Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
designed as dose range finding study
Deviations:
yes
Remarks:
designed as dose range finding study, thus, only limited ex aminations
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han'": Rccl-lant'': WIST
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Ltd., Oxon, UK
- Age at study initiation: time-mated females, delivered prior to day 3 of gestation
- Housing: individually in solid floor polypropylene cage with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): Rodent 2018C Teklad Global Certified Pelleted Diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: day of delivery until day 3 of gestation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG400
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure:purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
17 d (day 3 to 19 of gestation)
Frequency of treatment:
daily from day 3 to 19 of gestation
Duration of test:
20 d
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
- Time schedule:
mortality: Twice daily, early and late during the working period.
clinical signs: Individual clinical observations will be performed immediately before dosing, up to 30 minutes after dosing and one hour after dosing. In addition, post dosing observations will also be performed at approximately 4 hours after dosing during the normal working day (excluding weekends and Public Holidays). All observations will be recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights will be recorded on Days 3, 4, 5, 8, 11, 14, 17 and 20 of gestation.

FOOD CONSUMPTION:
Dietary intake will be recorded for individual animals on Days 3, 5, 8, 11, 14, 17 and 20 of gestation.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption will be monitored daily by visual inspection of water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- gross necropsy

Ovaries and uterine content:
The uterus of each adult female will be examined and where possible the following recorded;
i. Pregnancy status
ii. Number of corpora lutea
iii. Gravid uterus weight
iv. Number, status* and intra-uterine position of implantations
* = live fetus, dead fetus, late intra-uterine death or early intra-uterine death

Fetal examinations:
For each fetus/placenta, where possible, the following will be recorded:
i. External fetal abnonnalities
11. Fetal weight
iii. Fetal sex
iv. Placental weight

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Between Day 5 to 7 of gestation, the majority of females treated with 75 or 150 mg/kg bw/day showed instances of increased salivation and/or noisy respiration.
Incidental findings included one 75 mg/kg bw/day females showing observations of deceased respiratory rate, pilo-erection and diarrhoea between Days 5 to 7 with noisy respiration between Days 5 to 10 and 12 to16.
Mortality:
mortality observed, treatment-related
Description (incidence):
One 150 mg/kg bw/day female was found dead shortly after dosing on Day 5 of Gestation, there were no adverse effects on body weight, food consumption or clinical signs prior to death. Necropsy findings revealed darkened liver and kidneys, with dark patches in the lungs.
There were no further unscheduled deaths on the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
All female treatment groups exhibited lower body weight gains following the first dose (Day 3), with improvement evident thereafter.
During the first Week of treatment lower overall accumulative body weight gains were evident in females treated with 75 or 150 mg/kg bw/day in relation to controls. However, during the latter stage of gestation, overall body weight gains return to levels similar to controls. Body weight gains when adjusted for gravid uterus weight, revealed lower body weight gain of 17% and 40%, in females treated with 75 and 150 mg/kg bw/day, respectively. No such effect was detected at 25 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Instances of marginally lower food consumption were noted in 75 and 150 mg/kg bw/day dose groups between Days 3 to 8 and 3 to 5 of gestation, respectively. Thereafter food consumptions were generally similar to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water consumptions revealed no adverse effects.
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic findings in the decedent female (No. 58) findings revealed darkened liver and kidneys, with dark patches in the lungs.
No macroscopic abnormalities were detected in animals examined at study termination.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
Litter Data
There was no detrimental effect of maternal treatment on litter data as assessed by corpora lutea, numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size and sex ratio at 25, 75 or 150 mg/kg bw/day.
Females treated with 150 mg/kg bw/day showed slightly higher pre-implantation losses when compared to controls. Females at 25 or 150 mg/kg bw/day also showed slightly higher post-implantation losses in comparison with controls, without any dose response.

Litter Placental and Foetal weights
There were no effects of maternal treatment on mean foetal weights, placental weights or litter weight at 25, 75 or 150 mg/kg bw/day.

Effect levels (maternal animals)

Dose descriptor:
other: upper dose level for definitive OECD TG 422 study
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
body weight and weight gain

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): 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
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
External Examinations
There were no findings apparent for foetuses from treated females at external examination on Day 20 of gestation.
At 150 mg/kg bw/day incidental findings included one litter with five pale fetuses and a further one litter contained one fetus with encephaolcoele.

Effect levels (fetuses)

Dose descriptor:
other: upper dose level for definitive OECD TG 422 study
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: overall effects

Fetal abnormalities

Abnormalities:
no effects observed
Description (incidence and severity):
only external examinations performed in this dose range finding study

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Bodyweights

Group

 

Days

 

 

 

 

 

 

 

 

 

 

1

3

4

5

8

11

14

17

20

1

Mean

215.0

229.0

233.8

235.0

240.8

250.0

260.5

282.5

314.3

 

SD

15.4

17.2

18.5

15.7

17.5

18.6

21.0

19.8

16.5

 

n

4

4

4

4

4

4

4

4

4

2

Mean

211.0

226.6

227.2

229.8

237.8

249.6

261.0

287.4

323.2

 

SD

15.6

16.6

19.1

18.6

18.6

19.4

22.1

24.0

29.4

 

n

5

5

5

5

5

5

5

5

5

3

Mean

210.0

225.2

226.6

229.8

233.2

241.0

256.2

278.0

311.6

 

SD

15.2

16.2

16.3

14.5

15.2

14.4

16.3

18.3

22.6

 

n

5

5

5

5

5

5

5

5

5

4

Mean

209.8

223.0

222.3

225.8

229.0

243.3

256.3

280.3

308.0

 

SD

17.8

18.8

22.1

19.0

17.6

19.5

19.6

23.3

30.5

 

n

4

4

4

4

4

4

4

4

4


 

Body weight gains

Group

 

Days

 

 

 

 

 

 

 

 

 

 

1

3

4

5

8

11

14

17

 

 

3

4

5

8

11

14

17

20

1

Mean

14.0

4.8

1.3

5.8

9.3

10.5

22.0

31.8

 

SD

2.2

1.3

3.0

2.2

2.1

3.1

5.0

3.8

 

n

4

4

4

4

4

4

4

4

2

Mean

15.6

0.6

2.6

8.0

11.8

11.4

26.4

35.8

 

SD

6.8

4.0

2.4

1.4

0.8

3.6

2.5

5.8

 

n

5

5

5

5

5

5

5

5

3

Mean

15.2

1.4

3.2

3.4

7.8

15.2

21.8

33.6

 

SD

2.2

1.8

2.4

22.3

21.1

3.1

4.4

4.6

 

n

5

5

5

5

5

5

5

5

4

Mean

13.3

-0.8

3.5

3.3

14.3

13.0

24.0

27.8

 

SD

4.5

3.9

3.5

2.8

2.8

2.2

3.7

8.1

 

n

4

4

4

4

4

4

4

4

                       

 

Food consumption (g/rat/day)

Group

 

Days

 

 

3

5

8

11

14

17

 

 

5

8

11

14

17

20

1

Mean

16.0

14.8

18.3

21.5

19.8

22.1

 

SD

1.1

3.9

1.3

2.6

2.0

1.0

 

n

4

4

4

4

4

4

2

Mean

18.2

16.7

19.0

22.3

21.4

23.1

 

SD

1.9

1.6

1.1

1.6

2.3

0.5

 

n

5

5

5

5

5

5

3

Mean

15.1

11.6

16.9

21.3

19.7

22.5

 

SD

1.7

4.7

1.3

2.4

2.1

2.6

 

n

5

5

5

5

5

5

4

Mean

12.8

14.3

17.7

21.3

19.0

19.1

 

SD

1.9

1.8

1.7

2.3

2.5

3.4

 

n

4

4

4

4

4

4

 

 

Group

 

Copora Lutea

 

Number of Implants

Embryonic/Fetal Deaths

Implantation losses

Live young

 

 

Sex Ratio

(% male)

Mean Fetal Weight

 

 

Mean Placental

Weight

 

Litter Weight

Total Placental

Weight

 

Early

Late

Total

Pre-

Post-

Male

Female

Total

Male

Female

Combined

1

Mean

13.0

10.5

0.0

0.0

0.0

19.3

0.0

5.0

5.5

10.5

49.0

3.967

3.669

3.817

0.489

40.023

5.118

 

SD

1.4

1.3

0.0

0.0

0.0

4.3

0.0

2.6

3.3

1.3

25.8

0.250

0.295

0.255

0.023

4.887

0.538

 

n

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

2

Mean

14.5

11.8

0.2

0.0

0.2

19.0

1.3

5.2

6.6

11.8

43.2

4.017

3.856

3.933

0.500

46.534

5.908

 

SD

1.7

2.2

0.4

0.0

0.4

10.7

3.0

1.9

0.9

1.6

10.9

0.244

0.236

0.233

0.018

7.775

0.944

 

n

4

4

5

5

5

4

5

5

5

5

5

5

5

5

5

5

5

3

Mean

13.0

10.2

0.0

0.0

0.0

20.8

0.0

6.0

4.2

10.2

58.1

4.381

4.165

4.297

0.538

43.648

5.448

 

SD

1.9

1.6

0.0

0.0

0.0

13.7

0.0

1.6

0.4

1.6

6.9

0.196

0.248

0.194

0.078

5.933

0.937

 

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

4

Mean

14.5

11.0

0.3

0.0

0.3

24.7

2.5

5.3

5.5

10.8

45.2

4.462

4.176

4.281

0.565

45.493

6.153

 

SD

1.3

2.9

0.5

0.0

0.5

15.0

5.0

3.6

1.0

3.1

19.5

0.402

0.474

0.447

0.123

10.959

2.402

 

n

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

 

Applicant's summary and conclusion

Conclusions:
In consideration of the marked body weight effects on the males during the fourteen day repeated dose phase with non-pregnant animals (Phase I) at 200 mg/kg bw/day and the observed effects on body weight gains on females at 150 mg/kg bw/day during the repeated dose phase on pregnant animals (Phase II), these effects were considered not to be so significant to exclude 150 mg/kg bw/day as the high dose level for further investigating. Therefore, dose levels of 0, 25, 75 and 150 mg/kg bw/day are recommended for the forthcoming Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) (Study No. HD72JH).
Executive summary:

The study was designed to investigate the effects of oral administration of the test item on embryonic and foetal development during the period of organogenesis. The results provide supporting information to assist in the selection of dose levels for the subsequent longer term toxicological evaluation of the test item in a Combined Repeat Dose Toxicity Study with Reproductive/Developmental Toxicity Screening Test in the Rat (OECD 422); Study No. HD72JH.

In these investigations (Study Phase II) the test item was administered once daily by gavage to three groups, each of five time-mated Wistar Hanä: RccHanä: WIST strain female rats from Day 3 to Day 19 of gestation at dose levels of 25, 75 and 150 mg/kg bw/day. A control group of five time-mated females received vehicle alone (Polyethylene glycol 400) over the same treatment period. All females were killed on Day 20 of gestation.

 

Clinical signs, bodyweight development and food consumption were monitored during the study and all animals were subjected to gross necropsy examination including examination of the uterine contents and gross external necropsy of foetuses.

 

Adult Responses

Mortality

One 150 mg/kg bw/day female was found dead shortly after dosing on Day 5 of Gestation, there were no adverse effects on body weight, food consumption orclinical signs prior to death. Necropsy findings revealed darkened liver and kidneys, with dark patches in the lungs.

There were no further unscheduled deaths on the study.

 

Clinical Observations

Between Day 5 to 7 of gestation, the majority of females treated with 75 or 150 mg/kg bw/day showed instances of increased salivation and/or noisy respiration.

Incidental findings included one 75 mg/kg bw/day females showing observations of deceased respiratory rate, pilo-erection and diarrhoea between Days 5 to 7 with noisy respiration between Days 5 to 10 and 12 to16.

 

Body Weight

All female treatment groups exhibited lower body weight gains following the first dose (Day 3), with improvement evident thereafter.

During the first Week of treatment lower overall accumulative body weight gains were evident in females treated with 75 or 150 mg/kg bw/day in relation to controls. However, during the latter stage of gestation, overall body weight gains return to levels similar to controls. Body weight gains when adjusted for gravid uterus weight, revealed lower body weight gain of 17% and 40%, in females treated with 75 and 150 mg/kg bw/day, respectively. No such effect was detected at 25 mg/kg bw/day. 

 

Food Consumption

Instances of marginally lower food consumption were noted in 75 and 150 mg/kg bw/day dose groups between Days 3 to 8 and 3 to 5 of gestation, respectively. Thereafterfood consumptions were generally similar to controls.

 

Water consumption

Visual inspection of water consumptions revealed no adverse effects.

 

Post Mortem

The macroscopic findings in the decedent female (No. 58) findings revealed darkened liver and kidneys, with dark patches in the lungs.

No macroscopic abnormalities were detected in animals examined at study termination.

 

Litter Responses

Litter Data

There was no detrimental effect of maternal treatment on litter data as assessed by corpora lutea, numbers of implantations, in-uterooffspring survival (as assessed by the mean numbers of early or late resorptions), live litter size and sex ratio at 25, 75 or 150 mg/kg bw/day.

Females treated with 150 mg/kg bw/day showed slightly higher pre-implantation losses when compared to controls. Females at 25 or 150 mg/kg bw/day also showed slightly higher post-implantation losses in comparison with controls, without any dose response.

 

Litter Placental and Foetal weights

There were no effects of maternal treatment on mean foetal weights, placental weights or litter weight at 25, 75 or 150 mg/kg bw/day.

 

Foetal Evaluation

External Examinations

There were no findings apparent for foetuses from treated females at external examination on Day 20 of gestation.

At 150 mg/kg bw/day incidental findings included one litter with five pale fetuses and a further one litter contained one fetus with encephaolcoele.

 

Conclusion 

In consideration of the marked body weight effects on the males during the fourteen day repeated dose phase with non-pregnant animals (Phase I) at 200 mg/kg bw/day and the observed effects on body weight gains on females at 150 mg/kg bw/day during the repeated dose phase on pregnant animals (Phase II), these effects were considered not to be so significant to exclude 150 mg/kg bw/day as the high dose level for further investigating. Therefore, dose levels of 0, 25, 75 and 150 mg/kg bw/day are recommended for the forthcoming Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) (Study No.HD72JH).