1-[bis[3-(dimethylamino)propyl]amino]propan-2-ol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-08-19 - 2022-06-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021/02/28 - 2021/12/28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Dose range finding study

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KCC BIO LABS
- Age at study initiation: 6 to 7 Months
- Weight at study initiation: 2,5 - 3kg
- Fasting period before study: no
- Housing: The rabbits will be housed individually (except during cohabitation when the female rabbits will be cohabited with males in 1:1 ratio)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 (± 3) °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): 12-15/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 28/02/2021 - 05/04/2021

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: until visual conformation of mating
- Proof of pregnancy: visual confirmation of mating referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 6 to GD 28 of presumed gestation

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

45 mg/kg bw/day (nominal)

Dose / conc.:

90 mg/kg bw/day (nominal)

Dose / conc.:

180 mg/kg bw/day (nominal)

Remarks:

decreased to 120mg/kg bw/d from GD 9 onwards

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for clinical signs were performed twice a day, pre-dose and post-dose (30 minutes to 2 hour post dose, approximately) during treatment period and at least once on other days.
Each rabbit was observed twice daily during treatment for morbidity and mortality i.e., once in the morning and once in the afternoon. Based on the assessment if there wereno clinical signs of concern, then the observation was carried out once during weekend and public holidays.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The rabbits will be weighed on gestation days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29.

FOOD CONSUMPTION : Yes
- A known weight of food (food input) will be provided on Day 0. The food left over will be recorded and replenished to a known weight on Days 3, 6, 9, 12, 15, 18, 21, 24 and 27 and food output on Day 29 of presumed gestation will be recorded.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Gross necropsy, which involves an external observation and thoracic and abdominal viscera including uterine contents, will be performed on all animals in the study including dead, moribund and those sacrificed pre-term or at term

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
- Other: gross evaluation of placenta

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs or mortalities at the doses of 45 and 90 mg/kg/day. At the dose of 180 mg/kg/day (G4), there were 2/6 mortalities on GD 8. Hence the dose was reduced to 120 mg/kg/day
from GD 9 onwards. On GD 17, 2 rabbits were found dead.

Mortality:

mortality observed, treatment-related

Description (incidence):

At the dose of 180 mg/kg/day (G4), there were 2/6 mortalities on GD 8. Hence the dose was reduced to 120 mg/kg/day from GD 9 onwards. On GD 17, 2 rabbits were found dead.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean maternal body weights of rabbits at
tested dose of 45 mg/kg/day were statistically comparable to vehicle control group.
At 90 mg/kg/day, the mean body weights of rabbits were statistically comparable to vehicle control group. However, there was a tendency towards reduction in body weights and the body weight gain was significantly reduced during GD27-29 and the adjusted body weight gain was significantly reduced as compared to vehicle control group.
Due to only 2 remaining animals in the high dose group the analysis of body weight (change) was not done.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 45 mg/kg/day, the mean food consumption was statistically comparable to vehicle control group.
At 90 mg/kg/day, the food consumption was reduced (from -24 to 65%) during different periods of gestation with statistical significance at GD21-24, 24-27, 27-29, 6-29 and 0-29.
The reduction in body weight and food consumption at 90 mg/kg/day was considered treatment related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no gross pathological findings in any females of all dose levels, sacrificed at caesarean section except for an incidence of pale liver of all lobes in a dead rabbit at 180/ 120 mg/kg/day.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Mean or individual values of maternal parameters comprising of uterine weight, number of corpora lutea, implantations, early and late resorptions and pre and post implantation loss at 45 and 90 mg/kg/day were comparable to the vehicle control group. Gross evaluation of placenta revealed no findings. Due to only two surviving animals in the high dose group the statistical analysis was not possible.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 90 mg/kg/day, there was significant reduction in mean fetal weights of males (-14%), females (-18%) and male and female (-15%) as compared to vehicle control group. All other litter data parameters comprising of total number of fetuses, number of live fetuses were statistically comparable to vehicle control group at 45 and 90mg/kg bw/day.
Due to only one litter remaining at the high dose 180/120 mg/kg bw/d the data was not analysed statistically.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

No abnormality was noticed on external observations of fetuses at any of the dose levels.

Executive summary:

The objective of this preliminary study was to select the doses for the
definitive prenatal developmental toxicity study in New Zealand white rabbits. This study evaluated the maternal and developmental toxicity of the test item when administered orally to pregnant New Zealand white rabbits during gestation days (GD) 6 to 28. In this dose range finding study, 24 mated female rabbits were assigned to four groups (G1: Vehicle control, G2: low dose, G3: mid dose and G4: High dose). GD 0 for each individual female rabbit in the study was considered as the day on which mating had occurred. The rabbits in the high dose group were treated at 180 mg/kg/day from GD6 to GD8. Due to test item related mortalities, the dose was reduced to 120 mg/kg/day from GD 9 onwards.

Based on the mortalities of 4/6 females in the 180/120 mg/kg/day this dose is above the highest tolerable dose. Based on the reduction in body weight and food consumption and reduction in mean fetal weights at 90 mg/kg/day, the following doses are proposed for the definitive study: 90, 45, 15 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-[bis[3-(dimethylamino)propyl]amino]propan-2-ol

EC Number:

266-587-2

EC Name:

1-[bis[3-(dimethylamino)propyl]amino]propan-2-ol

Cas Number:

67151-63-7

Molecular formula:

C13H31N3O

IUPAC Name:

1-Bis(3-(dimethylamino)propylamino)-2-propanol

Test material form:

liquid

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KCC BIO LABS
- Age at study initiation: 6-10 months
- Weight at study initiation: XXXXXXXXXXXX
- Fasting period before study: no
- Housing: individually except during mating where 1 male and 1 female will be housed together
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-08-19 To: 2021-09-24

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

XXXXXXXXXXXXXXXXXXX

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: XXXXXXXXXX
- M/F ratio per cage: 1/1
- Proof of pregnancy: visual confirmation of mating referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Gestation day 6 until Gestation day 28

Frequency of treatment:

daily

Duration of test:

XXXXXXXXXX

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

45 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

90 mg/kg bw/day (nominal)

No. of animals per sex per dose:

23 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A preliminary dose-range-finding (DRF) in pregnant rabbits was carried out using 6 rabbits per group with the test item. The rabbits were treated at doses of 45, 90 and 180/120 mg/kg/day along with the concurrent vehicle control group at a dose volume of 4 mL/kg body weight from GD 6 to GD 28 and observed for clinical signs and mortality. Due to treatment related mortalities at 180 mg/kg/day, the dose was reduced to 120 mg/kg/day from GD 9 onwards.
The results were as follows: Due to treatment related deaths (4/6 rabbits), dose of 180/120 mg/kg/day was considered to have exceeded the maximum tolerable dose.
At the dose of 90 mg/kg/day, there was reduction in body weight, food consumption and reduction in fetal weights. No abnormality was noticed on external observations of fetuses.
Based on the results of DRF, the following doses are selected for the definitive study: Vehicle control (0 mg/kg/day), Low dose (15 mg/kg/day), Mid dose (45 mg/kg/day), High dose (90 mg/kg/day)
- Rationale for animal assignment (if not random): Pregnant rabbits on day 0 (day of coitus) obtained each day were randomly distributed to different groups by body weight stratification method using ProvantisTM software (Instem).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for clinical signs was performed twice a day, pre-dose and post-dose during treatment period and at least once on other days.

BODY WEIGHT: Yes
- Time schedule for examinations: The rabbits were weighed on gestation days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Gross necropsy, which involves an external observation and thoracic and abdominal viscera including uterine contents, was performed on all animals in the study including dead, moribund and those sacrificed pre-term or at term (caesarean section).

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
- Other: Gross evaluation of placenta

Fetal examinations:

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

Statistics:

Data will be captured using the ProvantisTM laboratory information management system (LIMS), parameters such as body weight, body weight change, food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, Pre/post implantation loss , number of implantations, sex ratio, number of corpora lutea, early and late resorptions will be evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. If data found to be homogeneous and of normal distribution, will be analysed by analysis of variance (ANOVA). If data found to be nonhomogeneous or of nonnormal data will be subjected for transformation and ANOVA will be done on transformed data. When ANOVA will be significant, pairwise comparisons of treated groups to the control group will be made using a parametric test, Dunnett, to identify statistical differences.

Fetal weight for male and female will be analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss will be analyzed using Kruskal Wallis test for group comparison. Mann-Whitney / Wilcoxon pairwise comparison of the treated groups with the control group is performed, when the group differences are significant.

The incidence of dams with resorptions will be tested for using Chi-square test followed by Fisher’s exact test for group association.

The incidence of fetuses and litter (incidence and percent) observations for external,visceral and skeletal will be tested for the Cochran-Armitage trend test and the pairwise comparison will be tested by Fisher’s exact test for group association.

All hypothesis testing will be carried out at the 5% (2-sided) significance level unless otherwise specified.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean maternal body weights, body weight gain and adjusted body weights during the different days of gestation in test item treated groups were statistically comparable to vehicle control group. However, there was a non significant reduction in body weight gains during different days of gestation (GD6-9, GD9-12 and GD27-29) at 90 mg/kg/day as compared to vehicle control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

As compared to the vehicle control group, there was no change in food consumption of rabbits dosed at 15 and 45 mg/kg/day except for significant increase in food consumption at 15 mg/kg/day during GD 3 to 6, which was considered an incidental finding.

At 90 mg/kg/day, there was a significant reduction in food consumption by 11 to 29 % during GD 6-9, 9-12, 12-15, 18-21, 21-24 and 27-29 as compared to vehicle control group. The reduction in food consumption at 90 mg/kg/day was considered treatment related

Food efficiency:

not examined

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

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The gravid uterine weight was statistically comparable between the vehicle control and rabbits treated at 15, 45 and 90 mg/kg/day.

Gross pathological findings:

no effects observed

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female abortion at 15 mg/kg/day on GD27, which was considered as an incidental finding.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The maternal parameters comprising pre and post implantation loss were statistically comparable between the vehicle control and rabbits treated at 15, 45 and 90 mg/kg/day.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

The maternal parameters comprising early and late resorptions were statistically comparable between the vehicle control and rabbits treated at 15, 45 and 90 mg/kg/day.

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were 2, 2, 3, 3 non pregnant females in the control, 15, 45 and 90mg/kg bw/d groups respectively.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

45 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test-item-related malformation was observed during external observation of the fetuses at any of the doses. Anomaly of umbilical hernia (1/144 fetuses) at 15 mg/kg/day and (3/136 fetuses) at 45 mg/kg/day and small fetus (1/144 fetuses) at 15 mg/kg/day were observed. Malformations of cranium excencephaly in vehicle (1/153 fetuses) control group and right forelimb bent inwards (1/119 fetuses) at 90 mg/kg/day were observed. These findings were randomly distributed across the treatment groups and were considered incidental.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test-item-related skeletal malformations observed in fetuses of does treated up to 90 mg/kg/day. Majority of variations and anomalies observed in various skeletal components across treated groups were comparable to the vehicle control group.

There was significant increase in extra 8 th lumbar centra and arch at 45 and
90 mg/kg/day and delayed ossification of 6th sternum at
45 mg/kg/day.However, these findings were not dose related and were randomly distributed across the treatment groups and hence were considered non-adverse.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test-item-related visceral malformations observed in fetuses of dams treated up to 90 mg/kg/day. Anomalies such as gall bladder hypoplastic in test-item-treated groups and hyperplastic gall bladder and bi-lobed gall bladder in vehicle control group were observed. Incidence of liver median lobe extra lobation at 90 mg/kg/day and slight kidney renal pelvis dilation at 15 mg/kg/day was observed. These findings were not considered adverse as these observations commonly occur in animals of this test model and the incidence of occurrence was consistent with vehicle control group.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Summary maternal data

Sex: Female				G1 0 mg/kg/day	G2 15 mg/kg/day	G3 45 mg/kg/day	G4 90 mg/kg/day
Group size			23		23	23		23
Number dams aborted			0		1	0		0
Number sacrificed at C/S			23		22	23		23
Pregnant at C/S		N	21		20	20		20
Gravid Uterus Weight (g)	[a]	Mean SD	425.8 115.7		444.2 111.5	392.1 87.7		359.6 96.2
Number of Corpora Lutea	[a]	Mean SD Sum	10.33 2.01 217.00		10.20 2.33 204.00	10.00 2.99 200.00		9.20 2.17 184.00
No. of Implantation	[a1]	Mean SD Sum	7.67 2.33 161.00		7.50 2.44 150.00	7.20 1.94 144.00		6.60 2.48 132.00
Dams with Early resorption		N	1		3	3		3
Number of Early Resorptions	[a1]	Mean SD Sum	0.0 0.2 1.0		0.2 0.4 3.0	0.2 0.5 4.0		0.2 0.4 3.0
% Early Resorption /Animal	[a1]	Mean SD	0.48 2.18		2.50 6.54	2.72 7.90		2.39 6.01
Dams with Late resorption		N	6		2	4		10
Number of Late Resorptions	[a1]	Mean SD Sum	0.3 0.6 7.0		0.2 0.5 3.0	0.2 0.4 4.0		0.5 0.5 10.0
% Late Resorptions /Animal	[a1]	Mean SD	5.09 8.68		2.08 6.55	2.39 5.06		12.01 15.87
Dams with Resorptions	[f]	N	7		5	6		12
Total Number of Resorption (early + late)	[f]	Mean SD Sum	0.4 0.6 8.0 .		0.3 0.6 6.0 .	0.4 0.7 8.0		0.7 0.6 13.0
Pre-implantation Loss/Animal	[a1]	Mean SD	2.67 1.96 56.00		2.70 1.38 54.00	2.80 2.26 56.00		2.60 1.96 52.00
% Pre-implantation Loss	[a]	Mean SD	25.72 17.94		27.47 13.57	24.99 18.21		28.60 21.50
Post-implantation Loss/Animal	[a1]	Mean SD	0.38 0.59 8.00		0.30 0.57 6.00	0.40 0.68 8.00		0.65 0.59 13.00
% Post-implantation Loss (%)	[a1]	Mean SD	5.57 8.66		4.58 8.65	5.11 9.21		14.39 16.42

C/S: Cesarean section

 

[a] - Anova & Dunnett

[a1] - Anova & Dunnett (Rank)

[f] - Chi-Squared & Fisher's Exact

 

Table 2: Summary litter data

 

Sex: Female			G1 0 mg/kg/day	G2 15 mg/kg/day	G3 45 mg/kg/day	G4 90 mg/kg/day
Total Number of fetuses		Sum	153	144	136	119
Total Number of litters		Sum	21	20	20	20
Number of dead fetuses		Sum	0	0	0	0
Total number of live fetuses		Sum	153	144	136	119
Mean litter size	[a]	Mean SD N	7.3 2.4 21	7.2 2.6 20	6.8 1.8 20	6.0 2.8 20
No. of Live Male fetuses		Sum	71	80	65	60
% Male Fetus		Mean SD	45.2 17.6	56.1 22.1	47.7 18.1	50.9 24.8
Mean Fetal Weight - Male (g)	[c]	Mean SD	40.836 5.758	41.944 3.431	38.849 5.728	38.526 7.595
No. of Live Female fetuses		Sum	82	64	71	59
% Female Fetus		Mean SD	54.8 17.6	43.9 22.1	52.3 18.1	49.1 24.8
Mean Fetal Weight - Female (g)	[c1]	Mean SD	39.794 5.501	42.204 5.808	38.737 4.739	38.770 4.847
Mean Fetal Weight- Male+Female (both) (g)	[c2]	Mean SD	40.423 4.636	42.172 3.627	38.854 5.122	38.213 6.617

 

[a] - Anova & Dunnett (Log)

[c] - Ancova/Anova & Dunnett: {Covariate(s): Number of Live Male Fetuses}

[c1] - Ancova/Anova & Dunnett: Covariate(s): Number of Live Female Fetuses}

[c2] - Ancova/Anova & Dunnett: Covariate(s): Number of Live Fetuses}

Applicant's summary and conclusion

Conclusions:

In this study, prenatal developmental toxicity of the test item was evaluated in New Zealand White rabbits following daily administration by oral gavage at 0, 15, 45, 90 mg/kg/day during gestation days 6 to 28.

The test item at 15 and 45 mg/kg/day was without effect on maternal body weights, weight gain, food consumption, and the maternal and litter parameters were comparable to vehicle control group. Gross evaluation of the placenta revealed no findings.

Treatment with the test item at 90 mg/kg/day resulted in treatment-related reduction in maternal body weight gain and food consumption indicating maternal toxicity.

There were no gross pathological changes at any dose level. External and visceral and skeletal examination of fetuses revealed no signs of teratogenicity up to the highest dose of 90 mg/kg/day.

Based on the above findings, under the test conditions used in this study, the following NOAEL values were derived:

• NOAEL for maternal toxicity was considered at 45 mg/kg/day as reduction in body weight gain and food consumption was observed at 90 mg/kg/day.

• NOAEL for developmental toxicity and teratogenicity was considered at
90 mg/kg/day since the other maternal and litter parameters were unaffected by treatment and there was no evidence of test item related fetal findings up to the dose of 90 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the prenatal developmental toxicity of the test item in New Zealand white rabbits. This study evaluated the developmental and maternal toxicity of the test item administered to pregnant rabbits by oral route during gestation days (GD) 6 to GD 28.  This study provided a rational basis for risk assessment in humans. The results of this study were used to establish the No Observed Adverse Effect Level (NOAEL) / No Observed Effect Level (NOEL) for maternal and developmental toxicity of the test item in rabbits.

The dose levels for this study were selected based on Dose Range Finiding study (DRF) study under study number N5369. Dose levels of 0, 45, 90 and 180/120 mg/kg/day were evaluated in pregnant rabbits (6/group) treated with the test item or vehicle from GD 6 to GD 28 at the dose volume of 2 mL/kg body weight. Due to test item related mortalities at 180 mg/kg/day, the dose was reduced to 120 mg/kg/day from GD 9 onwards.There was  reduction in body weight and food consumption and reduction in mean fetal weights at 90 mg/kg/day . Based on the results of of dose range finding study in pregnant rabbits, high dose of 90 mg/kg/day was selected for the definitive study. Mid dose of 45 mg/kg/day and low dose of
15 mg/kg/day was selected to provide a graded response to the test item.

In the definitive study, 92 mated female rabbits were assigned to four groups. Each group consisted of 23 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred. Test item dssolved in vehicle (Milli-Q water) was administered at 0, 15, 45 and 90 mg/kg/day at the dose volume of 10 mL/kg body weight. The control group received the vehicle only.

The following parameters and end points were evaluated in this study: Clinical signs, body weight, body weight gain, food consumption, caesarean section was performed for all the surviving rabbits on GD 29 and dams were examined for gross pathological changes. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses. The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed, and examined for external malformations. All the live fetuses were examined for visceral and skeletal variations and malformations.

Dose formulations were analysed twice i.e once during initiation and once at termination of treatment, the results of analysis indicated  within ± 10.0% of the nominal concentration and the relative standard deviation (%RSD) was less than 10%.

Main findings from the study are summarized below:

• Mortality, clinical signs, and gross necropsy changes: There were no unscheduled deaths, clinical signs, or any gross pathological findings that could be attributed to the test item.


• Body weight: No toxiclogically significant changes in maternal body weight or maternal body weight gain was observed at the doses of 15 and 45 mg/kg/day. At 90 mg/kg/day, there was a non-significant reduction in maternal body weight gain as compared to vehicle control group.


• Food intake: No toxiclogically significant changes in maternal food intake were observed at 15 and 45 mg/kg/day. At 90 mg.kg/day there was a significant reduction of food consumption by 17 to 29% associated with reduction in maternal body weight gain indicating maternal toxicity.


• Maternal developmental parameters: No significant changes in any maternal developmental toxicity endpoint were observed at 15, 45 and
  90 mg/kg/day. No gross pathological changes in the placenta were observed in any of the animals.

Litter Parameters: The total number of fetuses was 153, 144, 136 and 119 at 0, 15, 45, and 90 mg/kg/day, respectively. No dead fetuses were observed at any dose level. No significant changes in litter size, sex ratio, or fetal weight were observed up to the dose of 90 mg/kg/day.

• Fetal examination: The results from external, visceral, and skeletal examinations did not reveal any significant effects that could be attributed to the test item.

 

Based on the above findings, under the test conditions used in this study, the following NOAEL values were derived:

• NOAEL for maternal toxicity was considered at 45 mg/kg/day as reduction in body weight gain and food consumption was observed at 90 mg/kg/day.


• NOAEL for developmental toxicity and teratogenicity was considered at
  90 mg/kg/day since the other maternal and litter parameters were unaffected by treatment and there was no evidence of test item related fetal findings up to the dose of 90 mg/kg/day.