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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:
25 Sep 2014 - 11 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline and in compliance with GLP.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): 13-Docosenamide, (Z)-
- Physical state: beige solid
- Composition of test material, percentage of components: 100% active
- Lot/batch No.: MEM-369181
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Wistar rats, Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 11 - 12 weeks (females), 19 – 20 weeks (males)
- Weight at study initiation: 200 – 243 g (females), 390 – 522 g (males)
- Housing: The animals were kept individually in IVC cages (except during the pre-mating period when females were kept in groups of two animals and mating period when two females were paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 02102140509)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 1526), ad libitum
- Water: Sulphur acidified tap water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was ground to a fine powder with the help of a mortar and pestle. The powdered test item was weighed into a tarred plastic vial on a precision balance and the vehicle corn oil was added to give the appropriate final concentration of the test item. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration. The test item formulation was prepared freshly on each administration day before the administration procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 14.29 mg/mL (low dose, LD), 42.86 mg/mL (medium dose, MD), 142.86 mg/mL (high dose, HD)
- Amount of vehicle (if gavage): 7 mL/kg bw/day
- Lot/batch no. (if required): MHBP7039V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of 13-Docosenamide, (Z)- was performed using GC with FID detection. Formulation analysis was performed on samples of all dose groups collected at various intervals during the study. Concentration analysis of formulation samples was performed in the first and the last study week for all dose groups. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 98.2%, 96.3% and 110.8% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations did not differ from nominal concentrations by more than 20%. Stability of formulation samples was investigated in study week 1 based on concentration in the samples taken from the LD, MD and HD dose groups. After 3 hours storage at room temperature the recovery compared to the starting value was between 90.1% and 96.3%. All samples were stable, as concentration after storage did not differ from the start value by more than 20%. Homogeneity of formulation samples was determined in the first and the last study week for the LD, MD and HD dose groups. The mean recovery observed for the LD dose group was 113.5% and 109.2% of the nominal value, for the MD dose group 99.8% and 113.8% of the nominal value and for the HD dose group 107.0% and 110.1% of the nominal value. The coefficients of variation (COV) of the different sampling locations (top, middle, bottom) were 13.6% and 10.8% in the LD dose group, 8.9% and 13.0% in the MD dose group and 4.7% and 4.5% in the HD dose group. All samples were homogenous, as COV was below or equal to the acceptance criterion of 20%.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day. The subsequent morning and each morning thereafter, the vaginal smear of each female was checked until positive evidence of mating was confirmed.
- Proof of pregnancy: The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other.
- Any other deviations from standard protocol: The mating resulted in 23 sperm-positive females in the control and 24 each in the LD, MD and HD group. Non sperm-positive females were excluded from the study without any further observations.
Duration of treatment / exposure:
Day 5 - 19 of gestation
Frequency of treatment:
daily, 7 days/week
Duration of test:
Until Day 20 of gestation / post mating
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal dose
No. of animals per sex per dose:
22 pregnant females (control)
21 pregnant females (LD)
23 pregnant females (MD)
20 pregnant females (HD)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: According to the results of the dose range finding study (BSL study No. 143124) and in consultation with the sponsor doses of 100, 300 and 1000 mg/kg bw/day were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, preferably at the same time each day, animals were observed for spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Sperm-positive females on Gestation Days 0, 5, 8, 11, 14, 17 and 20. Males were only weighed once prior to mating.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption of pregnant females was measured on Gestation Day 5, 8, 11, 14, 17 and 20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: At the time of termination or death during the study, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Uteri that appeared non-gravid were further examined by staining with 10% ammonium sulphide solution to confirm the non-pregnant status.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
A statistical assessment of the body weight and food consumption results, prenatal parameters and litter weight data was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Fetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using a Fisher’s exact test. Litter incidence was the primary unit for the statistical analysis and interpretation. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Historical control data:
Historical control data from the Test Facility were not provided.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
All animals survived to the scheduled necropsy and no mortality was observed in this study. No test item-related clinical signs were observed in any of the treated groups. A few spontaneous clinical signs were observed in few animals, but no effect on overall health, body weight and food consumption was observed in these animals. The mean body weight and body weight gain were unaffected by test item administration during the gestation period when compared with the controls. Throughout the treatment period, body weights and body weight gain increased as the study progressed and were within the normal range of variation for this strain. No treatment-related effect on food consumption was observed during the treatment period. No treatment-related effects were observed in terminal body weight, gravid uterine weight, adjusted maternal weight, number of corpora lutea, implantations, resorptions, percent preimplantation loss and percent postimplantation loss. There were no statistically significant differences. Successful mating resulted in 21/24 pregnancies in the LD group, 23/24 in the MD group and 20/24 in the HD group, compared to 22/23 pregnancies in the control group. Low pregnancy rate in the LD and HD group was considered to be a biological variation and of no toxicological relevance. No gross pathological changes were observed during the macroscopic examination of females of the control, the LD and the MD group. However, in the HD group fluid distension of the uterus was observed in one female, and in another female white discoloured areas on the liver, white to yellow discolouration of the left side of the oesophagus/trachea, yellow fluid content in the thoracic cavity and enlarged adrenal glands were observed at necropsy. Due to the isolated incidence in only two animals, these gross pathological findings were considered to be spontaneous in nature and as such not considered to be a systemic effect due to test item administration.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Statistical analysis of litter data revealed no treatment-related effects on group mean number of live fetuses and dead fetuses, number of male and female fetuses, total number of fetuses or sex ratio when compared with the controls. No treatment-related effect on per litter data parameters including group litter mean weight, total litter weight and male litter weight was observed. However, statistically significantly lower female litter weight was observed in the LD group when compared with the controls. Due to lack of dose dependency and consistency, this effect on female litter weight was considered to be of no toxicological relevance. There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. The statistical analysis showed no significant differences to the control group. A range of visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower compared to the controls, and litter incidences were statistically insignificant except significantly lower discolouration of the dexter lobe of the liver in the MD group when compared to the controls. As the observed finding was minor and lacked dose dependency and consistency, no toxicological significance can be attributed to it; this finding was considered to be spontaneous in nature. All remaining fetal visceral findings revealed no significant alterations compared to the control group. Craniofacial examination by a razor blade serial sectioning technique revealed few findings (slightly dilated right or both lateral ventricles, right or left retinal fold and slightly dilated 3rd lateral ventricle) at frequencies generally comparable to or in some cases slightly higher or lower compared to the controls. Statistical analysis of the data revealed no significant effect in any of these findings as compared to the control group. Therefore, these findings are considered to be not treatment related and spontaneous in nature. Skeletal examination of Alizarin red stained fetuses revealed a range of abnormalities in the treated groups which were of a type comparable to the control group or which occurred at an incidence either comparable to or lower or sometimes higher than in the control group. A statistically significant increase in the litter incidence of incomplete ossification of the interparietal bone in the LD group was observed when compared to the control group. Due to lack of dose dependency and consistency this findings was not assumed to be test item related. All remaining skeletal examinations revealed no significant alterations compared to the control group.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Litter data – summary

Group

 

Mean litter weight (g)

Number of males

Number of females

Live foetuses

Dead foetuses

Sex ratio

Total litter weight (g)

Male litter weight (g)

Female litter weight (g)

 

C

 

Mean

3.86

5.18

5.68

10.86

0.00

0.98

42.26

20.46

21.80

SD

0.58

1.97

1.55

2.38

0.00

0.50

12.79

8.71

7.87

N

22

22

22

22

22

22

22

22

22

 

LD

 

Mean

3.67

5.33

4.62

9.95

0.00

1.40

35.84

20.00

15.84*

SD

0.44

2.24

2.16

3.23

0.00

1.12

10.99

9.07

6.63

N

21

21

21

21

21

20 a)

21

21

21

 

MD

 

Mean

3.83

5.26

5.17

10.43

0.00

1.18

39.67

20.27

19.40

SD

0.59

1.66

1.40

1.44

0.00

0.77

6.37

5.86

6.17

N

23

23

23

23

23

23

23

23

23

 

HD

 

Mean

3.83

4.95

5.70

10.60

0.05

1.01

40.34

19.44

20.90

SD

0.51

1.64

2.15

2.68

0.22

0.55

10.25

6.86

7.75

N

20

20

20

20

20

20

20

20

20

Asterisks indicate significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

C: Control

LD: Low dose

MD: Medium dose

HD: High dose

N: Number of pregnant females (dams)

a): One dam in the LD group with no female pup, the animal was therefore excluded from sex ratio calculation

Table 2: Fetal skeletal findings – summary

 

Skeletal findings

Group

1

2

3

4

C

LD

MD

HD

 

 

Skull interparietal

incomplete ossification

 

No of Incidences

21

30

30

19

Total No of Observed Foetuses

125

109

123

104

% Foetus Incidence

16.80

27.52

24.39

18.27

No of Litters with at least 1 incidence

12

15*

14

8

Total No of Observed Litters

22

21

23

20

% Litter Incidence

54.55

71.43

60.87

40.00

Asterisks indicate significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

C: Control

LD: Low dose

MD: Medium dose

HD: High dose

Table 3: Fetal visceral findings – summary

 

Visceral findings

Group

1

2

3

4

C

LD

MD

HD

 

 

Liver dexter lobe discolored (dark)

 

No of Incidences

0

0

6

1

Total No of Observed Foetuses

114

100

117

102

% Incidence of Abnormality

0.00

0.00

5.13

0.98

No of Litters with at least 1 incidence

0

0

5*

1

Total No of Observed Litters

22

21

23

20

% Litter Incidence

0.00

0.00

21.74

5.00

Asterisks indicate significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

C: Control

LD: Low dose

MD: Medium dose

HD: High dose

Applicant's summary and conclusion

Conclusions:
On the basis of this prenatal developmental toxicity study in pregnant female Wistar rats with 13-Docosenamide, (Z)- at dose levels of 100, 300, and 1000 mg/kg bw/day administered from gestation days 5 to 19 no test item related toxicological findings in dams or fetuses were revealed. Under the conditions of the study, 1000 mg/kg bw/day was considered as no observed adverse effect level (NOAEL) for both maternal and embryo-fetal toxicity of 13-Docosenamide, (Z)-.