Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
internal reference: SAT 070060
Batch No: Kn-Gi 9328-168

Test animals

Species:
rat
Strain:
Wistar

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The dose volume administered to the animals was 10 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Active ingredient and homogeneity of Kn 172 at 6, 10 and 14 mg/mL concentrations in distilled water was determined prior to the commencement of main study and at 6, 9 and 12 mg/mL concentrations in distilled water was determined prior to the commencement of treatment and during the treatment period from main study by HPLC/UV.
Details on mating procedure:
Post acclimatisation, male and female rats (1:1) were cohabited in cages (size: 41cm x 28.2cm x 18 cm) on a rack till the required numbers of sperm positive females were obtained. The females were examined each morning for evidence of mating by examination of the vaginal smear, collected after lavage. Females showing presence of spermatozoa in the smear were separated. The day of recording of spermatozoa in the vaginal smear was termed as Day " 0" of gestation. A total number of 25 sperm positive (E+) females were included in each dose group i.e. 0, 60, 90 and 120 mg/kg body weight/day.
Duration of treatment / exposure:
The pregnant females received Kn 172 for fifteen consecutive days, starting from 5th to 19th day of gestation at dose levels of 60, 90 and 120 mg/kg body weight/day.
Frequency of treatment:
daily
Duration of test:
until gestational day 20
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
60, 90 and 120 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:

Basis:

No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Mortality and Clinical Observations
Animals were observed daily for mortality and morbidity. Females, which died during the study, were weighed and subjected to post-mortem examination. During the study period, the details of clinical signs of toxicity including changes in skin and fur, eye and mucous membrane, respiratory, circulatory, autonomous and central nervous system and urinogenital system of the pregnant females were recorded daily.

Body Weight and Feed Consumption
The gestational body weights of the pregnant females were recorded on day 0, 3, 5, 8, 11, 14, 17 and 20 of gestation. Feed input and the quantity of feed left over of the pregnant females were recorded on day 0, 3, 5, 8, 11, 14, 17 and 20 of gestation. An amount of 150 g of rat pellet feed was given to each pregnant female on each occasion and the feed left over was recorded.
Ovaries and uterine content:
After necropsy, the gravid uterus was removed without separating the left and right horns, excess fat was trimmed off, blotted free of blood using absorbent filter paper and weighed. The ovaries were removed and the corpora lutea were counted. The uteri of any animal judged to be non-pregnant was stained with 5% aqueous (v/v) ammonium sulphide solution and examined for implantation sites (Salewski, 1964).

The following parameters were recorded:
Number of corpora lutea
Total weight (g) of uterus (gravid)
Number of implantations
Number of resorptions (early/late)
Number of dead foetuses
Number of live foetuses
Number of male and female foetuses
The sex of the foetus (The sex of the foetus was determined by examination of external genitalia and
the ano-genital distance)
Individual foetal weight (g)
Foetal gross external anomalies
Fetal examinations:
The foetuses were examined for gross external anomalies. The abnormal foetuses were recorded, preserved and stored as per their respective groups. After examination in each litter, the abdominal and thoracic cavity of approximately 50% of foetuses were dissected and examined for visceral anomalies (Wilson, 1965) under a Labomed stereozoom microscope (Model N° SZ-790; manufactured by N.K. Jain Instruments Pvt. Limited, Ambala Cantt, Punjab, India). In visceral examination, intestine, stomach, spleen and pancreas were examined for size and position. The liver was evaluated for size, colour and appearance. The reproductive organs were exposed by raising the intestine and attached viscera from the dorsal wall with a cotton swab. The kidneys and adrenal glands were observed for size, position and colour. After this observation, the liver was carefully lifted with a cotton swab to examine the diaphragm for herniation.

In lungs, the right and left lobes were counted and evaluated for size. The thymus gland was checked for size and position. The size and shape of the heart was also examined. The pericardial sac was opened, so that the heart is fully exposed and examined for the presence of aortic arch, pulmonary arch and ductus arteriosis. The heart was then repositioned and two cuts were made through the right ventricle, first starting from the right of the ventral midline surface at the apex to the base of the pulmonary artery and, second through the midline surface at the apex extending to the left ventricle into the ascending aorta. Incisions of the heart were made with fine microscissors and opened with fine forceps for the examination of interventricular septum. The oesophagus, trachea and thyroid glands were also checked for abnormality, if any (Hayes, 1994).

After completing the visceral examination, the foetuses were stored in alcohol-aceto formalin and the cephalic region of these foetuses were subjected to razor sectioning in order to visualize internal structures of the head including the symmetry of external nares, nasal conchae, nasal septum, palate, the development of the cerebellum and brain stem. Five transverse sections of the foetal cephalic regions were observed under Labomed stereozoom microscope. The deviations from the normal development of the cephalic region, if any, were recorded.

The remaining foetuses (50%) of each litter were subjected to skeletal examination (Dawson, 1926) using Alizarin Red S rapid single staining technique for a week duration after fixing in 70% isopropyl alcohol for one day. Then the foetuses were stained in alizarin red solution (0.0025%) for one day, macerated with 1% aqueous potassium hydroxide (KOH) solution for approximately one day and then proceeded for clearing and hardening in fresh solution of glycerol, 70% isopropyl alcohol and benzyl alcohol (2:2:1) for one day and finally stored in a mixture of 70% isopropyl alcohol and glycerol (1 : 1) (Gurr, 1956). After minimum 4 hours of preservation in 1:1 mixture, the stained foetuses were examined under Labomed stereozoom microscope. The skull was examined for size, shape and degree of ossification of nasal, frontal, parietal, interparietal, occipital, lacrimal, zygomatic, squamosal, pre-maxillary, maxillary, mandible, basisphenoid, exoccipital and tympanic bullae. Similarly, the vertebral arches and its centre (of thoracic, lumbar and sacral), ribs and sternal centre were also examined for size, shape and counted for the number of ossification centre. The thoracic, pelvic girdles, fore limbs and hind limbs were examined for the development of flat, long bones and the phalanges – for the number of metacarpal and metatarsals. Any deviation from the normal development of the above mentioned bones were recorded for each foetus.
Statistics:
Bartlett test, Student t-test and ANOVA with Dunnet’s t-test (Mean Body Weight, Mean Body Weight Change, Mean Feed Consumption, Mean Gravid uterine weight, Prenatal data, Foetal data)
Test of significance for difference of proportions and/or Chi-Square test (Mortality rate, Pregnancy rate, Incidence of foetuses with malformation/variation, Incidence of litters containing foetuses with malformation/variation)
Indices:
The data on the number of sperm positive animals in each group and number of animals, which were found pregnant at term, were compiled and the pregnancy rate was expressed in terms of percentage. The percentage of dead animals (mortality rate), percentage of sacrificed animals (survived) at term was evaluated. The maternal feed consumption was calculated for the gestational intervals, i.e., days 0-3, 3-5, 5-8, 8-11, 11-14, 14-17 and 17-20 of gestation. Similarly, the per cent maternal body weight change during these gestational intervals was also estimated from the gestational body
weights.
The gravid uterine weights were recorded and the per cent relative uterine weights were calculated. Pre-implantation loss was estimated from the total implants and the number of corpora lutea. Post implantation loss was calculated from the total implantation and resorption sites including dead foetuses.
The mean of female foetal weights, male foetal weights and foetal weights composite of both sex were calculated for each litter from which the group means were calculated. The male litter weights, female litter weights and the total litter weights for each pregnant female were calculated and the group means of these were compared with those of the controls. The male to female (male/female) sex ratio was also calculated for all groups.
Historical control data:
Historical control data of JAI Research Foundation was used.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical symptom of salivation was observed in rats from 90 mg/kg body weight/day dose group during treatment period, while salivation, lethargy, catalepsy, piloerection, lacrimation and tremors were observed in 120 mg Kn 172/kg body weight/day dose groups for some days during treatment period. The symptom was visible in rats immediately post dosing, however the treated rats recovered within 4 -5 hour post dosing.
The mean maternal body weights during 8th to 20th day of gestation were significantly decreased during the study at 120 mg/kg bodyweight/day dose group. The 20th day corrected body weight of the pregnant female rats was significantly decreased at the dose level of 90 and 120 mg/kg body weight/day dose group when compared with the control group. The percent body weight change data was significantly decreased during 5th to 8th day of gestation at the dose level of 60, 90 and 120 mg/kg body weight and during 8th to 11th day of gestation at the dose level of 90 and 120 mg/kg body weight. This effect was compensated to control data upto the end of the study.
The feed consumption was significantly decreased during 5th to 20th day of gestation at the dose level of 120 mg/kg body weight when compared with the control group.
The gross pathological changes/lesions observed during necropsy of live females (terminally sacrificed) were inconsistent and considered not be attributed to the treatment. Whereas, found dead rats (two animals at the 120 mg/kg dose) showed macroscopic lesions (lung - congestion, liver - mottling and stomach - congestion) could be correlated with the treatment.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The number of foetus and litter affected with short supernumerary rib was statistically significantly increased at 60, 90 and 120 mg/kg body weight/day dose groups when compared with the concurrent control. However, significant increase was observed at 90 and 120 mg/kg body weight/day dose groups when compared with the historical control data of this strain. As such incidence of supernumerary ribs is well known for rats, especially in such kind of studies (Chernoff et al., 1991), they are considered to be of minor relevance. This consideration is even based on the limited number of in-house historical control data by now, due to the change of the animal breeder.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
From the present study, it is concluded that the "No Observed Adverse Effect Level" (NOAEL) for maternal toxicity of Kn 172 is 60 mg/kg body weight/day due to clinical symptoms, decrease body weight change and feed consumption seen in the 90 and 120 mg/kg body weight/day. The NOAEL for foetal toxicity of Kn 172 is 60 mg/kg body weight/day due to the occurrence of foetus with an increased number of short supernumerary rib at 90 and 120 mg/kg body weight/day.
The results of the present study indicate that Kn 172 is non-teratogenic in rats upto the dose level of 120 mg/kg body weight/day.
Executive summary:

This study was performed to evaluate the possible prenatal developmental toxicity of Kn 172 by oral gavage in pregnant Wistar rats. The method followed was as per the guideline of OECD N° 414 (January 2001).

Pregnant Wistar rats were exposed during gestational days (GD) 5 to 19, to Kn 172 suspended in distilled water once daily, by oral gavage at the dose levels of 60, 90 and 120 mg/kg body weight/day, based on test results of a dose-range finder study. Pregnant rats from the control group were administered distilled water only. Maternal body weights, clinical symptoms and feed consumption were recorded throughout the gestation period. The treated and control rats were sacrificed on GD 20, the uteri were removed, weighed and examined for the number of implantation, resorption and for live and dead foetus. The ovaries were checked for corpora lutea. The foetus were weighed and examined for external, visceral, head razor section and skeletal abnormalities.

Two treatment related mortalities were observed in the 120 mg/kg body weight/day dose groups from the pregnant female rats. Pregnancy data remained comparable between rats from various treated groups and concurrent control group. Clinical symptom of salivation was observed in rats from 90 mg/kg body weight/day dose group during treatment period, while salivation, lethargy, catalepsy, piloerection, lacrimation and tremors were observed in 120 mg Kn 172/kg body weight/day dose groups for some days during treatment period. The symptom was visible in rats immediately post dosing, however the treated rats recovered within 4 -5 hour post dosing.

The mean maternal body weights during 8th to 20th day of gestation were significantly decreased during the study at 120 mg/kg bodyweight/day dose group. The 20th day corrected body weight of the pregnant female rats was significantly decreased at the dose level of 90 and 120 mg/kg body weight/day dose group when compared with the control group. The percent body weight change data was significantly decreased during 5th to 8th day of gestation at the dose level of 60, 90 and 120 mg/kg body weight and during 8th to 11th day of gestation at the dose level of 90 and 120 mg/kg body weight. This effect was compensated to control data upto the end of the study.

The feed consumption was significantly decreased during 5th to 20th day of gestation at the dose level of 120 mg/kg body weight when compared with the control group.

The gross pathological changes/lesions observed during necropsy of live females (terminally sacrificed) were inconsistent and considered not be attributed to the treatment. Whereas, found dead rats showed macroscopic lesions (lung - congestion, liver - mottling and stomach - congestion) could be correlated with the treatment.

No treatment related effects were observed in the mean prenatal and foetal data upto the dose level of 120 mg/kg body weight/day.

No significant difference in the incidences of malformation or birth defects was recorded during external, visceral and head razor examination of foetus from the control and various treated groups. The number of foetus and litter affected with short supernumerary rib was statistically significantly increased at 60, 90 and 120 mg/kg body weight/day dose groups when compared with the concurrent control. However, significant increase was observed at 90 and 120 mg/kg body weight/day dose groups when compared with the historical control data of this strain. As such incidence of supernumerary ribs is well known for rats, especially in such kind of studies (Chernoff et al., 1991), they are considered to be of minor relevance. This consideration is even based on the limited number of in-house historical control data by now, due to the change of the animal breeder.

From the present study, it is concluded that the " No Observed Adverse Effect Level (NOAEL)" for maternal toxicity of Kn 172 is 60 mg/kg body weight/day due to clinical symptoms, decrease of body weight and feed consumption seen in 90 and 120 mg/kg body weight/day. The NOAEL for foetal toxicity of Kn 172 is 60 mg/kg body weight/day due to the occurrence of foetus with an increased number of short supernumerary rib at 90 and 120 mg/kg body weight/day.

The results of the present study indicate that Kn 172 is non-teratogenic in rats up to the dose level of 120 mg/kg body weight/day.