[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Male fertility index, female fertility index, gestation index, parturition index, percentage of pregnant rats and mating index were comparable with that of the control group. The duration of the gestation and pre-coital interval was also comparable with that of the control group.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March - May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

RccHan: WIST. Female, Nulliparous and non pregnant. 8-9 weeks old

Sex:

female

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The route of vehicle and test item administration was oral through gavage and was selected after discussion with the sponsor.

Duration of treatment / exposure:

The duration of dosing was for 14 days. The first day of dose administration was designated as day 1 for each rat.

Frequency of treatment:

Dose formulation was administered once daily by oral gavage at approximately the same time each day using intubation cannula attached to a graduated syringe.

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals of each sex at each dose.

Control animals:

yes, concurrent vehicle

Positive control:

No positive control

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All rats belonging to vehicle control group were found normal throughout the treatment period. The o
bserved clinical signs were: weakness, polyuria,piloerection, salivation,

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

No morbidity and mortality was observed in rats from vehicle control and low dose groups.
Three mortalities were observed in high dose group. Two mortalities qwere observed in mid dose
group.
Three male and four female rats were sacrificed under moribund condition in high dose group. Three
male and five female rats were sacrificed udner moribund condition in mid dose group.
The rats sacrificed under moribund condition and mortality observed in mid and high dose groups
were considered as treatment related as the mortality observed after showing severe clinical sign.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease in mean body weight was observed in male and female rats of low d
ose group on treatment days 8, 11 and 14 as compared with that of the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease in mean body weight was observed in male and female rats of low d
ose group on treatment days 8, 11 and 14 as compared with that of the control group.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

External examination of found dead, moribundly sacrificed, and termianlly sacrificed rats of either sex
belonging to all groups did not revaeal any abnormality.
Internal examination of found dead male rats revelaed gaseous distension of stomach (in 3 of 4 rats)
gaseous distension of itnestine (1in 4 rats) and whitish foci - non glandular stomach (in 1 of 4 rats)
whereas internal examination of found dead female rat revealed gaseous distension of stomach.
Internal examination of moribundly sacrificed male reats revealed gaseous distension of stomach (in 2
of 6 rats), whitish foci - non glandular stomach (in 6 of 6 rats), and reddish foci -non glandular stoamc
h (in 1 o f 6 rats) whereas internal examination of moribundly sacrifcie female rats revelaed gaseous
distension of stomach (in 5 of 9 rats) and whitish foci -n on glandular sotamch (in 8 of 9 rats).
Internal examination of terminally sacrificed rats of either sex belonging to vehicle control and low
dose groups did not reveal any abnormality.
Changes observed during internal examination of found dead and moribundly sacrificed rats were
considered as effects of test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

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:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Conclusions:

Based on the results of present study, under the condition and procedures followed, dose levels of 1000 and
500 mg/kg b. wt. produced severe toxicity viz. treatment related mortality, morbidity, and adverse clinical
signs whereas a dose level of 250 mg/kg b. wt. produced mild toxicity viz. decreased body weight change
(7.90% in males), food consumption and treatment related changes in absolute and relative organ weight.
Hence, following dose levels are suggested for reproduction/ developmental toxicity screening study (dose
range finding study):
Options Low dose Mid dose High dose
I 15 50 150
II 15 60 240
III 20 60 180
(mg/kg b. wt/day)

Executive summary:

EXECUTIVE SUMMARY

Method:

Test Item

POLIOL MB 600

Test System

Wistar strain (RccHan:WIST)

Route of Administration

Oral through gavage

Dose Levels (mg/kg b. wt./day)

G1 (vehicle control): 0; G2 (low dose): 250; G3 (mid dose): 500; G4 (high dose): 1000

Duration of Treatment

14 days

N° of Rats

5 rats/sex/group

Frequency of Dose Formulation Preparation

Daily fresh preparation

All rats were observed twice a day for mortality, morbidity and visible clinical signs throughout the

treatment period. Body weight of all surviving rats was recorded on days 1, 4, 8, 11, 14 and 15. Food

consumption was calculated during treatment period for all surviving rats. At terminal sacrifice, surviving

rats were sacrificed by carbon dioxide asphyxiation and subjected to gross pathological examination.

Absolute organ weights for all surviving rats were recorded and relative organ weights were calculated for

the organs: liver, kidneys, adrenals, testes, epididymis, prostate, LABC, seminal vesicle with coagulating

gland, thymus, heart, brain, spleen, uterus with cervix, and ovaries.

Results:

POLIOL MB 600 at 250 mg/kg b. wt. /day

No mortality was observed.

Salivation was observed in all male and female rats from treatment day 8.

Treatment related decrease in body weight, body weight change, food consumption and

terminal body weight were observed in male and female rats.

In male rats, statistically significant decrease in absolute weight of thymus, prostate, and LABC

and relative weight of prostate were observed. In female rats, statistically significant increase

in absolute and relative weight of spleen was observed. These changes in organ weights

(absolute and relative) were considered as treatment related however adversity of effect could

not be established due to limited end-points of this study.

External and internal examination of rats of either sex did not reveal any abnormality.

POLIOL MB 600 at 500 mg/kg b. wt. /day

Two mortalities (two male rats) were observed.

Three male and five female rats were sacrificed under moribund condition.

Treatment related clinical signs viz. weakness, salivation, piloerection (only in male rats) and

polyuria were observed in all male and female rats.

Internale xaminationo ff oundd eadm aler atsr evealedg aseousd istensiono fs tomach( in1 o fr ats)a ndw hitishf oci– n onglandular

stomach (in 1 of 2 rats).

Internal examination of moribundly sacrifice male rats revealed gaseous distension of stomach

(in 1 of 3 rats) and whitish foci – non-glandular stomach (in 3 of 3 rats).

Internal examination of moribundly sacrifice female rats revealed gaseous distension of

stomach (in 2 of 5 rats) and whitish foci – non-glandular stomach (in 5 of 5 rats).

POLIOL MB 600 at 1000 mg/kg b. wt. /day

Three mortalities (2 male and 1 female rats) were observed.

Three male and four female rats were sacrificed under moribund condition.

Treatment related clinical signs viz. weakness, salivation, piloerection and polyuria were

observed in all male and female rats.

Internal examination of found dead male rats revealed gaseous distension of stomach (in 2 of 2

rats) and gaseous distension of intestine (in 1 of 2 rats).

Internal examination of found dead female rat revealed gaseous distension of stomach.

Internal examination of moribundly sacrificed male rats revealed gaseous distension of stomac

(in 1 of 3 rats), whitish foci – non-glandular stomach, (in 3 of 3 rats) and reddish foci – nonglandular

stomach (in 1 of 3 rats).

Internal examination of moribundly sacrificed female rats revealed gaseous distension of

stomach (in 3 of 4 rats) and whitish foci – non-glandular stomach, (in 3 of 4 rats).

In male rats, statistically significant decrease in absolute weight of thymus, prostate, and LABC

and relative weight of prostate were observed. In female rats, statistically significant increase

in absolute and relative weight of spleen was observed. These changes in organ weights

(absolute and relative) were considered as treatment related however adversity of effect could

not be established due to limited end-points of this study.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2019 to February 2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dose formulations and vehicle were administered to male and female rats daily up to and including
the day before scheduled sacrifice. The dose-volume for administration was 10 mL/kg body weight.
The dose-volume was adjusted according to the most recently recorded body weight. The control
group was received vehicle alone. The first day of dosing was designated as day 1 for all rats.

Details on mating procedure:

During the mating period, male and female rats were mated in a 1:1 ratio to obtain the F1 pups. Each morning, female rats were examined for the presence of sperm and the ‘day 0’ of pregnancy was recorded. ‘Day 0’ of pregnancy is defined as the day on which vaginal plug or sperm was observed in the vaginal smear of rats.
For the purpose of mating, each female rat was placed with a male rat from the same dose level (1:1 mating) until the mating occurred or 2 weeks had elapsed. Acyclic female rats were assumed as pregnant after the completion of two week mating period. A detailed record of the mating pairs was maintained in the raw data. Care was taken to avoid mating amongst siblings.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The active ingredient (a.i.) concentration and homogeneity of the test item were analysed once befor
e initiation of treatment and twice during the treatment period. Two sets of samples (10 samples pe
r set) were collected by sampling three aliquots (upper, middle and lower layers) from each concent
ration except control (only one aliquot). One set of samples was used for analyses and the other set
of samples was stored at 2 - 8 °C. On each occasion, the mean concentration was determined and
compared with the nominal value. The acceptance criteria were ± 15% deviation from nominal value
and %CV < 10. The samples were analysed at JRF using a validated analytical method (JRF Study N
° 228-2-13-21964). Results are appended in the study report (APPENDIX 35). Stored samples will be
disposed of upon finalisation of the report.

Duration of treatment / exposure:

Dosing of both sexes was initiated 2 weeks prior to the mating and continued during the mating perio
d. After mating, the male rats were further dosed up to and including the day before scheduled sacr
ifice. Female rats were dosed during pregnancy and up to post-partum day 14.
Rats belonging to recovery groups were kept for 14 days after the first scheduled sacrifice of dams,
without treatment.

Frequency of treatment:

Dose formulations and vehicle were administered to male and female rats daily up to and including th
e day before scheduled sacrifice

Details on study schedule:

Dosing of both sexes was initiated 2 weeks prior to the mating and continued during the mating perio
d. After mating, the male rats were further dosed up to and including the day before scheduled sacr
ifice. Female rats were dosed during pregnancy and up to post-partum day 14.
Rats belonging to recovery groups were kept for 14 days after the first scheduled sacrifice of dams,
without treatment.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Dose / conc.:

37.5 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

mid dose

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose:

15 rats/sex per dose, plus 5 rats per sex for the control and high dose groups of the recovery groups.

Control animals:

yes, concurrent vehicle

Positive control:

No

Parental animals: Observations and examinations:

Mortality and Morbidity
Rats were observed daily, twice, for mortality and morbidity
Clinical Sign
Rats were observed daily twice, during treatment and recovery periods, for any visible clinical signs.
Body Weight
Body weight of all-male rats was recorded on the first day of dosing and at weekly intervals thereafter.
Body weight of all female rats was recorded on the first day of dosing and at weekly intervals during the pre-mating period. During the gestation period, female rats were weighed on gestation day 0, 7, 14, 20, and 25. During the lactation period, female animals were weighed within 24 hours of parturition (day ‘0’ post-partum/lactation day), and on post-partum days 4, 7, and 14. Parturition day ‘0’ was defined as the day on which the female littered.
Body weight of all rats belonging to recovery groups was recorded on the first day of dosing and at weekly intervals thereafter.
On the day of fasting, body weights of all surviving rats were recorded. Body weights of all rats were also recorded on the day of sacrifice.
Food Consumption
The food consumption was determined by differentiating the weight of food input and leftover.
Food weights of male rats were determined weekly during the pre-mating and post-mating periods.
In female rats, during the pre-mating period, food weights were recorded at weekly intervals. During the gestation period, food weights were measured on day 0, 7, 14, and 20. During the lactation period, food weights were measured on day 0, 4, 7, and 14. Additional food was offered as and when required.
Food consumption was not measured during the mating period.
Food weights of male and female rats belonging to recovery groups were determined weekly throughout treatment and recovery periods.
Neurobehavioral Observation (NBO)
To assess the behavioural and neurological status of each rat, the below-mentioned parameters of NBO were evaluated prior to initiation of treatment and at weekly intervals, thereafter. As per JRF/TOX/SOP-375 (Issue I), on day of Functional battery observation (FOB), NBO was performed before the start of FOB.
Home Cage Observation
In-home cage, rats were observed for posture and presence or absence of convulsions as mentioned below:
Text Table 3: Home cage observation
Parameters
Observations
Posture
Curled up often asleep (Asleep)
Vertical jumping
Writhing (twisting, squirming or contorted motion)
Flattened, limbs may be spread out
Rearing
Sitting normally, feet tucked in (Sitting B)
Sitting but with head hung down (Sitting A)
Lying on side
Circling
Sitting or standing alert, watching (Sitting C)
Parameters
Observations
Clonic Movement
Present
Absent
Tonic Movement
Present
Absent
Handling Observation
After completion of home cage observations, each rat was picked up by the observer and observed for below-mentioned parameters:
Text Table 4: Handling observation
Parameters
Observations
Ease of removing the animal from the cage
Very easy (sits quietly) – V.easy
Easy (vocalisations without resistance)
Moderately difficult (rears)
Difficult (runs around cage)
Very difficult (aggressive, attempts to bite with or without vocalisations)
Handling reactivity
Difficult (squires, twists, attempts to bite with or without vocalisations)
Freezes (rigid in hand and totally inactive)
Moderately easy (vocalisations without resistance)
Easy (alert, limbs put against the body)
Parameters
Observations
Palpebral closure
Eyelids slightly closed
Ptosis - drooping of eyelids half
Eyelids completely closed
Eyelids wide open (W.open)
Lacrimation
None (no external lacrimation)
Severe (drooping of tears)
Slight (wetness of lower eyelids)
Moderate (wetness of eyelids and its surrounding area)
Eye examination
Normal
Discharge (draining of normal or pathological content)
Conjunctivitis (inflammation of conjunctival mucous membrane)
Chemosis (swelling of the conjunctiva)
Cataract (opacity of the lens)
Corneal opacity (an opaque spot or area on the cornea)
Microphthalmos (abnormally small eyeball)
Exophthalmos (abnormal protrusion of the eyeball)
Piloerection
Absent
Present
Skin examination
Alopecia (absence or loss of hair)
Rough coat (ungroomed or greasy hair coat)
Dermatitis (inflammation of the skin)
Normal
Salivation
Severe (drooping of saliva)
Moderate (wetness of lower mandible and its surrounding areas)
Slight (wetness of lower mandible)
None (no external salivation)
Open Field Observation
For open-field observations, rats were placed (one at a time) in an open arena (size: 495× 495 × 280 mm) with a flat surface covered with clean absorbent paper and observed for a period of 2 minutes. During the 2 minutes period, each rat was observed for the below-mentioned parameters:
Text Table 5: Open field observation
Parameters
Observations
Gait
Normal
Slightly abnormal
Moderately abnormal
Severely abnormal
Mobility
Slightly impaired
Moderately impaired
Normal
Totally impaired
Arousal
Very low (V.low)
High
Low
Very high (V.high)
Vocalisations
Vocalisations (actual number)
Rears
Rears (actual number)
Respiration
Snuffles (accumulation of secretion with respiratory noise)
Normal
Dyspnoea (abnormally difficult or laboured breathing)
Tachypnoea (quick and usually shallow respiration)
Abdominal breathing (breathing by diaphragm)
Gasping (convulsive catching of breath with the wide-open mouth)
Clonic movement
Mild clonic tremors of limbs
Absent
Chewing, clonus of the jaws
Repetitive clonic tremors of the whole body
Tonic movement
Opisthotonos (head, body and limbs rigidly arched backwards)
Absent
Tonic contraction or extension of hind limbs
Emprosthotonos (head, body and limbs extended forward)
Urination
Urination (actual number of urine pool in open field)
Defecation
Defecation (actual number of the faecal bolus in open field)
Stereotypy
Absent
Circling
Excessive grooming
Other (actual observations)
Bizarre behaviour
Other (actual observations)
Self-destructive biting (e.g., tail, paws) or self-mutilation
Biting of open field/standard arena
Absent
Retropulsion (moving backwards
Sensory Reactivity Measurements
For sensory reactivity measurements, rats were placed in an open arena (size: 495 × 495 × 203 mm) with a flat surface covered with clean absorbent paper. The below-mentioned parameters were performed and recorded for rats.
A) Approach Response
Each rat was approached at nose level with the end of a blunt object. The object was held approximately 3 cm away from the face of the rat for approximately 4 seconds to allow time for the rat to respond. The degree of the elicited response was recorded as absent, slow, moderate or fast response.
B) Touch Response
Approaching the rat from the side, the rump of the rat was gently touched with a blunt object. The contact was brief (approximately 1 to 2 seconds) and deliberate but not forceful. The degree of the elicited response was recorded as the absent, slight, normal or exaggerated response.
C) Click Response
A clicker was positioned approximately 5 cm above the back of the rat with care taken not to have the clicker in the rat’s field of vision. The clicker was held in the palm of the hand to ensure consistency of sound from test to test. The degree of the elicited response of the rat to the click sound was recorded as the absent, slight, normal or exaggerated response.
D) Pupil Response
The beam of a pocket-sized flashlight was brought from a lateral position medially towards the centre of the face of the rat. Constriction of the pupil was observed as a positive response. The degree of elicited response was recorded as normal or abnormal.
E) Tail-pinch Response
The tail was squeezed approximately 2 to 3 cm from the tip using forceps (always applying about the same amount of force for each rat). The degree of the elicited response was recorded as absent, slight, flinch (normal) or exaggerated response.
F) Air Righting Reflex
Each rat was held supine, with the hands of the observer under the back and shoulders of rat for support. The rat was dropped from a height of approximately 30 cm. The ease and uprightness of the landing were recorded as normal, slightly abnormal, moderately abnormal or severely abnormal.
Hind Limb Foot Splay
The landing hind limb feet of each rat were marked with a non-permanent, non-toxic ink just prior to testing. Each rat was suspended in a prone position and then dropped on to a recording sheet from a height of approximately 30 cm. This procedure was repeated three times. The distance between two-foot prints was measured and the average of the three-foot splay values was calculated.
7.7.3 Grip Strength
Grip strength of both forelimb and hindlimb was measured with a grip strength meter to determine the ability of each rat to grasp and hold on the mesh platform. The grip strength of each rat was measured for 3 consecutive times and the results were averaged separately for the forelimb and hindlimb.
7.7.4 Motor Activity
Motor activity of each rat was monitored using an automated photobeam activity system equipped with a computer analyser. Rats were monitored for three consecutive 10 minutes intervals (total 30 minutes for each rat) allowing for examination of both exploratory and acclimation activity levels. The motor activity parameters i.e., total activity, ambulatory activity and fine activity were evaluated and reported.

Oestrous cyclicity (parental animals):

Oestrous cycle length and pattern were evaluated by vaginal smears observation of individual female rats during the pre-treatment period of two weeks. Vaginal smear was monitored daily from the beginning of the treatment period until evidence of mating. Vaginal smear, from each pregnant rat, was also observed on the day of terminal sacrifice. Care was taken to avoid disturbance to mucosa while obtaining vaginal cells.

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number of pups, sex of pups, stillbirths, live birth, runts, and the presence of gross anomalies. Pups which died during lactation were weighed and subjected to a post-mortem examination. Pups found dead on the day of littering were examined for possible defects and cause of death and discarded in the absence of gross findings.
Each pup was observed for the presence of milk in the stomach on PND 0 to ensure nursing care.
AGD of each pup was measured on PND 0. Male pups were observed for the retention of nipples/areolae on PND 13.
Pup Body Weight
Individual pup body weight was recorded on PND 0, 4, 7, and 14.

Postmortem examinations (parental animals):

Blood and Urin Collection
At the time of terminal sacrifice, blood (approximately 3.5 mL) was collected from all surviving rats/sex/group under anaesthesia (isoflurane) by orbital plexus puncture. Rats were deprived of food overnight (allowed water ad libitum) prior to blood collection. Blood samples were collected for haematology (in vials containing 4% EDTA), coagulation parameters PT and APTT (in vials containing 3.2% sodium citrate), clinical chemistry, and thyroid hormone analysis (in vials without anticoagulant). Samples were collected at approximately the same time per day and finished by

Postmortem examinations (offspring):

Surviving rats (including pups of PND 14) were sacrificed by carbon dioxide asphyxiation. Male rats were sacrificed after 65% of females have delivered. Culled pups on PND 4, which were not subjected for blood collection, were sacrificed through intraperitoneal administration of thiopentone sodium. Pups were sacrificed on PND 14. Female rats were sacrificed on LD 15. Female rats which did not deliver by day 25 post-coitum were sacrificed on post-coitum day 25. Rats belonging to recovery groups will be sacrificed after 15 days after the first scheduled sacrifice of dams.
Gross necropsy was conducted under the direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out the blood from the rat. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened, and a thorough examination of organs was carried out to detect abnormalities. Special attention was paid to organs of the reproductive system.
At the time of sacrifice or death during the study, all adult rats and pups were examined macroscopically for any structural abnormalities or pathological changes. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.
The uteri of all cohabited female rats were examined for the presence and number of implantation sites. The number of corpora lutea was recorded from those female rats which are sacrificed on gestation day 25 and implants were observed.
Pups which were found dead on PND 0 were subjected for gross examination and portion of lung was immersed in water for confirmation of live and dead status at the time of delivery (stillbirth or dead). Pups which were found dead were observed for the presence of milk band. Pups without gross lesions were discarded after the examination.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation (mild to moderate) was observed approximately 3 to 5 minutes after dosing in all male
and female rats of all test item treated groups and persisted for approximately 20 to 25 minutes.
This finding is considered a response to the dose solution and toxicologically non-adverse in nature.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related mortality or morbidity was observed during the study period in the control
and test item treated groups.
One female rat (Rat N° 108) was found dead on treatment day 10. Necropsy findings in this rat
(reddish discolouration in lungs during gross observation and haemorrhages and granuloma during
histological examination) suggest that this death was due to gavage accident.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male:
Marginal decrease in mean body weight of male rats, belonging to 150 mg/kg b. wt./day dose group
(main and recovery), was observed.
The mean body weight of male rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups was
comparable with that of the control group.
Female:
Marginal decrease in mean body weight of female rats, belonging to 150 mg/kg b. wt./day dose
group was observed during the pre-mating period.
Marginal decrease in mean body weight of female rats of recovery group, belonging to 150 mg/kg
b. wt./day dose group was also observed.
During gestation and lactation periods, the mean body weight of female rats belonging to 150
mg/kg b. wt./day dose groups was comparable with that of the control group.
The mean body weight of female rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups, was
comparable with that of the control group during pre-mating, gestation and lactation periods except
statistically significant decrease in the mean body weight of female rats, belonging to 75 mg/kg b.
wt./day dose group, on lactation day 14.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male
Statistically significant decrease in mean food consumption of male rats, belonging to 150 mg/kg
b. wt./day dose group was observed during treatment days 1-8 (main and recovery), 8-15, 29-36,
36-43, 43-50, and 1-50 when compared with that of the control group.
Mean food consumption of male rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups was
comparable with that of the control group.
Female
Statistically significant decrease in mean food consumption of female rats, belonging to 150 mg/kg
b. wt./day dose group, was observed during pre-mating days 1-8 (main and recovery), 8-15, and 1-
15 when compared with that of the control group.
During the gestation period, the mean food consumption of female rats was comparable with that
of the control group.
Statistically significant decrease in mean food consumption of female rats, belonging to 150 mg/kg
b. wt./day dose group was observed during lactation days 0-4, 7-14, and 0-14 when compared with
that of the control group. In the absence of supporting findings in lactation body weight and body
weight gain, decreased mean food consumption during lactation period was considered as
incidental.
During pre-mating, gestation, and lactation periods, mean food consumption of female rats,
belonging to 37.5 and 75 mg/kg b. wt./day dose groups, was comparable with that of the control
group except for a statistically significant decrease in mean food consumption of female rats,
belonging to 37.5 mg/kg b. wt./day dose group, during pre-mating days 8-15.
A decrease in the mean food consumption of male and female (pre-mating) rats was considered as the effect of the test item treatment but non-adverse in nature.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In male rats of high dose group, statistically significant increase in WBC, neutrophil, monocyte
and platelets was observed, while statistically significant decrease in haemoglobin, haematocrit,
MCV and MCH was noted. In female rats of high dose group, statistically significant decrease in
RBC, while statistically significant increase in lymphocytes was noted. These effects were related
to test item treatment as it was well correlated with inflammatory reactions observed in stomach.
After 14-days of recovery period, statistically significant decrease in MCH and statistically
significant increase in reticulocytes was noted in male rats of high dose recovery group. Increase
in reticulocytes was due to decrease in haemoglobin and haematocrit at terminal sacrifices. Results
shows that effects were recovered almost completely after 14-days of recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In high dose male rats, statistically significant decrease in albumin was noted, while in high dose
female rats decrease in albumin was observed without statistical significance. Statistically
significant decrese in albumin: globulin ratio was noted in both sexes of high dose group. It was
related to decrease in albumin. Effects were considered as related to test item treatment and were
recovered after 14-days of recovery period.
In high dose group, statistically significant decrease was noted BUN and urea in females. Effect
was less likely to be related to treatment as it was not observed in males.
Statistically significant decreases in LDH and CK in high dose male rats and in ALT in mid dose
male rats were noted. Similarly, in treated recovery group, statistically significant decrease was
noted in ALT in both sexes. These effects were considered as toxicologically insignificant.
Statistically significant decrease was observed in glucose in low dose and high dose female rats
and increase in AST in low dose female rats. These effects were unrelated to test item treatment
due to lack of dose dependency and consistency between sexes.
In treated recovery group male rats, statistically significant decrease and increase was noted in
creatinine and phosphorus, respectively. It was unrelated to test item treatment in absence of similar effects at the end of treatment.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

Test item treatment did not lead to any alteration in any urinalysis parameters.
Statistically significant increase in specific gravity was noted in G6 females, which was not
considered as related to test item treatment due to absence of similar findings in terminally
sacrificed animals and consistency between sexes.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home Cage Observations (TABLES 13 and 14; APPENDICES 21 and 22)
In the home cage, all rats from treatment and control groups revealed normal postures asleep (curled
up often asleep), sitting A (sitting but with head hung down), sitting B (sitting normally, feet tucked
in), sitting C (sitting or standing alert, watching) and rearing. Clonic and tonic movements were
absent in the home cage during NBO.
9.11.2 Handling Observations (TABLES 13 and 14; APPENDICES 21 and 22)
NBO performed during handling of rats did not reveal any abnormality related to treatment. All the
rats revealed a normal behaviour during removal (very easy - animals sit quietly) and handling
(easy - alert, limbs put against the body). None of the rats showed lacrimation, salivation or
piloerection. Eyelids were wide open in all rats. Eye and skin examinations of rats from all groups
did not reveal any abnormality.
9.11.3 Open Field Observations (TABLES 13 and 14; APPENDICES 21 and 22)
In the open field, all rats from treatment and control groups showed normal gait, mobility, arousal
and respiration during the two minutes observation period. Clonic and tonic movements, stereotypy
and bizarre behaviour were absent. No treatment-related significant changes were observed in
vocalisation, rearing, urination, and defecation counts of male and female rats from treatment
groups when compared with that of the control group.
Some incidental changes were observed in male rats of high dose group (statistically significant
decrease in rearing count during week 5 (main and recovery groups) and urination count during
week 2) and low dose group (statistically significant increase in rearing count during week 1).
These changes were considered incidental in the absence of other supporting findings.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Sensory reactivity parameters viz., approach response, touch response, click response, pupil
response, tail pinch response and air righting reflex in all treatment groups were comparable with
that of the control group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment of the test item was associated with histopathological lesions (ulceration, hyperplasia,
hyperkeratosis and inflammatory cells) in the stomach at all 3 dose levels in dose-dependent
manner. After 14-days of the recovery period, though some of the lesions were continued, marked
recovery was observed in incidence and severity.
Lesions noted in bone marrow (increased cellularity) and spleen (EMH) in mid dose and high dose
were considered as related to treatment. These were more likely to be owed to inflammatory lesions in the stomach (supported by haematology and lesion in the stomach). Lesions were recovered completely after the recovery period.
Similarly, treatment also caused tubular hypertrophy in kidneys at all dose levels. Marginal
recovery was observed after 14-days of the recovery period.
Microscopic lesions observed in other organs were at a lower rate of occurrence and with minimal
to mild severity with an almost similar incidence between control and high dose group. At
instances, though differences observed in the incidence of lesions between treated and control
group, those lesions were considered as spontaneous or incidental in nature representing the normal physiological/metabolic or congenital changes and not treatment related.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effect of the test item treatment was observed on the mean oestrous cycle length and the mean
number of oestrous cycles.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Male fertility index, female fertility index, gestation index, parturition index, percentage of
pregnant rats and mating index were comparable with that of the control group. The duration of the
gestation and pre-coital interval was also comparable with that of the control group.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male fertility index, female fertility index, gestation index, parturition index, percentage of
pregnant rats and mating index were comparable with that of the control group. The duration of the
gestation and pre-coital interval was also comparable with that of the control group.

Dose descriptor:

LOAEL

Effect level:

ca. 37.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Clinical signs:

no effects observed

Description (incidence and severity):

All pups belonging to control and test item treated groups were found normal throughout the study
period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Mortality index of male, female and composite of male and female pups, belonging to 150 mg/kg
b. wt./day dose group was statistically significantly increased during post-natal days 0-4 when
compared with that of the control group.
This increased in mortality index was considered as the adverse effect of test item treatment.
Mortality index of male, female and composite of male and female pups, belonging to 37.5 and 75
mg/kg b. wt./day dose groups, was comparable with that of the control group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight and body weight gain of male, female and composite of male and female
pups, belonging to 37.5, 75, and 150 mg/kg b. wt./day dose groups were comparable with that of
the control group.

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):

no effects observed

Description (incidence and severity):

The anogenital distance of male and female pups, belonging to 37.5, 75, and 150 mg/kg b. wt./day
dose groups was comparable with that of the control group.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

None of the male pups belonging to either control or the test item treated groups showed retention
of nipples.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In male pups, a statistically significant increase was noted in the relative weight of thyroid gland in
the high dose group (G4). It was considered as unrelated to test item treatment due to lack of effect in absolute weight and lack of consistency between sexes.
Significant decrease noted in weight of thyroid gland in mid dose (G3) female rats was not related
to test item treatment in absence of dose dependency.

Gross pathological findings:

no effects observed

Description (incidence and severity):

External and internal examination of pups (found dead and sacrificed terminally at PND 4 and 14)
did not reveal any abnormality.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Litter Size and Male Sex Ratio (TABLE 9; APPENDIX 12)
Male sex ratio and the mean counts of male pups, female pups, and a composite of male and female pups, belonging to 37.5, 75, and 150 mg/kg b. wt./day dose groups were comparable with that of the control group.
Live Birth and Survival Index (TABLE 12)
Live birth index of male, female, and composite of male and female pups, belonging to 150 mg/kg
b. wt./day dose group was statistically significantly decreased when compared with that of the
control group.
Survival index of male, female, and composite of male and female pups, belonging to 150 mg/kg
b. wt./day dose group was statistically significantly decreased during the postnatal days 0-4 when
compared with that of the control group.
This decrease in the live birth index and survival index were considered as adverse effects of the
test item treatment.
Live birth index and survival index of male, female and composite of male and female pups,
belonging to 37.5 and 75 mg/kg b. wt./day dose groups were comparable with that of the control
group.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: post-natal loss, live birth index, mortality index, and survival index

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

CONCLUSION
Based on the results of this study, it is concluded that:
- Test item treatment produced whitish foci in the non-glandular stomach, histopathological
lesions (ulceration, hyperplasia, hyperkeratosis and inflammatory cells) in the stomach, and
tubular hypertrophy in kidneys at all three dose levels. Based on these findings, stomach and
kidneys were identified as target organs and No Observed Adverse Effect Level (NOAEL) for
parental toxicity was established below 37.5 mg/kg b. wt./day.
- Test item treatment produced adverse effects on developmental parameters i.e., post-natal loss,
live birth index, mortality index, and survival index at 150 mg/kg b. wt./day dose level whereas
no treatment-related effects were observed on sexual function and fertility endpoints. Hence,
No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was established at 75
mg/kg b. wt./day.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Read across from structural analogue, justification can be found attached in section 13.2.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All rats belonging to vehicle control group were found normal throughout the treatment period. The o
bserved clinical signs were: weakness, polyuria,piloerection, salivation,

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

No morbidity and mortality was observed in rats from vehicle control and low dose groups.
Three mortalities were observed in high dose group. Two mortalities qwere observed in mid dose
group.
Three male and four female rats were sacrificed under moribund condition in high dose group. Three
male and five female rats were sacrificed udner moribund condition in mid dose group.
The rats sacrificed under moribund condition and mortality observed in mid and high dose groups
were considered as treatment related as the mortality observed after showing severe clinical sign.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease in mean body weight was observed in male and female rats of low d
ose group on treatment days 8, 11 and 14 as compared with that of the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease in mean body weight was observed in male and female rats of low d
ose group on treatment days 8, 11 and 14 as compared with that of the control group.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

External examination of found dead, moribundly sacrificed, and termianlly sacrificed rats of either sex
belonging to all groups did not revaeal any abnormality.
Internal examination of found dead male rats revelaed gaseous distension of stomach (in 3 of 4 rats)
gaseous distension of itnestine (1in 4 rats) and whitish foci - non glandular stomach (in 1 of 4 rats)
whereas internal examination of found dead female rat revealed gaseous distension of stomach.
Internal examination of moribundly sacrificed male reats revealed gaseous distension of stomach (in 2
of 6 rats), whitish foci - non glandular stomach (in 6 of 6 rats), and reddish foci -non glandular stoamc
h (in 1 o f 6 rats) whereas internal examination of moribundly sacrifcie female rats revelaed gaseous
distension of stomach (in 5 of 9 rats) and whitish foci -n on glandular sotamch (in 8 of 9 rats).
Internal examination of terminally sacrificed rats of either sex belonging to vehicle control and low
dose groups did not reveal any abnormality.
Changes observed during internal examination of found dead and moribundly sacrificed rats were
considered as effects of test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

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:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Conclusions:

Based on the results of present study, under the condition and procedures followed, dose levels of 1000 and 500 mg/kg b. wt. produced severe toxicity viz. treatment related mortality, morbidity, and adverse clinical signs whereas a dose level of 250 mg/kg b. wt. produced mild toxicity viz. decreased body weight change (7.90% in males), food consumption and treatment related changes in absolute and relative organ weight.
Hence, following dose levels are suggested for reproduction/ developmental toxicity screening study (dose range finding study):
Options Low dose Mid dose High dose
I 15 50 150
II 15 60 240
III 20 60 180
(mg/kg b. wt/day)

Reference 4

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Read across from structural analogue, justification can be found attached in section 13.2.

Reason / purpose for cross-reference:

read-across source

Positive control:

No

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation (mild to moderate) was observed approximately 3 to 5 minutes after dosing in all male
and female rats of all test item treated groups and persisted for approximately 20 to 25 minutes.
This finding is considered a response to the dose solution and toxicologically non-adverse in nature.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related mortality or morbidity was observed during the study period in the control
and test item treated groups.
One female rat (Rat N° 108) was found dead on treatment day 10. Necropsy findings in this rat
(reddish discolouration in lungs during gross observation and haemorrhages and granuloma during
histological examination) suggest that this death was due to gavage accident.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male:
Marginal decrease in mean body weight of male rats, belonging to 150 mg/kg b. wt./day dose group
(main and recovery), was observed.
The mean body weight of male rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups was
comparable with that of the control group.
Female:
Marginal decrease in mean body weight of female rats, belonging to 150 mg/kg b. wt./day dose
group was observed during the pre-mating period.
Marginal decrease in mean body weight of female rats of recovery group, belonging to 150 mg/kg
b. wt./day dose group was also observed.
During gestation and lactation periods, the mean body weight of female rats belonging to 150
mg/kg b. wt./day dose groups was comparable with that of the control group.
The mean body weight of female rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups, was
comparable with that of the control group during pre-mating, gestation and lactation periods except
statistically significant decrease in the mean body weight of female rats, belonging to 75 mg/kg b.
wt./day dose group, on lactation day 14.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male
Statistically significant decrease in mean food consumption of male rats, belonging to 150 mg/kg
b. wt./day dose group was observed during treatment days 1-8 (main and recovery), 8-15, 29-36,
36-43, 43-50, and 1-50 when compared with that of the control group.
Mean food consumption of male rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups was
comparable with that of the control group.
Female
Statistically significant decrease in mean food consumption of female rats, belonging to 150 mg/kg
b. wt./day dose group, was observed during pre-mating days 1-8 (main and recovery), 8-15, and 1-
15 when compared with that of the control group.
During the gestation period, the mean food consumption of female rats was comparable with that
of the control group.
Statistically significant decrease in mean food consumption of female rats, belonging to 150 mg/kg
b. wt./day dose group was observed during lactation days 0-4, 7-14, and 0-14 when compared with
that of the control group. In the absence of supporting findings in lactation body weight and body
weight gain, decreased mean food consumption during lactation period was considered as
incidental.
During pre-mating, gestation, and lactation periods, mean food consumption of female rats,
belonging to 37.5 and 75 mg/kg b. wt./day dose groups, was comparable with that of the control
group except for a statistically significant decrease in mean food consumption of female rats,
belonging to 37.5 mg/kg b. wt./day dose group, during pre-mating days 8-15.
A decrease in the mean food consumption of male and female (pre-mating) rats was considered as the effect of the test item treatment but non-adverse in nature.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In male rats of high dose group, statistically significant increase in WBC, neutrophil, monocyte
and platelets was observed, while statistically significant decrease in haemoglobin, haematocrit,
MCV and MCH was noted. In female rats of high dose group, statistically significant decrease in
RBC, while statistically significant increase in lymphocytes was noted. These effects were related
to test item treatment as it was well correlated with inflammatory reactions observed in stomach.
After 14-days of recovery period, statistically significant decrease in MCH and statistically
significant increase in reticulocytes was noted in male rats of high dose recovery group. Increase
in reticulocytes was due to decrease in haemoglobin and haematocrit at terminal sacrifices. Results
shows that effects were recovered almost completely after 14-days of recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In high dose male rats, statistically significant decrease in albumin was noted, while in high dose
female rats decrease in albumin was observed without statistical significance. Statistically
significant decrese in albumin: globulin ratio was noted in both sexes of high dose group. It was
related to decrease in albumin. Effects were considered as related to test item treatment and were
recovered after 14-days of recovery period.
In high dose group, statistically significant decrease was noted BUN and urea in females. Effect
was less likely to be related to treatment as it was not observed in males.
Statistically significant decreases in LDH and CK in high dose male rats and in ALT in mid dose
male rats were noted. Similarly, in treated recovery group, statistically significant decrease was
noted in ALT in both sexes. These effects were considered as toxicologically insignificant.
Statistically significant decrease was observed in glucose in low dose and high dose female rats
and increase in AST in low dose female rats. These effects were unrelated to test item treatment
due to lack of dose dependency and consistency between sexes.
In treated recovery group male rats, statistically significant decrease and increase was noted in
creatinine and phosphorus, respectively. It was unrelated to test item treatment in absence of similar effects at the end of treatment.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

Test item treatment did not lead to any alteration in any urinalysis parameters.
Statistically significant increase in specific gravity was noted in G6 females, which was not
considered as related to test item treatment due to absence of similar findings in terminally
sacrificed animals and consistency between sexes.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home Cage Observations (TABLES 13 and 14; APPENDICES 21 and 22)
In the home cage, all rats from treatment and control groups revealed normal postures asleep (curled
up often asleep), sitting A (sitting but with head hung down), sitting B (sitting normally, feet tucked
in), sitting C (sitting or standing alert, watching) and rearing. Clonic and tonic movements were
absent in the home cage during NBO.
9.11.2 Handling Observations (TABLES 13 and 14; APPENDICES 21 and 22)
NBO performed during handling of rats did not reveal any abnormality related to treatment. All the
rats revealed a normal behaviour during removal (very easy - animals sit quietly) and handling
(easy - alert, limbs put against the body). None of the rats showed lacrimation, salivation or
piloerection. Eyelids were wide open in all rats. Eye and skin examinations of rats from all groups
did not reveal any abnormality.
9.11.3 Open Field Observations (TABLES 13 and 14; APPENDICES 21 and 22)
In the open field, all rats from treatment and control groups showed normal gait, mobility, arousal
and respiration during the two minutes observation period. Clonic and tonic movements, stereotypy
and bizarre behaviour were absent. No treatment-related significant changes were observed in
vocalisation, rearing, urination, and defecation counts of male and female rats from treatment
groups when compared with that of the control group.
Some incidental changes were observed in male rats of high dose group (statistically significant
decrease in rearing count during week 5 (main and recovery groups) and urination count during
week 2) and low dose group (statistically significant increase in rearing count during week 1).
These changes were considered incidental in the absence of other supporting findings.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Sensory reactivity parameters viz., approach response, touch response, click response, pupil
response, tail pinch response and air righting reflex in all treatment groups were comparable with
that of the control group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment of the test item was associated with histopathological lesions (ulceration, hyperplasia,
hyperkeratosis and inflammatory cells) in the stomach at all 3 dose levels in dose-dependent
manner. After 14-days of the recovery period, though some of the lesions were continued, marked
recovery was observed in incidence and severity.
Lesions noted in bone marrow (increased cellularity) and spleen (EMH) in mid dose and high dose
were considered as related to treatment. These were more likely to be owed to inflammatory lesions in the stomach (supported by haematology and lesion in the stomach). Lesions were recovered completely after the recovery period.
Similarly, treatment also caused tubular hypertrophy in kidneys at all dose levels. Marginal
recovery was observed after 14-days of the recovery period.
Microscopic lesions observed in other organs were at a lower rate of occurrence and with minimal
to mild severity with an almost similar incidence between control and high dose group. At
instances, though differences observed in the incidence of lesions between treated and control
group, those lesions were considered as spontaneous or incidental in nature representing the normal physiological/metabolic or congenital changes and not treatment related.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effect of the test item treatment was observed on the mean oestrous cycle length and the mean
number of oestrous cycles.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Male fertility index, female fertility index, gestation index, parturition index, percentage of
pregnant rats and mating index were comparable with that of the control group. The duration of the
gestation and pre-coital interval was also comparable with that of the control group.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male fertility index, female fertility index, gestation index, parturition index, percentage of
pregnant rats and mating index were comparable with that of the control group. The duration of the
gestation and pre-coital interval was also comparable with that of the control group.

Dose descriptor:

LOAEL

Effect level:

ca. 37.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Clinical signs:

no effects observed

Description (incidence and severity):

All pups belonging to control and test item treated groups were found normal throughout the study
period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Mortality index of male, female and composite of male and female pups, belonging to 150 mg/kg
b. wt./day dose group was statistically significantly increased during post-natal days 0-4 when
compared with that of the control group.
This increased in mortality index was considered as the adverse effect of test item treatment.
Mortality index of male, female and composite of male and female pups, belonging to 37.5 and 75
mg/kg b. wt./day dose groups, was comparable with that of the control group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight and body weight gain of male, female and composite of male and female
pups, belonging to 37.5, 75, and 150 mg/kg b. wt./day dose groups were comparable with that of
the control group.

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):

no effects observed

Description (incidence and severity):

The anogenital distance of male and female pups, belonging to 37.5, 75, and 150 mg/kg b. wt./day
dose groups was comparable with that of the control group.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

None of the male pups belonging to either control or the test item treated groups showed retention
of nipples.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In male pups, a statistically significant increase was noted in the relative weight of thyroid gland in
the high dose group (G4). It was considered as unrelated to test item treatment due to lack of effect in absolute weight and lack of consistency between sexes.
Significant decrease noted in weight of thyroid gland in mid dose (G3) female rats was not related
to test item treatment in absence of dose dependency.

Gross pathological findings:

no effects observed

Description (incidence and severity):

External and internal examination of pups (found dead and sacrificed terminally at PND 4 and 14)
did not reveal any abnormality.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Litter Size and Male Sex Ratio (TABLE 9; APPENDIX 12)
Male sex ratio and the mean counts of male pups, female pups, and a composite of male and female pups, belonging to 37.5, 75, and 150 mg/kg b. wt./day dose groups were comparable with that of the control group.
Live Birth and Survival Index (TABLE 12)
Live birth index of male, female, and composite of male and female pups, belonging to 150 mg/kg
b. wt./day dose group was statistically significantly decreased when compared with that of the
control group.
Survival index of male, female, and composite of male and female pups, belonging to 150 mg/kg
b. wt./day dose group was statistically significantly decreased during the postnatal days 0-4 when
compared with that of the control group.
This decrease in the live birth index and survival index were considered as adverse effects of the
test item treatment.
Live birth index and survival index of male, female and composite of male and female pups,
belonging to 37.5 and 75 mg/kg b. wt./day dose groups were comparable with that of the control
group.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: post-natal loss, live birth index, mortality index, and survival index

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

CONCLUSION
Based on the results of the source substancestudy, it is concluded that:
- MBC 450 is likely to produce whitish foci in the non-glandular stomach, histopathological
lesions (ulceration, hyperplasia, hyperkeratosis and inflammatory cells) in the stomach, and
tubular hypertrophy in kidneys at all three dose levels. Based on these findings, stomach and
kidneys were identified as target organs and No Observed Adverse Effect Level (NOAEL) for
parental toxicity was established below 37.5 mg/kg b. wt./day.
- MBC 450 is likely to produce adverse effects on developmental parameters i.e., post-natal loss,
live birth index, mortality index, and survival index at 150 mg/kg b. wt./day dose level whereas
no treatment-related effects were observed on sexual function and fertility endpoints. Hence,
No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was established at 75
mg/kg b. wt./day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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

Description of key information

Live birth index of male, female, and composite of male and female pups, belonging to 150 mg/kg
b. wt./day dose group was statistically significantly decreased when compared with that of the control group.

Survival index of male, female, and composite of male and female pups, belonging to 150 mg/kg
b. wt./day dose group was statistically significantly decreased during the postnatal days 0-4 when
compared with that of the control group.
This decrease in the live birth index and survival index were considered as adverse effects of the
test item treatment.
Live birth index and survival index of male, female and composite of male and female pups, belonging to 37.5 and 75 mg/kg b. wt./day dose groups were comparable with that of the control
group.

Based on the results of this study, it is concluded that:

- Test item treatment produced adverse effects on developmental parameters i.e., post-natal loss,live birth index, mortality index, and survival index at 150 mg/kg b. wt./day dose level whereasno treatment-related effects were observed on sexual function and fertility endpoints. Hence, No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was established at 75 mg/kg b. wt./day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

As our study strongly manifest that there are toxic effects on the offspring, we consider that the substance should be classified. However, as the OECD 422 is not the optimal choice for assess the toxicity to reproduction, and considering that is not possible to determine if the adverse effects on the offspring are related to toxicity to reproduction or a consequence of other toxic effects of the substance. Therefore, we classify the substance as Toxicity for reproduction, category 2.