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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 August 2015 -- 16 September 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
range-finding study
GLP compliance:
no
Remarks:
(not required for a range-finding study)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:

- Lot/batch No.: A27Z0R010101
- Expiry date: 19 February 2017
- Storage condition: at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France.
- Age/Weight: at the beginning of the treatment period, the animals were 10-11 weeks old and had a mean body weight of 305 g (range: 270 g to 336 g).
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 4 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 31 August 2015 to 16 September 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Type of formulation
(visual observation): Solution in the vehicle

Preparation procedure: According to the validation study describing the preparation procedure (homogeneity and stability testing) for a range of concentrations covering the lowest and highest used in this study.
The test item was weighed and mixed with the required quantity of vehicle.
The pH of the dose formulation was adjusted to 8.0 (± 0.5) with a solution of Hydrochloric acid 30 35%, TraceSELECT® Ultra, for ultratrace analysis. The volume of HCl added was recorded for each concentration.
The final pH was checked and recorded by using a pH-meter
Frequency of preparation: Test item dose formulations
Based on test item dose formulation stability and vehicle expiry
Vehicle preparation
Based on the CiToxLAB France in-house procedures (frequency documented in study raw data)
Storage condition
(control dose formulation(s)): At room temperature
Storage conditions
(test item dose formulations): At room temperature and protected from light
Delivery conditions: At room temperature and protected from light
Vehicle
Name: Drinking water treated by reverse osmosis using ELIX 5 (Millipore SA)
Used for preparation of test item dose formulations and administered to control group (control dose formulation)
Preparation: By CiToxLAB France Pharmacy
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No chemical analysis was performed.
Details on mating procedure:
- Proof of pregnancy: vaginal plug, referred to as Day 0 post-coitum
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum
Frequency of treatment:
Daily
Duration of test:
21 days
No. of animals per sex per dose:
8 time-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous study performed in the same species.
In the previous dose-range finding study by the oral route (gavage), male and female Sprague-Dawley rats received Diethylaminopropylamine (as pH-neutralized dose formulations) daily by gavage at dose-levels of 100, 300 or 1000 mg/kg bw/day for 2 weeks. At 1000 mg/kg bw/day, in-life observations were limited to ptyalism in both sexes and lower body weight gain at treatment initiation resulted in a final lower body weight in males. Histopathology findings consisted of minimal to slight hyperplasia of squamous cells associated with minimal to slight hyperkeratosis within the forestomach in all males and females given 1000 mg/kg bw/day.

Thus, based on these available data, the selected dose-levels of the present study are: 100, 300 and 1000 mg/kg bw/day.

Examinations

Maternal examinations:
MORBIDITY AND MORTALITY:
- Time schedule: at least twice a day during the treatment period, including weekends.

CLINICAL OBSERVATIONS:
- Time schedule: once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT (GAIN):
- Time schedule: on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c., and prior to premature sacrifice.

FOOD CONSUMPTION:
- Time schedule: on Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 p.c.,
- Organs examined: the principal thoracic and abdominal organs and the weight of the gravid uterus .
Ovaries and uterine content:
The ovaries and uterus of the females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

The following classification was used to record:
- uterine scar: uterine implantation without implant,
- early resorption: evidence of implant without recognizable embryo,
- late resorption: dead embryo or fetus with external degenerative changes,
- dead fetus: non live fetus with no external degenerative changes.
Fetal examinations:
Each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices. All fetuses were then discarded.
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
Indices:
% of pre-implantation loss =
Number of corpora lutea - Number of implantations
_______________________________________________________________ x 100
Number of corpora lutea

% of post-implantation loss =
Number of implantation sites - Number of live fetuses
__________________________________________________________________________ x 100
Number of implantation sites

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
One female given 1000 mg/kg bw/day was not pregnant.
All other mated females were pregnant.
One female given 1000 mg/kg bw/day was sacrificed prematurely on Day 15 p.c. for ethical reasons. Prior to sacrifice, signs of poor clinical condition were observed (i.e. piloerection, round back, pallor of extremities, hypoactivity, half-closed eyes and reddish vaginal discharge). A body weight loss of 66 g was recorded between Days 6 and 15 p.c. together with reduced food consumption.
At necropsy, thymus was reduced in size, whitish nodules were seen in the forestomach, blackish contents were observed in the cecum and reddish contents were noted in the vagina. Seventeen corpora lutea and fourteen implantation sites were recorded.
No other unscheduled deaths occurred in any other groups.
Ptyalism was observed in all surviving females given 1000 mg/kg/day during the first and/or the second weeks of treatment for 1 to 6 days duration. This was considered to be of minor toxicological significance.
Piloerection in 1/7 females given 1000 mg/kg bw/day was noted on Day 14 p.c. only.
Reflux at dosing was observed on two occasions in 2/8 and 1/7 females at 300 and 1000 mg/kg/day, respectively.
One palpable mass, measuring 2 cm in diameter, was seen on mammary gland in 1/8 females at 300 mg/kg/day from Day 18 p.c.. This was not considered to be test item-related as it was of isolated occurrence and not dose-related (Table 1).
At 1000 mg/kg bw/day and when compared with controls, mean body weight loss was noted over the first 3 days of treatment (-13 g vs. +13 g), followed by a lower mean body weight gain until Day 15 p.c. (+12 to +14 g vs. +15 to +25 g). This trend was accentuated by the body weight loss (-50 g) recorded in the prematurely sacrificed female between Days 9 and 15 p.c.. A statistically significantly lower body weight gain was therefore observed over the whole study period (+105 g vs. +140 g).
At 300 mg/kg bw/day and when compared with controls, a lower mean body weight gain was noted over the first 3 days (+9 g vs. +13 g).
At 100 mg/kg bw/day and when compared with controls, there were no effects on mean body weight or mean body weight change (Table 2).
At 1000 mg/kg bw/day and when compared with the controls, lower mean food consumption (-14 to -52%) was recorded between Days 6 and 15 p.c.. This effect was associated with reduced food consumption recorded in the prematurely sacrificed female between Days 9 and 15 p.c..
At 300 mg/kg bw/day, a comparable same trend was observed over the first three days of the study, but to a lesser extent (-11% vs. controls). These differences correlated with the mean body weight loss or the lower mean body weight gain also observed at 300 mg/kg bw/day.
At 100 mg/kg bw/day, there were no effects of treatment with the test item.
When compared with the control group, dams given 1000 mg/kg bw/day had a lower gravid uterus weight observed in high-dose dams (-9% compared to controls). Lower net weight change (+16 g vs. +41 g in controls) was considered to be a consequence of the lower body weight gain in these females.
Gestation parameters were not impacted by the test item treatment at any dose-levels.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no effect on the fetal body weight and on the percentage of male fetuses (sex ratio).
There were no treatment-related fetal external malformations or variations in any groups.

Effect levels (fetuses)

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: clinical signs

Dose-level (mg/kg bw/day)

0

100

300

1000

Piloerection

0

0

0

1

Ptyalism

0

0

0

7

Reflux at dosing

0

0

2

1

Mass on mammary gland

0

0

1

0

Total affected animals

0/8

0/8

3/8

7/7

Table 2: mean body weights and body weight changes

Dose-level (mg/kg bw/day)

0

100

300

1000

Body weight (g)

 

 

 

 

Day 6 p.c.

306

-

304

(-1)

304

(-1)

304

(-1)

Day 21 p.c.

445

-

444

(0)

452

(+2)

410

(-8)

Body weight change (g)

 

 

 

 

Days 6 - 9 p.c.

+13

+14

+9

-13#

Days 9-12 p.c.

+25

+24

+25

+14

Days 12-15 p.c.

+15

+16

+19

+12

Days 6 - 21 p.c.

+140

+140

+148

+105**

Statistical significance; **: p<0.01 and #: p<0.001. p.c.:post-coitum.

-: not applicable.

( ): in brackets, percentage difference vs. controls.

Table 3: Mean food consumption changes (expressed in g/animal/day) 

Dose-level (mg/kg bw/day)

0

100

300

1000

Days 6 - 9p.c.

27

-

27

(0)

24

(-11)

13#

(-52)

Days 9-12p.c.

28

-

27

(-4)

28

(0)

16#

(-43)

Days 12-15p.c.

29

-

29

(0)

30

(+3)

25

(-14)

Statistical significance:#: p<0.001.

p.c.:post-coitum.

-: not applicable.

( ): in brackets, percentage differencevs.controls.

Table 4: mean carcass, net change and gravid uterus weights (a) (g)

Dose-level (mg/kg bw/day)

0

100

300

1000

Gravid uterus weight

98

-

95

(-4)

109

(+11)

89

(-9)

Carcass weight

347

-

350

(+1)

343

(-1)

321

(-7)

Net weight change from Day 6.p.c.

+41

+45

+39

+16#

Statistical significance:#: p<0.001.

( ): in brackets, percentage differencevs.controls.

p.c.:post-coitum.

(a): weights are rounded values.

-: not applicable.

Table 5: Hysterectomy data 

Dose-level (mg/kg bw/day)

0

100

300

1000

Number of females with live fetuses at term

8

8

8

6

Mean number of corpora lutea

16.5

14.3

14.9

15.0

Mean number of implantation sites

13.4

13.0

14.3

13.3

Mean pre-implantation loss (%)

17.0

8.8

4.2

8.9

Mean number of fetuses

12.5

11.8

13.9

12.0

Percentage of dead fetuses (%)

0

0

0

0

Mean number of uterine scars + resorptions

0.9

1.3

0.4

1.3

Mean number of early resorptions

0.8

1.3

0.4

1.3

Mean number of late resorptions

0.1

0.0

0.0

0.0

Mean post-implantation loss (%)

6.3

10.4

2.7

10.2

  

Table 6: Fetal data 

Dose-level (mg/kg bw/day)

0

100

300

1000

Mean fetal body weight (g)

5.69

-

5.77

(+1.4)

5.72

(+0.5)

5.36

(-5.8%)

Mean percentage of male fetuses (%)

49.5

53.7

47.4

40.8

( ): in brackets, percentage differencevs.controls.

-: not applicable.

Applicant's summary and conclusion