[No public or meaningful name is available]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, dodecyl ester, potassium salt

EC Number:

254-414-3

EC Name:

Phosphoric acid, dodecyl ester, potassium salt

Cas Number:

39322-78-6

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of potassium didodecylphosphate and dipotassium dodecylphosphate and water

Details on test material:

Chemical name (IUPAC): Reaction mass of potassium didodecylphosphate and dipotassium dodecylphosphate and water

CAS No.: 39322-78-6



Manufactured date: 23.10.2015

Expiry date: 22.10.2017

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species: Rat (Rattus norvegicus)

Strain: Wistar rats – Han Tac: WH rats

Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311,Gajwel,Mandal, Medak District, Andhra Pradesh(A.P)

Justification for selection of species: Rat is a standard laboratory rodent species used for prenatal developmental toxicity assessment and also preferred by various regulatory authorities for toxicity assessment. The Wistar rat was selected due to the large amount of background data accumulated for this strain.

No. of groups: 4 groups
• Vehicle control : 0 mg/kg/day (G1)
• Low dose: 50 mg/kg/day (G2)
• Mid dose: 250 mg/kg/day (G3)
• High dose: 1000 mg/kg/day (G4)

No. of Day 0 mated females (sperm positive in vaginal smears)/ group: 24 /group, Total = 96 females

Age at the start of treatment: 14 to 15 weeks

Mean Body weight (g) and body weight range of Day 0 mated females: The weight variation did not exceed ± 20 % of the mean body weight in each group.

Mean body weight(g) / Body weight range(g)
G1 : 227.336 ± 16.88 / 202.16 to 257.40
G2 : 227.491 ± 16.67 / 202.87 to 259.32
G3 : 227.466 ± 16.78 / 203.12 to 256.62
G4 : 227.158 ± 16.66 / 202.69 to 256.17

Identification: Temporary Identification; Before mating, the rats were allotted temporary identification numbers and were identified by crystal violet body marking.

Permanent Identification; After mating, the rats were identified by the last three digits of the permanent accession numbers written on the tail. The permanent accession number was included on the cage cards.

Acclimatization: After physical examination for good health and suitability for the study, the rats were acclimatized for 12 days. During the acclimatization period, all rats were observed once daily. Females used in this study were nulliparous and non-pregnant.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analyzed for active ingredient concentration (a.i.) and homogeneity at the initiation of treatment and at termination of treatment period.

The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another was kept as back up set which was stored in the experimental room depending upon the obtained stability results. For each set, two replicate samples were drawn from top, middle and bottom layers of each dose formulation. In case of control, two replicate samples from middle layer were drawn. Dose formulations were sent to Analytical R&D Department of Advinus Therapeutics Limited for formulation analysis to determine the concentration and homogeneity of the test item in dose formulations.

The dose formulations collected at initiation of treatment (23 May 2016) and at termination of treatment (06 June 2016) were initially analyzed by adopting an in-house developed and validated method under Advinus
Study No. G11296.

As per sponsor’s suggestion, in addition, on each day of treatment, a back-up sample (a composite sample of approximately 50 mL) from each dose formulation was stored at frozen conditions at -10 to -20°C for sampling at a later date. Later another analytical method was provided by sponsor and the revised analytical method was also validated in-house, after suitable modifications. The stored formulation samples corresponding to those collected on 23 May 2016 and 06 June 2016 are being analyzed using the revised analytical method.

Formulations were considered acceptable when the overall mean result (calculated using all the 6 replicate values) of all the layers and mean of each layers was within ± 15.0 % of the claimed concentration and the relative standard deviation (% RSD, calculated using all the 6 replicate values) of assay of top, middle and bottom layers was equal to or less than 10.0 %.

The unused back up samples (analyzed initially) were discarded as the results of first set of analysis were within the acceptable limits.

Details on mating procedure:

- Impregnation procedure: [cohoused]

- If cohoused:
- M/F ratio per cage: [1:1]
- Length of cohabitation: During the mating period, female rats were cohabited with males in a 1:1 ratio. When sperm was detected in a vaginal smear or vaginal plug was observed in the morning, the animal was considered to be mated. This day was considered as day 0 of gestation.

The mated female rats obtained each day were assigned to the treatment groups and vehicle control groups by body weight stratification. This procedure was continued till the required numbers of Day 0 mated females were obtained (24 per group).

- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy

Duration of treatment / exposure:

The dose formulations of Leomin PN pa was administered orally by gavage using disposable plastic syringe attached with a metal feeding/intubation cannula to rats of low dose (G2), mid dose (G3) and high dose (G4) groups once daily from GD 5 to GD 19 of presumed gestation, at approximately the same time each day (varying by± 2 hours).

Frequency of treatment:

Gestation Day 5 to 19

Duration of test:

15 Days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Doses / Concentrations:
Group 1
Basis:
nominal conc.
0 mg/mL

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Doses / Concentrations:
Group 2
Basis:
nominal conc.
5 mg/mL

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Doses / Concentrations:
Group 3
Basis:
nominal conc.
25 mg/mL

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Doses / Concentrations:
Group 4
Basis:
nominal conc.
100 mg/mL

No. of animals per sex per dose:

No. of Day 0 mated females (sperm positive in vaginal smears)/ group: 24 /group, Total = 96 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As per the Sponsor’s suggestion, based on the results of a dose range finding study the following doses were selected for main embryo-fetal developmental toxicity study with Leomin PN pa in Wistar rats by oral route:

• G1 - Vehicle control - 0 mg/kg/day
• G2 - Low dose - 50 mg/kg/day
• G3 - Mid dose - 250 mg/kg/day
• G4 - High dose - 1000 mg/kg/day

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The rats were subjected to physical examination after mating i.e., on day ‘0’, of gestation and at weekly interval during the presumed gestation period and findings were recorded.

- Cage side observations checked in table 2 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations for clinical signs were performed twice a day - pre dose and post dose (within 1-2 hours of administration) during treatment days and once on non-treatment days.

BODY WEIGHT: Yes /
- Time schedule for examinations: All females included in the study were weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
About 200 g (food input) was provided on Day 0. The food output was recorded and replenished to about 200 g on Days 3, 5, 8, 11, 14 and 17 and food output on Day 20 of presumed gestation was recorded. There was no spillage of food.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:

OTHER:

- Dam examination
The following maternal data were recorded.
a. Pregnancy status
b. Gravid uterine weight
c. No. of corpora lutea
d. No. of implantation sites
e. No. of early resorptions
f. No. of late resorptions

- Fetal examination
The following litter data was recorded:
a. Total number of fetuses
b. Number of live fetuses
c. Number of dead fetuses
d. Individual fetal body weight (g)
e. Fetus sex

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter]

Statistics:

The following statistical tests were used:

The data on maternal body weight, body weight change, gravid uterine weight, corrected body weight gain, maternal food consumption, number of corpora lutea , number of implantations, total number of fetuses, male and female fetus number and weight were analyzed using ANOVA model, after testing for homogeneity for intra group variance using Levene’s test.

Incidence of pre-implantation loss, post implantation loss, Number of early, late and total resorptions were analyzed using Kruskal Wallis test.

Overall percentage of normal and minor external, visceral and skeletal malformations, Sex ratio and number of dams with any resorptions were analyzed using 2 X 2 Contingency Table.

Statistically significant differences (p < 0.05), indicated by the aforementioned tests were designated by symbol ‘*’ throughout the report.

Historical control data:

Enclosed in Annexure 7

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There was treatment-related significant reduction in maternal body weights and food consumption at 250 and 1000 mg/kg/day as compared to vehicle control group along with significant reduction in corrected body weight gain at 1000 mg/kg/day indicating maternal toxicity.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the NOAEL for maternal toxicity was established at 50 mg/kg bw/day due to treatment-related significant reduction in maternal body weight and food consumption at 250 and 1000 mg/kg bw/day. The NAOEL for fetal/developmental toxicity was established at 250 mg/kg bw/day due to treatment-related significant reduction in fetal weights at 1000 mg/kg bw/day. The NOAEL for teratogenicity was set at the highest dose of 1000 mg/kg bw/day, due to the absence of any signs of teratogenicity at the test doses.

Executive summary:

A study was conducted to evaluate the prenatal developmental toxicity of the test substance, mono- and di- C12 PSE, K+ in rats, according to OECD guideline 414, in compliance with GLP. In this study, each group consisted of 24 presumed pregnant Wistar rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal smear was found sperm positive. The test substance was dissolved in vehicle [Milli-Q water] and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 50, 250 and 1000 mg/kg bw/day. The rats in the vehicle control group received the vehicle alone. A constant dose volume of 10 mL/kg body weight was administered to all groups. The dose formulation solutions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits. The mated females were observed twice daily for clinical signs, mortality and morbidity. Body weights were recorded on GD 0, 3, 5, 8, 11, 14, 17 and 20. The intermittent body weight gain and food intake was calculated and presented for rats found pregnant at caesarean section. Caesarean section was performed for all the rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the dams were removed (by laparotomy) and the contents were examined. The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each dam was examined for visceral malformations and the remaining half was evaluated for skeletal malformations. There were no mortalities and clinical signs at all the doses tested. There was treatment-related significant reduction in maternal body weights and food consumption at 250 and 1000 mg/kg bw/day as compared to vehicle control group along with significant reduction in corrected body weight gain at 1000 mg/kg bw/day indicating maternal toxicity. Gross necropsy findings of small thymus and spleen along with scanty mucoid ingesta in stomach and intestines were observed at 1000 mg/kg bw/day which were considered as treatment-related findings. There was treatment-related significant reduction in fetal weights of males, females and total fetuses at 1000 mg/kg bw/day indicating developmental toxicity which is likely to be associated with secondary effects of maternal toxicity at this dose. The other maternal and litter data parameters were comparable to vehicle control group up to the high dose of 1000 mg/kg bw/day. Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses. Under the study conditions, the NOAEL for maternal toxicity was established at 50 mg/kg bw/day due to treatment-related significant reduction in maternal body weight and food consumption at 250 and 1000 mg/kg bw/day. The NAOEL for fetal/developmental toxicity was established at 250 mg/kg bw/day due to treatment-related significant reduction in fetal weights at 1000 mg/kg bw/day. The NOAEL for teratogenicity was set at the highest dose of 1000 mg/kg bw/day, due to the absence of any signs of teratogenicity at the test doses (Rao, 2017).