to lack of dose dependency. No other malformations or variations were observed in any other foetuses of control or treated groups. The head razor examination did not show any malformation or variation in any of the foetuses in any of the groups.

Other effects:

no effects observed

Description (incidence and severity):

A significant decrease in crown to rump length (CRL) was observed in foetuses of G4 (mean, 3.29 cm) as compared to control group (mean, 3.42 cm). The observed decrease in CRL of G4 foetuses was within the historical control range of test facility (3.23-3.91 cm) and this effect was therefore considered to be non-adverse. A significant increase in normalized AGD was observed in male foetuses of G4 group (mean, 2.10) as compared to G1 (mean, 2.04). The mean normalized AGD was within the historical control range of facility (1.83-2.29) and thus the observed increase in normalized AGD in G4 males was not considered to be an adverse effect of treatment. No significant changes in normalized AGD were observed for the female foetuses between the doses.

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

other: crown to rump length, AGD

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

no effects observed

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Conclusions:

In this OECD 414 conducted with Wistar rats, the NOAELs for maternal developmental toxicity and foetal developmental toxicity were established at 1000 and 500 mg/kg bw/day, respectively. NOAEL for maternal general toxicity could not be established due to minor but statistically significant decreases in body weight and body weight gain at 250 mg/kg bw/day. The foetal effects observed at 1000 mg/kg bw/day included significant decreases in body weight (sex-combined [i.e. 11.0% decrease vs controls] and of females [i.e. 11.0% decrease vs controls]) that were minimally or slightly below the historical control range of the test facillity. These foetal effects were considered secondary to maternal general toxicity, which at 1000 mg/kg bw/day included significant decreases in body weight (on GD 8, by 5.3%; on GD 14, by 5.5%; on GD 17, by 6.1%; and on GD 20, by 7.3%) and significant decreases in body weight gain (on GD 0-8, by 53.5%; on GD 0-11, by 32.4%; on GD 0-14, by 28.6%; on GD 0-17, by 24.0%; and on GD 0-20, by 22.0%). A significant positive correlation was found between test item dose and the incidence of unossified sternebrae (skeletal variation) at 500 and 1000 mg/kg bw/day. The increased incidence of this skeletal variation was considered treatment-related but non-adverse.

Executive summary:

The present study aimed to provide information on systemic, maternal, and developmental toxicological endpoints associated with the repeated oral administration of graduated doses of Rose crystal ex benzaldehyde (CAS No. 90-17-5) to Wistar rats. For this purpose, the OECD Test Guideline 414 (Adopted 25 June 2018) was followed. A total of 100 pregnant female rats were randomised into 4 experimental groups each containing 25 animals. The groups, viz., G1, G2, G3 and G4, received 0, 250, 500 and 1000 mg/kg bw/day of Rose crystal ex benzaldehyde (CAS No. 90-17-5), respectively. The test item was administrated via oral gavage and the vehicle used in this study was 0.5% w/v methyl cellulose. All groups were administered with vehicle or with dose formulation once daily from gestation day (GD) 5 to 19 (i.e. to one day prior to scheduled sacrifice). To ensure the accurate administration of the test item, the concentrations and homogeneity of the dose formulation were verified through formulation analyses in the first and last week of treatment. The results of these analyses were within the acceptable limits on both occasions. Observations on the animals included mortality/morbidity, onset of clinical signs/symptoms, body weight, body weight changes, feed consumption, pregnancy observations, foetal developmental parameters, serum hormone levels, gross pathology and histopathology. All animals survived to planned death. No clinical signs or symptoms were observed in G1, G2 or G3 during the study period. Clinical signs in the G4 animals included hypothermia and incoordination (on the first day of treatment) and numerous cases of chromodacryorrhea between the 2nd and 10th day of treatment. Significant decreases in the mean body weight and % change in body weight with respect to GD 0 were observed in G2, G3, and G4 as compared to the control group. The decreases were dose-dependent and attributed to the test item. Significant decreases in feed consumption were observed in G3 and G4 as compared to the control group. No significant differences were observed in triiodothyronine (T3) and Thyroxin (T4) levels in pregnant females of any group as compared to the G1 control group. Thyroid stimulating hormone (TSH) level was significantly decreased in the G4 group (mean, 0.16 microlitre IU/ml) as compared to control group (mean 0.21 microlitre IU/ml)). Since no associated changes were observed in triiodothyronine (T3) or Thyroxin (T4) levels and no associated gross or histopathological alterations of the thyroid were noted; the observed small decrease in TSH in G4 animals was considered incidental/non-adverse. The absolute and organ weight relative weight to body weight of ovary and thyroid-parathyroid remained statistically comparable between all the groups. A significant reduction in absolute uterus weight was observed in G4 pregnant dams (mean 55.68 g.) as compared to the control group (mean, 67.15 g). No other significant changes in uterus weight (either absolute or relative to body weight) were observed in the study. Since also no gross findings were made in the uteri from G4, the significant decrease in absolute uterus weight was considered incidental/non-adverse. A significant increase in placenta weight was observed in G3 (mean, 0.47 g) and G4 (mean, 0.48 g) as compared to G1 (mean, 0.44g) group. The observed placenta weights in G3 and G4 group were well within the historical control range of the facility (0.40-0.52 g); hence the significant changes in placenta weights were not considered to be adverse effects of treatment. No treatment-related changes were observed during the gross or histopathologic examination. The incidences of total litter resorption were 1 of 22 dams with confirmed pregnancy at necropsy at 500 mg/kg and 2 of 24 dams with confirmed pregnancy at necropsy at 1000 mg/kg. All three cases were attributed to early resorptions. This effect was considered to be incidental/non-adverse since no significant changes in pregnancy rate with live foetuses were observed. The pregnancy rate with live foetuses were 96, 92, 84, and 88% at G1, G2, G3, and G4, respectively, and all of these values were within the historical control range of the test facility (i.e. of 72-96%). Maternal parameters including pregnancy ratio, number of corpora lutea, implantation sites, resorptions and percentage of pre- and post-implantation loss remained comparable between all groups. Significant decreases in the mean body weight of foetuses (both sex-combined) were observed in G3 (mean, 3.18 g, i.e. 5.9% decrease vs. controls) and G4 group (mean, 3.01 g, i.e. 11.0% decrease vs controls) as compared to G1 (mean, 3.38 g). Significant decreases in mean body weight of male foetuses were observed in G3 (mean, 3.29 g, i.e. 5.7% decrease vs controls) and G4 groups (mean, 3.14 g, i.e. 10.5% decrease vs controls) as compared to control group (mean, 3.49 g). Furthermore, significant decreases in the mean body weight of female foetuses were observed in G3 (mean, 3.03 g, i.e. 7.6% decrease vs controls) and G4 groups (mean, 2.92 g, i.e. 11.0% decrease vs controls) as compared to G1 (mean, 3.28 g). The mean body weights of foetuses from the G3 group were well within the historical control range (male & female combined weight: 3.02-3.9 g; male: 3.11-4.03 g, female: 2.99-3.75 g). The significant decreases in foetal weight in G4 for sex-combined and females were minimally/slightly below the historical control range and were therefore considered treatment-related. Mean numbers of live foetuses per dam were 12.44, 10.24, 9.56, and 10.00 in G1, G2, G3, and G4, respectively with no significant differences were observed. M/F sex ratios were 1.02, 1.26, 1.48, and 0.83 in G1, G2, G3 and G4, respectively with no significant differences were observed. Mean litter sizes were 12.96, 11.13, 11.38, and 11.36 in G1, G2, G3, and G4, respectively with no significant differences were observed. A significant decrease in crown to rump length (CRL) was observed in foetuses of G4 (mean, 3.29 cm) as compared to control group (mean, 3.42 cm). The observed decrease in CRL of G4 foetuses was within the historical control range of test facility (i.e. of 3.23 - 3.91 cm) and this effect was therefore considered to be incidental/non-adverse. A significant increase in normalized AGD was observed in male foetuses of G4 group (mean, 2.10) as compared to G1 (mean, 2.04). The mean normalized AGD was within the historical control range of facility (i.e. of 1.83-2.29) and thus the observed increase in normalized AGD in G4 males was not considered to be an adverse effect of treatment. No significant changes in normalized AGD were observed for the female foetuses between the doses. No treatment-related effects were observed during the gross, visceral or skeletal examination except for a significant positive correlation found between the doses of test item and the incidence of unossified sternebrae (skeletal variation). The increased incidence of this skeletal variation, which was notable at 500 and 1000 mg/kg bw/day, was considered treatment-related but non-adverse.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Additional information

OECD 414 study:

The present study aimed to provide information on systemic, maternal, and developmental toxicological endpoints associated with the repeated oral administration of graduated doses of Rose crystal ex benzaldehyde (CAS No. 90-17-5) to Wistar rats. For this purpose, the OECD Test Guideline 414 (Adopted 25 June 2018) was followed. A total of 100 pregnant female rats were randomised into 4 experimental groups each containing 25 animals. The groups, viz., G1, G2, G3 and G4, received 0, 250, 500 and 1000 mg/kg bw/day of Rose crystal ex benzaldehyde (CAS No. 90-17-5), respectively. The test item was administrated via oral gavage and the vehicle used in this study was 0.5% w/v methyl cellulose. All groups were administered with vehicle or with dose formulation once daily from gestation day (GD) 5 to 19 (i.e. to one day prior to scheduled sacrifice). To ensure the accurate administration of the test item, the concentrations and homogeneity of the dose formulation were verified through formulation analyses in the first and last week of treatment. The results of these analyses were within the acceptable limits on both occasions. Observations on the animals included mortality/morbidity, onset of clinical signs/symptoms, body weight, body weight changes, feed consumption, pregnancy observations, foetal developmental parameters, serum hormone levels, gross pathology and histopathology. All animals survived to planned death. No clinical signs or symptoms were observed in G1, G2 or G3 during the study period. Clinical signs in the G4 animals included hypothermia and incoordination (on the first day of treatment) and numerous cases of chromodacryorrhea between the 2ndand 10thday of treatment. Significant decreases in the mean body weight and % change in body weight with respect to GD 0 were observed in G2, G3, and G4 as compared to the control group. The decreases were dose-dependent and attributed to the test item. Significant decreases in feed consumption were observed in G3 and G4 as compared to the control group. No significant differences were observed in triiodothyronine (T3) and Thyroxin (T4) levels in pregnant females of any group as compared to the G1 control group. Thyroid stimulating hormone (TSH) level was significantly decreased in the G4 group (mean, 0.16 microlitre IU/ml) as compared to control group (mean 0.21 microlitre IU/ml)). Since no associated changes were observed in triiodothyronine (T3) or Thyroxin (T4) levels and no associated gross or histopathological alterations of the thyroid were noted; the observed small decrease in TSH in G4 animals was considered incidental/non-adverse. The absolute and organ weight relative weight to body weight of ovary and thyroid-parathyroid remained statistically comparable between all the groups. A significant reduction in absolute uterus weight was observed in G4 pregnant dams (mean 55.68 g.) as compared to the control group (mean, 67.15 g). No other significant changes in uterus weight (either absolute or relative to body weight) were observed in the study. Since also no gross findings were made in the uteri from G4, the significant decrease in absolute uterus weight was considered incidental/non-adverse. A significant increase in placenta weight was observed in G3 (mean, 0.47 g) and G4 (mean, 0.48 g) as compared to G1 (mean, 0.44g) group. The observed placenta weights in G3 and G4 group were well within the historical control range of the facility (0.40-0.52 g); hence the significant changes in placenta weights were not considered to be adverse effects of treatment. No treatment-related changes were observed during the gross or histopathologic examination. The incidences of total litter resorption were 1 of 22 dams with confirmed pregnancy at necropsy at 500 mg/kg and 2 of 24 dams with confirmed pregnancy at necropsy at 1000 mg/kg. All three cases were attributed to early resorptions. This effect was considered to be incidental/non-adverse since no significant changes in pregnancy rate with live foetuses were observed. The pregnancy rate with live foetuses were 96, 92, 84, and 88% at G1, G2, G3, and G4, respectively, and all of these values were within the historical control range of the test facility (i.e. of 72-96%). Maternal parameters including pregnancy ratio, number of corpora lutea, implantation sites, resorptions and percentage of pre- and post-implantation loss remained comparable between all groups. Significant decreases in the mean body weight of foetuses (both sex-combined) were observed in G3 (mean, 3.18 g, i.e. 5.9% decrease vs. controls) and G4 group (mean, 3.01 g, i.e. 11.0% decrease vs controls) as compared to G1 (mean, 3.38 g). Significant decreases in mean body weight of male foetuses were observed in G3 (mean, 3.29 g, i.e. 5.7% decrease vs controls) and G4 groups (mean, 3.14 g, i.e. 10.5% decrease vs controls) as compared to control group (mean, 3.49 g). Furthermore, significant decreases in the mean body weight of female foetuses were observed in G3 (mean, 3.03 g, i.e. 7.6% decrease vs controls) and G4 groups (mean, 2.92 g, i.e. 11.0% decrease vs controls) as compared to G1 (mean, 3.28 g). The mean body weights of foetuses from the G3 group were well within the historical control range (male & female combined weight: 3.02-3.9 g; male: 3.11-4.03 g, female: 2.99-3.75 g). The significant decreases in foetal weight in G4 for sex-combined and females were minimally/slightly below the historical control range and were therefore considered treatment-related.Mean numbers of live foetuses per dam were 12.44, 10.24, 9.56, and 10.00 in G1, G2, G3, and G4, respectively with no significant differences were observed. M/F sex ratios were 1.02, 1.26, 1.48, and 0.83 in G1, G2, G3 and G4, respectively with no significant differences were observed. Mean litter sizes were 12.96, 11.13, 11.38, and 11.36 in G1, G2, G3, and G4, respectively with no significant differences were observed. A significant decrease in crown to rump length (CRL) was observed in foetuses of G4 (mean, 3.29 cm) as compared to control group (mean, 3.42 cm). The observed decrease in CRL of G4 foetuses was within the historical control range of test facility (i.e. of3.23-3.91 cm) and this effect was therefore considered to beincidental/non-adverse. A significant increase in normalized AGD was observed in male foetuses of G4 group (mean, 2.10) as compared to G1 (mean, 2.04). The mean normalized AGD was within the historical control range of facility (i.e. of1.83-2.29) and thus the observed increase in normalized AGD in G4 males was not considered to be an adverse effect of treatment. No significant changes in normalized AGD were observed for the female foetuses between the doses. No treatment-related effects were observed during the gross, visceral or skeletal examination except for a significant positive correlation found between the doses of test item and the incidence of unossified sternebrae (skeletal variation). The increased incidence of this skeletal variation, which was notable at 500 and 1000 mg/kg bw/day, was considered treatment-related but non-adverse.

Justification for classification or non-classification

In the OECD 414 study with Wistar rats, adverse effects on development were limited to lower foetal weights at 1000 mg/kg/day in both females (mean, 2.92 g. vs. mean, 3.28 g. in controls) and in male and females combined (mean 3.01 g. vs. mean, 3.38 in controls). Each decrease in foetal weight at 1000 mg/kg amounted to an 11.0% decrease compared to the control data. The average foetal weight for females at 1000 mg/kg/day (i.e. 2.92 g) was slightly below the historical control range of the test facility (i.e. 2.99 – 3.75 g) and the average foetal weight for male and females combined at 1000 mg/kg (i.e. 3.01 g) was minimally below the historical control range of the test facility (i.e. 3.02 – 3.9 g). The significant decrease in foetal weight were observed in the presence of maternal general toxicity, which at 1000 mg/kg/day included significant decreases in body weight (on GD 8, by 5.3%; on GD 14, by 5.5%; on GD 17, by 6.1%; and on GD 20, by 7.3%) and significant decreases in body weight gain (on GD 0-8, by 53.5%; on GD 0-11, by 32.4%; on GD 0-14, by 28.6%; on GD 0-17, by 24.0%; and on GD 0-20, by 22.0%). Considering that the average foetal weights at 1000 mg/kg/day were only slightly or marginally outside of the historical control data and that they were observed in the presence of reduced maternal body weight gains, which can directly influence foetal growth (see Chernoff et al. Reproductive Toxicology 2008; 25: 192-202), the substance was not considered to be a primary developmental toxicant.  

In the OECD 407 study with Sprague Dawley rats, no treatment-related gross or histopathological findings were observed in any of the primary or secondary reproductive organs that were examined.

Based on the presented data, the substance is regarded to be classified as Not Classified for Toxicity to reproduction according to Regulation EC No 1272/2008.