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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:
key study
Study period:
28 Feb 2018 to 09 Jul 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Toxicological Studies, ul. Doświadczalna 27, 43–200 Pszczyna, Poland
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Cmdb: Wi; outbred
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Experimental Medicine Centre at the Medical University in Białystok kept behind the breeding barrier.
- Weight at study initiation: Mean body weight 231.7 g (200 to 268 g)
- Housing: The animals were housed in plastic cages with metal wire lid and dimensions: 58 x 37 x 21 cm (length x width x height). Autoclaved, and additionally UV-irradiated wood chips were used as bedding. At mating one female was placed in one cage with one male. At pregnancy the females were housed individually. The environment of the animals was enriched by placing wooden blocks, tunnels and nesting materials for laboratory animals in the cages.
- Diet: “ALTROMIN” standard laboratory fodder, Altromin Spezialfutter GmbH & co.KG, Seelenkamp, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: at least 5 days.
- A general medical-veterinary examination was performed on the day of the introduction of the animals to the acclimatisation, whereas a detailed medical-veterinary examination was performed prior to the beginning of the experiment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 to 24.0
- Humidity (%): 33.5 to 58.5
- Air changes (per hr): about 16
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
28 Feb 2018 to 09 Jul 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared every two days based on results of the stability test. To ensure the appropriate concentration for doses, the right quantity of test item was dissolved for each dose. The following establishments was fulfilled during preparation of doses:
Group 1: 1 g of solution contained 20 mg of test item at dose of 80 mg/kg bw
Group 4: 1 g of solution contained 31,25 mg of test item at dose of 125 mg/kg bw
Group 2: 1 g of solution contained 50 mg of test item at dose of 200 mg/kg bw
Group 3: 1 g of solution contained 125 mg of test item at dose of 500 mg/kg bw
The test item was given from the 5th to the 19th day of gestation using a stomach tube. The control group of pregnant females (group 0) was given sunflower oil at the same volume as groups treated with the test item. In order to maintain a constant dose level appropriate to the animals’ body weight, the doses were adjusted on days of body weight determination

VEHICLE
- Amount of vehicle: A constant volume of the solution, i.e. 0.4 mL/100 g bw was used at each dose level.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to confirm correct preparation of the test item in the study, the solutions of the test item in sunflower oil were chemically analysed. Samples of the test item solution were transferred for chemical analyses to the Laboratory of Analytical Chemistry, which is a part of the Department of Ecotoxicological Studies at the Institute of Industrial Organic Chemistry, Branch Pszczyna.
Before the beginning of the study analytical method validation was conducted. During the study the samples were transferred thrice for chemical analysis: at the beginning, in the middle and at the end of the study. The concentration of tes substance in sunflower oil was chemically determined with a High Performance Liquid Chromatography (HPLC), Shimadzu, Prominence LC-2030C with Diode Array Detector (DAD).
Details on mating procedure:
- Impregnation procedure: cohoused, not related males used for mating with nulliparous female rats came from the same husbandry and stock and had a similar age.
- M/F ratio per cage: 1/1, one male was used to mate with to females.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of gestation.
Duration of treatment / exposure:
The test item/medium were administered to the females from the 5th to the 19th day of gestation.
Frequency of treatment:
Once daily
Duration of test:
On the 20th day of gestation the animals were euthanised.
Doses / concentrationsopen allclose all
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Group 4 (new group after termination of treatment goup 3)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Group 3 (group terminated for humanitatian reasons)
No. of animals per sex per dose:
Group 0,1: 22 females per group
Group 2,4: 23 females per group
Group 3: 10 females per group (terminated)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 80, 200 and 500 mg/kg bw and also the vehicle were selected on the basis on the results of Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening test (OECD 422). The high dose group was terminated due to occurrence of clinical signs (such as respiratory murmurs, difficult respiration, porphyrin discharge around nostrils, porphyrin discharge around the eyes, bristled coat, rounded back, dejection, decrease of locomotor activity, diarrhoea, urination, moisture around the snout, tremors, hypothermia and more than 20% decrease of body weight) and high maternal mortality. A new dose group, 125 mg/kg bw/day, was introduced.
- Rationale for animal assignment: Random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day during labour week and once a day on days off.
- Cage side observations included: general evaluation of their health state and observations of morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day.
- Detailed clinical observations included: skin changes, coat changes, changes in eyes and mucous membranes, respiratory system, circulatory system, nervous system, somatic activity and behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: 0, 5th, 8th, 11th, 14th, 17th and 20th day of gestation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 0, 5th, 8th, 11th, 14th, 17th and 20th day of gestation.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100 g body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20. Also females which died before gestation day 20 were macroscopically examined for any structural abnormalities or pathological changes which could have influenced gestation.
- Organs examined: Lungs, stomach, liver, small intestine, duodenum, jejunum, cecum, whole intestines, spleen, ovaries and area around the anus.
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: the left and right uteri horn were examined. The uteri of non-pregnant females and non-pregnant horns of the uteri were fixed between two glass plates and overexposed by electric lamp to confirm the non-pregnant status.
Fetal examinations:
- External examinations: Yes: all per litter, sex, body weight with and without placenta, placenta weight alone, body lenght with and without tail.
During gross evaluation attention was paid to: reactions to tactile impulses, formation of body coverings, formation of limbs, number of fingers and toes, shape of head, formation of auricular conchas, presence of nasal apertures, formation of oral cavity, presence of anus and tail.
- Soft tissue examinations: Yes: half per litter. Fetuses were cut in sagittal planes and the formation of particular cavities and presence of internal organs was evaluated.
- Skeletal examinations: Yes: half per litter. The stained fetuses were examined and formation of skull bones, back-bone, ribs, acromial and pelvic girdles with limbs was evaluated. Points of ossification in sternum, metacarpus of fore limbs and metatarsus of hind limbs were counted during evaluation
- Head examinations: Yes: half per litter, the skull bones were examined.
Statistics:
When discussing the results of the prenatal developmental toxicity study, all females used in the study were taken into consideration. The evaluation of results included the relationship, or lack thereof, between the exposure of the animals to the test item and the incidence and severity of all findings, and also, when appropriate, historical control data to enhance interpretation of study results.
Only females whose pregnancy status is confirmed were taken into consideration when conducting statistical analyses with the use of STATISTICA 10, with p ≤ 0.05 concerning the following elements: body weight of pregnant females, food consumption of pregnant females, number of corpora lutea, number of fetuses in litter, number of males and females in litter, weight of uteri, weight of fetuses, length of fetuses, number of ossification points in sternum and limbs. For each quantitative parameter mean and standard deviation were determined.
The results obtained in the treated groups (group 1, group 4, group 2) were compared with the control group (group 0).
The course of the statistical analysis was as follows:
- first of all, the normality of distribution with the Shapiro-Wilk test was examined and the homogeneity of variance with the Brown-Forsythe test.
- if the test results were characterized by normal distribution and homogeneous variances, a one-way analysis of variance was used, if necessary confirmed with Dunnett’s test.
- in the absence of normality of distribution or non-homogeneous variances, the nonparametric Kruskal-Wallis test was used, if necessary confirmed with Dunnett’s test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Respiratory murmurs were observed in all treatment groups and the incidence increased with higher doses, no similar changes were observed in the control group. In Group 2 (200 mg/kg bw/day), one female suffered from difficulty breathing, accelerated respiration and intensifying dyspnoea. Another female suffered from porphyrin discharge around the nostrils.
In the terminated group 3 (dose of 500 mg/kg bw) clinical signs consisted of: respiratory murmurs, difficult respiration, porphyrin discharge around nostrils, porphyrin discharge around the eyes, bristled coat, rounded back, dejection, decrease of locomotor activity, diarrhoea, urination, moisture around the snout, tremors, hypothermia and more than 20% decrease of body weight, and high maternal mortality.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
There was no mortality in group 0 (0 mg/kg bw/day), group 1 (80 mg/kg bw/day) or group 4 (125 mg/kg bw/day).
Four animals died and one was euthanised in group 2 (200 mg/kg bw/day).
Four animals died and two were euthanised in group 3 (500 mg/kg bw/day; group was terminated).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decrease of average body weight of pregnant females from 8th to 20th day of experiment was observed in all treated groups and statistically significant decrease stated in group 2 (200 mg/kg bw) indicated influence of test item (Table 1 in 'Any other information on results incl. tables'). Individual body weight losses of pregnant females in treated groups also indicated influence of the test item because there were no similar body weight losses in control group. Mild body weight losses stated in two non-pregnant females of control groups were interpreted as accidental however in case of similar body weight losses in one non-pregnant female of group 2 (200 mg/kg bw) and one non-pregnant female of group 4 (125 mg/kg bw) influence of test item cannot be excluded.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decrease of average food consumption from 8th to 20th day of experiment and statistically significant decreases were stated in all treated groups compared to control group which indicated influence of test item (Table 2 in 'Any other information on results incl. tables'). On the basis of results, it can be seen that decrease of average food consumption increased with increasing dose.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
In females, which were euthanised for humanitarian reasons or died in group 2 (200 mg/kg bw) and 3 (500 mg/kg bw), many gross lesion were stated (Table 4 in 'Any other information on results incl. tables'). Extension of intestines or stomach, bloating, ulcers and erosions of the mucosa of the stomach, congestion of intestines, intestinal gas, content of the intestines tinged with blood, area around the anus contaminated with faeces and also haemorrhagic inflammation of intestines, indicated influence of test item on the gastrointestinal track. The circulatory disorders (i.e. congestion of lungs, ovaries and also intestines) and foci of emphysema, enlargement of the lungs (as a results of emphysema) and fragile spleen most likely represent euthanasia-related agonal and/or death-related events.
The circulatory disorders (i.e. congestion of stomach and intestines) which appeared in females euthanised on the 20th day of pregnant from control and all treated groups, most likely represent euthanasia-related agonal events (Table 3 in 'Any other information on results incl. tables'). However, increase in the numbers of females with these lesions in group 1, 4 , 2 and 3 compared to control group can be observed.
Extension of intestines, bloating and gas in intestines, ulcers and erosions of the mucosa of the stomach and thickened wall with an inflammatory character observed in females euthanised on the 20th day of pregnant from group 1, 4, 2 and 3 indicated influence of test item on the gastrointestinal track.
Spread, bright lesion with a firm consistency in the caudate lobe of the liver (probably of a cancerous nature) and lesion in size 6x1,5 mm, red, pedunculated in adipose tissue around the left ovary observed in 2 females from group 0 and bright red-yellow lesion, size 3x1 mm in the right corner of the uterus at the height of the first fetus observed in female from group 1 (80 mg/kg bw) can be considered as a spontaneous lesions. The lack of association with the influence of the test item is evidenced by the presence of these changes in the control group.
Other lesions observed in the liver i.e. fragile lesion with a clear lobular structure and with bright foci and two bright lesion with granular consistency are probably associated with the administration of the euthanasia dose to the liver instead of to the peritoneal cavity and should not be combined with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Pre- and post-implantation losses were observed in control and all experimental groups (Table 6 in 'Any other information on results incl. tables'). The percentage of pre- and post-implantation losses was comparable in all groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no implantation sites in non pregnant females, indicating that there was no total litter loss by resorption (Table 6 in 'Any other information on results incl. tables').
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Resorptions were observed in control and all experimental groups (Table 6 in 'Any other information on results incl. tables'). The number of resorptions was comparable for all groups.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Intrauterine mortality was equal in control group 0, 1 and 2 . No intrauterine mortality was stated in group 4 (Table 7 in 'Any other information on results incl. tables').
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of non-pregnant females was equal in control group and group 4 (125 mg/kg bw) and 2 (200 mg/kg bw). In the remaining groups, no non-pregnant females were stated (Table 6 in 'Any other information on results incl. tables').
Other effects:
not examined
Details on maternal toxic effects:
An overview of the result of mating can be found in Table 6 in 'Any other information on results incl. tables'.

Effect levels (maternal animals)

Key result
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
gross pathology

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase of weight of the fetuses without placenta and fetal membranes in group 4 (125 mg/kg bw) should be interpreted as accidental because there were no changes in group 2 (with higher dose - 200 mg/kg bw) (Table 8 in 'Any other information incl. results').
The weight of placenta was statistically significantly lower in group 2 (200 mg/kg bw) compared with the control group but weight of fetuses with and without placenta and fetal membranes were in the normal range so this change should not be use in isolation as the only criterion for assessment and should not be combined with the test item.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Intrauterine mortality was equal in control group 0, 1 and 2 . No intrauterine mortality was stated in group 4 (Table 7 in 'Any other information on results incl. tables').
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The average numbers of the males in litter were statistically significantly lower in group 4 compared with the control group but there were no changes in group 2 (with higher dose - 200 mg/kg bw) so it should be interpreted as accidental and should not be combined with the test item (Table 7 in 'Any other information on results incl. tables').
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No changes in litter size were observed (Table 7 in 'Any other information on results incl. tables').
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
An overview of the external malformations can be found in Table 9 in 'Any other information on results incl. tables'.
No statistically significant difference in lengths of the fetuses with tail and lengths of the fetuses without tail in all treated groups in comparison with the control group were stated.
Subcutaneous haemorrhages in different parts of the fetal bodies which occurred in the treated groups and in the control group, should not be perceived as the effects of the test item. Subcutaneous haemorrhages in fetuses were often observed in other prenatal developmental toxicity studies performed at the Institute of Industrial Organic Chemistry Branch Pszczyna and should be considered as typical disturbances in fetuses. Similarly, three case of internal haemorrhage in fetuses were probably accidental and should not be considered as the test item related effect.
Omphalocele which occurred in one fetus of group 2 (200 mg/kg bw) is a serious defect in the abdominal wall at the umbilicus, through which the intestines protrude and is regarded as a serious malformation, but it occurred also in control group in one fetus and hence should not be connected with the influence of the test item. Significantly smaller fetuses occurred in all groups (control and treated) and because there were no statistically significant decrease of weight of fetuses and no statistically significant difference in lengths of the fetuses from treated groups, this lesion should not be combined with the test item.
In group 1 (80 mg/kg bw), occurrence of two smaller fetuses with deformed tail and 4-fingered forelimbs from the same litter, one fetus with diaphragmatic hernia and one, dead fetus with hypoplasia of the right ventricle should be treated as accidental due to the significantly smaller number of fetuses with malformation in groups with higher dose of the test item (groups 4 and 2). In groups with higher dose of the test item there were no increased number of resorptions and intrauterine mortality of fetuses, which confirms the randomness of malformation in group 1 (80 mg/kg bw). Additionally, based on historical data (previous studies conducted at Institute of Industrial Organic Chemistry Branch Pszczyna), occurrence of 4 fetuses with serious malformations per 294 fetuses in group are within the normal range.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The average number of ossification points in the sternum and metacarpus can be found in Table 10 and 11 in 'Any other information of results incl. tables'. An overview of these findings is represented in Table 12 in the same field.
Variations in sternum ossification centres were of unilateral, bipartite and misaligned character and occurred in all treated groups and in control group. The lack of one of ossification point occurred in all treated groups and in control group. Changes in skull, vertebra column and ribs were observed in one fetus from group 1 (80 mg/kg bw). In gross examination of the same fetus, the deformed tail and 4-fingered forelimbs were observed and, as mentioned, this changes should be treated as accidental.
Analysis of percentage of fetuses with particular number of ossification points of sternum showed no difference between control group and treated groups.
Statistically significantly greater number of ossification points in metacarpus was stated in group 1 (80 mg/kg bw), group 4 (125 mg/kg bw) and group 2 (200 mg/kg bw) compared with the control group. It is possible that the average number of ossification points in metacarpus in control group was accidentally and exceptionally low because the highest average number of ossification points in metacarpus was reported in group 1 (80 mg/kg bw) and the averages were lower in groups 4 (125 mg/kg bw) and 2 (200 mg/kg bw) which may indicate a lack of connection between the dose level and the average number of ossification points in metacarpus. No statistically significant difference of ossification points in sternum and in metatarsus in treated groups compared with the control group indicates the randomness of statistical significant changes in the average number of ossification point in metacarpus in treated groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Evaluation of sagittal sections of fetuses from control group confirmed findings from gross evaluation.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

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

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1. Body weight of pregnant females [g] (mean ± SD).

 

Day of gestation

Group 0

0 mg/kg bw

n=20

Group 1

80 mg/kg bw

n=22

Group 4

125 mg/kg bw

n=21

Group 2

200 mg/kg bw

n=16

Group3 500 mg/kg bw

n=4 #

0

227.5 ± 14.57

230.7 ± 16.70

228.3 ± 13.06

231.6 ± 16.12

234.3 ± 17.35

5

247.0 ± 15.06

252.5 ± 17.91

247.7 ± 15.46

252.3 ± 18.30

256.8 ± 16.82

8

254.8 ± 15.39

252.2 ± 22.49

243.5 ± 16.01

247.2 ± 19.46

238.8 ± 16.58

11

266.7 ± 17.19

264.3 ± 21.33

254.8 ± 18.60

255.9 ± 21.78

240.3 ± 29.15

14

280.0 ± 17.54

277.0 ± 20.80

270.4 ± 15.26

261.0 ± 19.20 *

245.3 ± 23.91

17

304.3 ± 17.41

302.8 ± 22.89

290.8 ± 16.82

279.3 ± 25.52 *

267.0 ± 21.37

20

339.3 ± 24.63

336.5 ± 26.64

323.0 ± 19.93

308.0 ± 25.92 *

300.8 ± 23.47

n – number of animals in group

* - statistically significant difference with p ≤ 0.05

# - no statistical analysis

 

Table 2. Food consumption [g/100g of bw/day] (mean ± SD).

Day of gestation

Group 0

0 mg/kg bw

n=20

Group 1

80 mg/kg bw

n=22

Group 4

125 mg/kg bw n=21

Group 2

200 mg/kg bw n=16

Group3 500 mg/kg bw

n=4 #

5

8.51 ± 0.73

8.07 ± 0.94

7.93 ± 0.72

8.21 ± 0.80

8.38 ± 0.54

8

7.05 ± 0.76

6.52 ± 0.82

5.80 ± 0.92 *

5.67 ± 1.19 *

3.45 ± 1.57

11

7.27 ± 0.60

6.32 ± 0.59 *

5.95 ± 0.60 *

5.50 ± 0.71 *

3.63 ± 1.28

14

7.31 ± 0.54

6.57 ± 0.76 *

6.33 ± 0.65 *

5.46 ± 1.06 *

4.83 ± 1.73

17

7.05 ± 0.75

6.45 ± 0.55

6.45 ± 0.56

5.42 ± 1.39 *

6.63 ± 0.61

20

6.22 ± 0.59

5.90 ± 0.53

5.67 ± 0.59

5.55 ± 1.16 *

6.63 ± 0.38

n – number of animals in group

* - statistically significant difference with p ≤ 0.05

# - no statistical analysis

 

Table 3. Gross lesions – euthanised animals on the 20th day of experiment.

 

Examined organ

 

Type of change

Group

0

0 mg/kg bw n=22

1

80 mg/kg bw n=22

4

125 mg/kg bw n=23

2

200 mg/kg bw n=18

3

500 mg/kg bw n=4

 

Stomach

Congestion

2

[81 ,9]

2

[9,19]

-

4

[7,81,9,18]

1

[9]

Erosions of the mucosa

-

4

[6,9,10,12]

4

[7,9,181,21]

5

[1,7,9,16,21]

1

[3]

Ulcers

-

-

-

2

[4,14]

3

[3,6,9]

 

Jejunum

Extension

-

-

-

2

[10,20]

-

Thickened wall with an inflammatory

character

 

-

 

-

 

-

1

[20]

 

-

(Intestinal) gas

-

-

-

1

[10]

-

 

Cecum

Slight congestion

2

[15,21]

6

[1,2,5,6,11,21]

2

[15,21]

2

[1,10]

1

[6]

 

Congestion

2

[17,181]

4

[9,10,12,19]

3

[9,161,181]

7

[2,6,7,81,9,131

,18]

2

[1,3]

Extension

-

-

1

[181]

2

[10,131]

1

[3]

Small amount of (intestinal) gas

-

-

-

-

1

[6]

(intestinal) gas /

bloating

-

1

[17]

6

[1,4,6,7,8,181]

2

[10,131]

2

[3,9]

 

Colon

Slight congestion

-

1

[11]

-

-

-

Congestion

-

-

-

1

[6]

-

Extension

-

-

-

-

1

[9]

Bloating

-

-

1

[6]

-

-

Whole intestines

Congestion

2

[81,9]

-

-

-

-

 

Liver

Fragile liver with a

clear lobular structure and with bright foci

 

-

1

[10]

 

-

 

-

 

-

Bright lesion, size 5x5 mm with a granular consistency in the left

lateral lobe

 

-

 

1

[8]

 

-

 

-

 

-

Lesion spread, bright with a firm consistency in the caudate lobe,

probably of a cancerous nature

 

1

[14]

 

-

 

-

 

-

 

-

Bright lesion with granular consistency (10x 6 mm) in

caudate lobe

 

-

 

-

 

1

[5]

 

-

 

-

Lesion in size 6x1,5 mm, red, pedunculated in adipose tissue

around the left ovary

1

[81]

 

-

 

-

 

-

 

-

Bright red-yellow lesion, size 3x1 mm in the right corner of the uterus

at the height of the first fetus

 

-

1

[10]

 

-

 

-

 

-

[ ] – computer numbers of animals with gross lesions

n – number of tested animals

X1 - females not pregnant

 

 

Table 4. Gross lesions – dead and euthanised for humanitarian reason animals.

Examined organ

 

Type of change

Group

0

0 mg/kg bw n=0

1

80 mg/kg bw n=0

4

125 mg/kg bw n=0

2

200 mg/kg bw n=5

3

500 mg/kg bw n=6

 

Lungs

Strong congestion

-

-

-

2

[11,12]

1

[7]

Congestion

-

-

-

3

[15,221,23]

-

Enlargement

-

-

-

2

[11,12]

4

[2,5,7,10]

Foci of emphysema

-

-

-

3

[11,12,15]

4

[2,5,7,10]

Foci of atelectasis

-

-

-

1

[23]

-

 

Stomach

Strong extension

-

-

-

3

[11,12,15]

2

[7,10]

Extension

-

-

-

1

[221]

4

[2,41,5,81]

 

Gas (bloating)

 

-

 

-

 

-

3

[12,15,221]

6

[2,41,5,7,81,10

]

Erosions of the mucosa

-

-

-

3

[12,15,23]

4

[41,5,7,10]

Ulcers

-

-

-

1

[15]

2

[7,10]

 

Small intestine

Congestion

-

-

-

1

[12]

-

Extension

-

-

-

-

3

[2,5,81]

Content tinged with blood

-

-

-

1

[12]

-

Duodenum

Haemorrhagic

inflammation

-

-

-

1

[23]

-

Jejunum

Haemorrhagic inflammation

-

-

-

1

[23]

-

 

Cecum

Congestion

-

-

-

-

3

[2,41,81]

Extension

-

-

-

-

5

[2,41,5, 81,10]

(Intestinal) gas/bloating

-

-

-

1

[15]

5

[2,41,5,81,10]

 

Whole intestines

Extension

-

-

-

2

[11,12]

3

[41,7,10]

(Intestinal) gas

-

-

-

2

[11,12]

1

[7]

Content slightly tinged with blood

-

-

-

1

[11]

-

Haemorrhagic inflammation

-

-

-

1

[12]

-

Spleen

Fragile

-

-

-

1

[221]

-

Ovaries

Congestion

-

-

-

1

[11]

-

Area around the anus contaminated with light / yellow and rare / liquid faeces

 

-

 

-

 

-

 

-

3

[2,41,81]

[ ] – computer numbers of animals with gross lesions

n – number of tested animals

X1 - euthanized for humanitarian reason animal

 

 

Table 5. Number of corpora lutea, implantations, resorptions, pre- and post-implantation losses (mean ± SD)

 

Group

Number of  pregnant females

Average number of corpora lutea per one female

Number of implantations in not pregnant

females

Number of resorptions

Preimplantation losses

Postimplantation losses

total

in females min - max

average in one female

number

percent

number

percent

0

20

16.100 ± 2.198

0

24

0 – 5

1.200 ± 2.198

38

11.801

25

8.803

1

22

15.591 ± 2.062

0

16

0 – 5

0.727 ± 1.202

33

9.621

17

5.484

4

21

15.238 ± 3.015

0

29

0 – 8

1.381 ± 2.085

34

10.625

29

10.140

2

16

14.313 ± 1.352

0

13

0 – 3

0.813 ± 0.911

21

9.170

14

6.731

 

Table 6. Result of mating.

 

Parameter

Number of females

Group 0

0 mg/kg bw

Group 1

80 mg/kg bw

Group 4

125 mg/kg bw

Group 2

200 mg/kg bw

Group 3

500 mg/kg bw

Number of females per group

22

22

23

23

10

Number of pregnant females

20

22

21

21

10

Number of not pregnant females

2

[8,18]

0

2

[16,18]

2

[8, 13]

0

Number of died females

0

0

0

4

[11, 12, 15, 23]

4

[2, 5, 7, 10]

Number of euthanized females

0

0

0

1

[22]

2

[4, 8]

 

Females with resorptions

11

[2(2),4(1),5(2),

6(3),9(1),10(1),

11(2),13(2),15(5),

17(4),21(1)]

10

[1(5),3(1),9(1),

12(1),13(1),17(1),

18(1),19(3),

21(1)22(1)]

11

[2(4),4(1),5(1),

7(8),8(1),9(1),

12(2),13(2),17(5),

20(1),23(3)]

9

[1(1),2(1),3(3),

4(2),7(2),9(1),

14(1),19(1),20(1)]

 

no calculations were performed due to too small group size

Number of females for statistical analysis

20

22

21

16

0

[ ] computer number of female

() number of resorptions

 

Table 7. Number of fetuses (mean ± SD).

 

Group

Number of  pregnant females

Number of fetuses

 

Number of resorptions

 

total

 

dead

in litters min.max.

average in litter in 1 female

 

females

 

males

0

20

260

1

5 - 18

12.950 ± 3.517

5.700 ± 2.179

7.250 ± 2.573

24

1

22

294

1

7 - 19

13.318 ± 2.784

6.636 ± 1.840

6.682 ± 2.418

16

4

21

257

0

7 - 16

12.238 ± 2.548

7.000 ± 2.258

5.238 ± 1.841 *

29

2

16

195

1

8 - 16

12.125 ± 2.391

5.688 ± 2.056

6.438 ±1.931

13

* statistically significant difference with p ≤ 0.05

 

Table 8. Average weight of fetuses and placenta [g] (mean ± SD).

 

Group

 

Number of examined fetuses

Weight of fetuses

 

Weight of placenta

with placenta and fetal membranes

without placenta and fetal membranes

0

259

4.774 ± 0.338

3.386 ± 0.304

0.542 ± 0.077

1

293

4.745 ± 0.474

3.450 ± 0.326

0.531 ± 0.086

4

257

4.793 ± 0.420

3.491 ± 0.352*

0.543 ± 0.099

2

194

4.664 ± 0.507

3.449 ± 0.391

0.515 ± 0.096*

* statistically significant difference with p ≤ 0.05

 

Table 9. Gross lesions in fetuses.

 

Pathological changes

Number of fetuses

Group 0

0 mg/kg b.w.

Group 1

80 mg/kg b.w.

Group 4

125 mg/kg b.w.

Group 2

200 mg/kg b.w.

Total number of fetuses

260

294

257

195

Number of alive fetuses

259

293

257

194

Number of dead fetuses

1[17]

1[8]

0

1[5]

Number of fetuses with haemorrhages

12

12

7

4

Haemorrhage on the lateral, left elbow region

1

[2]

-

-

-

Haemorrhage on left metatarsus

1

[3]

-

1

[3]

-

Haemorrhage on right metatarsus

-

1

[13]

-

-

Haemorrhage on interscapular region

3

[3,13,19]

3 [3x3]

-

2

[16,17]

Haemorrhage on the right side of the thoracic

1

[3]

-

-

-

Haemorrhage on the left glenohumeral joint

1

[12]

-

-

-

Haemorrhage on the right, dorsal side of the neck

1

[15]

-

-

1

[5]

Haemorrhage on the right, ventral side of the neck

1

[16]

-

1

[20]

-

Haemorrhage on the ventral side of the neck

1

[19]

-

-

1

[2]

Haemorrhage at the base of the tail

-

3  [2x2,4]

-

-

Haemorrhage on the tail

1

[16]

3

[6,21,22]

3

[3,13,17]

-

Haemorrhage on temporomandibular joint region

-

1

[3]

-

-

Haemorrhage on the right forearm

-

-

1

[9]

-

Haemorrhage on the skin of the abdomen - abdominal cavity strongly enlarged, probably part of the euthanasia dose for the mother was

given to the fetus

 

1

[14]

 

-

 

-

 

-

Haemorrhage on the entire abdominal wall - abdominal cavity enlarged

-

1

[15]

-

-

Haemorrhage on the entire torso and

abdomen - abdominal cavity enlarged

 

-

 

-

1

[3]

 

-

Number of fetuses with other changes

2

9

3

3

Omphalocele

1

[13]

-

-

1

[10]

Significantly smaller

1 [17*]

4 [8,8*,2x17]

3 [3x7]

1

[2]

Deformed (shortened) tail

-

2 [2x17]

-

-

4-fingered forelimbs

-

2 [2x17]

-

-

Anasarca and amniotic fluid tinged with blood

-

1 [8*]

-

1 [5*]

[ ] computer numbers of females

2x, 3x etc.- number of fetuses from one female, with the same lesion

* dead fetus

 

Table 10. Percentage of fetuses with a given number of ossification points of sternum (%).

 

Group

Number of   examined fetuses

Number of ossification points

0

1

2

3

4

5

6

0

134

-

-

-

0.75

6.72

16.42

76.12

1

152

0.66

0.66

-

-

4.61

9.21

84.87

4

132

-

-

0.76

-

6.82

15.15

77.27

2

100

-

-

-

-

2.00

15.00

83.00

 

Table 11. Percentage of fetuses with a given number of ossification points of limbs (%).

 

Group

Number of   examined fetuses

Metacarpus of forelimbs Number of ossification points

Metatarsus of hindlimbs Number of ossification points

3

2.5*

3.5*

4

3

4

0

134

29.10

-

5.22

65.67

-

100.00

1

152

9.21

0.66

4.61

85.53

0.66

99.34

4

132

16.67

-

5.30

78.03

-

100.00

2

100

13.00

-

-

87.00

-

100.00

*value 3.5 means 3 ossification points in one limb and 4 in second limb; value 2.5 means 2 ossification points in one limb and 3 in second limb

 

Table 12. Average number of ossification points of sternum and limbs (mean ± SD).

 

Group

Number of

examined fetuses

 

Sternum

Metacarpus of forelimbs

Metatarsus of hindlimbs

0

134

5.679 ± 0.632

3.683 ± 0.453

4.000 ± 0.000

1

152

5.743 ± 0.785

3.875 ± 0.322*

3.993 ± 0.081

4

132

5.682 ± 0.669

3.807 ± 0.379*

4.000 ± 0.000

2

100

5.810 ± 0.443

3.870 ± 0.338*

4.000 ± 0.000

* statistically significant difference with p ≤ 0.05

Applicant's summary and conclusion

Conclusions:
In this GLP compliant OECD 414 study, the possible toxic effects of Rokopol RF-151 to pregnant Wistar rats (Cmdb: WI; outbred) and their fetuses was evaluated. On the basis of the clinical examinations and gross examination the NOAEL for maternal toxicity cannot be defined. On the basis of the results of the evaluation of fetuses the NOAEL for developmental toxicity can be defined as 200 mg/kg bw.
Executive summary:

In this GLP compliant OECD 414 study, the possible toxic effects of Rokopol RF-151 to pregnant Wistar rats (Cmdb: WI; outbred) and their fetuses was evaluated. The study was conducted on 100 females divided into five experimental groups: four treated groups and one control group. From day 5 to 19 of gestation the test item, dispersed in sunflower oil (solution), was administered via oral gavage at doses of 80 mg/kg bw (group 1), 125 mg/kg bw (group 4), in 200 mg/kg bw (group 2) and 500 mg/kg bw (group 3). Due to severe toxicity in the high dose group, this group was terminated for humanitarian reasons and a dose group of 125 mg/kg bw (group 4) was introduced. Group 0 served as control and was treated with sunflower oil.

During the entire study clinical observations for mortality and signs of toxic influence of the test item were performed in females, their body weight and food consumption were controlled. One day before an expected delivery, on day 20 of gestation, all females were killed with the use of Euthasol and subjected to caesarean section with gross examination. Moreover gross examinations were performed in all females which died or were euthanised for humane reason before the end of the study.

The following endpoints were determined in each pregnant female: number of alive and dead fetuses and number of resorptions. After the removing of fetuses each uterus was weighed. The non-gravid uteri and non-gravid horns were overexposed by electric lamp and were stained using ammonium sulphide in order to confirm the non-pregnant status. The number of corpora lutea was determined in fixed ovaries. The following endpoints were determined in the study: sex, body weight with and without placenta, weight of placenta, body length with and without tail. Each fetus was subjected to gross examination. Half of fetuses from each litter were stained with alizarin and subjected to evaluation of skeleton. The rest of them was fixed in 10% solution of formalin and evaluated for formation of body cavities and internal organs.

For the maternal generation, there was no mortality in group 0, 1 and 4. Four females died and one female was euthanised during the experiment in group 2. Four females died and two females were euthanised during the experiment in group 3. During the entire experiment, no clinical signs were observed in the control group. Respiratory murmurs were observed in all treatment groups and the incidence increased with higher doses. Average body weight of the pregnant females of group 1 and 4 did not differ statistically significantly from average body weight of the control group. In group 2, the average body weight was statistically significantly lower in comparison with the pregnant females of control group on 14th ,17th and 20th day of gestation. Decrease of average food consumption from 8th to 20th day of experiment and statistically significant decreases were stated in all treated groups compared to control group which indicated influence of test item. At necropsy, multiple effects were observed in females euthanised on the 20th day of pregnant from group 1, 4, 2 and 3 on the gastrointestinal track: congestion, extension of intestines, bloating and gas in intestines, ulcers and erosions of the mucosa of the stomach and thickened wall with an inflammatory character. These effects intensified with increasing concentrations.

Six females were non pregnant in the study (two in control group, two in group 4 and two in group 2). In the remaining groups, no non-pregnant females were stated. Resorptions and pre- and post-implantation losses were observed in control and all experimental groups. The number of resorption and number and percent of pre- and post-implantation losses in all groups was comparable. Intrauterine mortality was equal in control group and group 1 (80 mg/kg bw) and 2 (200 mg/kg bw). No intrauterine mortality was stated in group 4 (125 mg/kg bw). The average number of corpora lutea in all treated groups were comparable with the number of corpora lutea in the control group.

Due to too small number of females in group 3, evaluation of fetuses was not performed. Subcutaneous haemorrhages in different parts of the fetal bodies which occurred in the treated groups and in the control group, should not be perceived as the effects of the test item. Other external observations were incidental and not treatment- related. Variations in sternum ossification centres were of unilateral, bipartite and misaligned character and occurred in all treated groups and in control group. The lack of one of ossification point occurred in all treated groups and in control group. Changes in skull, vertebra column and ribs were observed in one fetus from group 1 (80 mg/kg bw). In gross examination of the same fetus, the deformed tail and 4-fingered forelimbs were observed and, as mentioned, this changes should be treated as accidental. Analysis of percentage of fetuses with particular number of ossification points of sternum showed no difference between control group and treated groups. Statistically significantly greater number of ossification points in metacarpus was stated in group 1, 4 and 2 compared with the control group. It is possible that the average number of ossification points in metacarpus in control group was accidentally and exceptionally low because the highest average number of ossification points in metacarpus was reported in group 1 and the averages were lower in groups 4 and 2, which may indicate a lack of connection between the dose level and the average number of ossification points in metacarpus. No statistically significant difference of ossification points in sternum and in metatarsus in treated groups compared with the control group indicates the randomness of statistical significant changes in the average number of ossification point in metacarpus in treated groups.

Based on the study and analysis of the results it can be stated that the test item Rokopol RF-151 affected results of the clinical examinations and gross examination of pregnant females in all treated groups which indicated that the NOAEL for maternal toxicity can not be defined. On the basis of the results of the evaluation of fetuses the NOAEL for developmental toxicity can be defined as >= 200 mg/kg bw.