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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:
2016-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: violet/dark blue solid
stable at room temperature
Specific details on test material used for the study:
Batch: P 112045
Purity: The elementary analysis shows 100.2g/100g.
Expiry date: February 2021
Storage conditions: room temparature

Test animals

Species:
rat
Strain:
other: Crl:WI (Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks
- Weight at study initiation: average 165 g (gestation day 0), average 196g (beginning of treatment period)
- Housing: single caging
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2016-05-11 To: 2016-06-02

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The specific amount of test substance was weighed, topped up with vehicle in a graduated flask and intensely mixed. The mixture was continuosly stirred until dosing.


VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item can be suspended in the vehicle. It is not soluble in water.
- Amount of vehicle (if gavage): 10 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance preparations was demonstrated over a period of 7 days at room temperature.
The homogeneous distribution of the test substance in the vehicle (drinking water) was confirmed.
The correctness of the prepared concentrations was shown.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
The day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory.
Duration of treatment / exposure:
gestation days 6- 19
Frequency of treatment:
daily
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on the results of the OECD 422 study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Morbidity, pertinent behavioral changes and/or signs of overt toxicity. were checked twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GD 0 to 20).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.


POST-MORTEM EXAMINATIONS: Gross pathology
- Sacrifice on gestation day: GD 20

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: Site of implantations in the uterus
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (1999) and SOLECKI et al. (2001, 2003):

Malformation
A permanent structural change that is likely to adversely affect the survival or health.

Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.
Statistics:
DUNNETT's test: Food consumption, body weight, body weight change, DUNNETT's test
corrected body weight gain, carcass weight, weight of the unopened uterus, weight of the placentas and
fetuses, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses

FISHER's exact test
Number of pregnant animals at the end of the study, FISHER's exact test mortality rate (of the dams) and number of litters with fetal findings

WILCOXON test
Proportion of fetuses with findings per litter
Indices:
sex ratio
conception rate (in %)
preimplantation loss (in %)
postimplantation loss (in %)
Historical control data:
Historical control data is included in the study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
The test item is a blue colorant. This caused the feces of the high dose group animals to be stained blue. At necropsy, the content of the gastrointestinal tract was blue; this reflects presence of the blue test item as expected for oral dosing.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed at the limit dose

Maternal abnormalities

Abnormalities:
no effects observed

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:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Individual skeletal malformations
One male control fetus had a shortened humerus. One low dose male fetus had a severely malformed vertebral column and/or ribs. There were no skeletal malformations in the mid and high dose fetuses.

Individual skeletal variations (Table 8)
High dose group fetuses had an incidence of a hole in the tuberositas deltoidea which slightly exceeded the historical control (2.6% versus a range of 0.2 - 2.3%). The incidence of supraoccipital hole(s) was not dose-related and within the historical control group range.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects at the limit dose.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1 (FC = Food consumption; BW = Body weight; BWC = Body weight change)

Parameter Test group 0
0
Test group 1
100 mg/kg bw
Test group 2
300 mg/kg bw
Test group 3
1000 mg/kg bw
Females mated/ pregnant/ animals with viable fetuses: 25/24/24 25/25/25 25/24/24 25/23/23
Clinical observations:

no adverse

effects

 no adverse effects

 no adverse

effects

Feces discolored (blue)

(25/25)

FC (0 - 6)
FC (6 - 19)
FC (0 - 20)
16.5 g
21.1 g
19.9 g
16.3 g (99%)
20.9 g (99%)
19.6 g (99%)
16.1 g (98%)
20.9 g (99%)
19.6 g (99%)
15.9 g (96%)
20.4 g (97%)
19.2 g (97%)
BW (0)
BW (6)
BW (19)
BW (20)
167.3 g
201.6 g
276.6 g
287.4 g
165.5 g (99%)
198.9 g (99%)
276.8 g (100%)
288.4 g (100%)
167.8 g (100%)
201.5 g (100%)
282.7 g (102%)
295.4 g (103%)
165.2 g (99%)
195.0 g (97%)
273.8 g (99%)
286.5 g (100%)
BWC (0 - 6)
BWC (6 - 19)
BWC (0 - 20)
BWC (0 - 1)
BWC (17 - 19)
34.3 g
75.1 g
120.2 g
14.9 g
20.0 g
33.4 g (97%)
78.0 g (104%)
123.0 g (102%)
13.2 g (88%)
22.5 g (113%)
33.7 g (98%)
81.1 g (108%)
127.6 g (106%)
14.3 g (96%)
23.1 g (116%)*
29.8 g (87%)**
78.7 g (105%)
121.3 g (101%)
12.3 g (83%)*
22.6 g (113%)
Uterus weight: 48.9 g 53.4 g (109%) 55.7 g (114%) 55.6 g (114%)
Carcass: 238.6 g 235.1 g (99%) 239.8 g (101%) 230.9 g (97%)
Corrected body weight gain: 37.0 g 36.2 g (98%) 38.2 g (103%) 35.8 g (97%)

Necropsy findings (dams): no effects observed Stomach: content discolored (5/25)
Caecum: content discolored (1/25)
Large intestine: content discolored (1/25)
Dilated renal pelvis (1/25)
Stomach: content discolored (9/25)
Small intestine: content discolored (2/25)
Caecum: content discolored (3/25)
Large intestine: content discolored (4/25)
Stomach: content discolored (6/25)
Small intestine: content discolored (1/25)
Caecum: content discolored (13/25)
Large intestine: content discolored (5/25)
Rectum: content discolored (11/25)
Postimplantation loss: 9.0% 9.5% 8.3% 2.3%
Total Resorptions: 0.9 1.0 0.9 0.3
Live fetuses/dam: 8.5 9.2 9.7 9.7
Placental weights: 0.50 g 0.52 g (104%) 0.50 g (99%) 0.51 g (102%)
Fetal weights: 3.6 g 3.7 g (101%) 3.7 g (102%) 3.7 g (101%)
Total external malformationsFetuses:
Litter: Affected fetuses/litter:
0/204 (0.0%)
0/24 (0.0%)
0.0%
0/230 (0.0%)
0/25 (0.0%)
0.0%
0/233 (0.0%)
0/24 (0.0%)
0.0%
0/223 (0.0%)
0/23 (0.0%)
0.0%

Table 3

Group 0 Group 1 Group 2 Group 3
0 100 mg/kg bw 300 mg/kg bw 1000 mg/kg bw
Pregnant 24 25 24 23
Aborted 0 0 0 0
Premature birth 0 0 0 0
Dams with viable fetuses 24 25 24 23
Dams with all resorptions 0 0 0 0
Pregnant at terminal sacrifice 24 25 24 23
Corpus Lutea (mean) 10.5 D 10.9 11.2 10.6
SD 1.14 1.86 1.28 1.92
Total 253 272 269 244
Implantation sites (mean) 9.4 D 10.2 10.6 10
SD 2.19 2.3 1.77 2.14
Total 226 256 254 229
preimplantation loss (mean) 10.6 D 6.8 5.7 6.8
SD 19.38 14.41 11.03 10.18
postimplantation loss (mean) 9 D 9.5 8.3 2.3
SD 12.21 11.54 9.58 4.75
Early resorptions (mean)  0.8 D 1 0.9 0.3
SD 1.02 1.31 1.08 0.54
total 19 24 21 6
Late resorptions (mean)  0.1 D 0.1 0 0
SD 0.45 0.28 0 0
total 3 2 0 0
Dead fetuses 0 0 0 0
% female fetuses 53.4 54.8 55.8 52.5
% male fetuses 46.6 45.2 44.2 47.5

D = Dunnett-test (two-sided)*: p<=0.05

Table 4: Total soft tissue malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

24
94

25
108

24
111

23
105

 

Fetal incidence

 

N (%)

 

0.0

 

1 (0.9)

 

0.0

 

0.0

 

Litter incidence

 

N (%)

 

0.0

 

1 (4.0)

 

0.0

 

0.0

Affected fetuses/litter

 

Mean%

 

0.0

 

0.8

 

0.0

 

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 5: Total soft tissue variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

24
94

25
108

24
111

23
105

 

Fetal incidence

 

N (%)

 

3 (3.2)

 

5 (4.6)

 

4 (3.6)

 

4 (3.8)

 

Litter incidence

 

N (%)

 

3 (13)

 

5 (20)

 

4 (17)

 

2 (8.7)

Affected fetuses/litter

 

Mean%

 

2.7

 

4.4

 

4.7

 

3.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 6: Total fetal skeletal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

24
110

25
122

24
122

23
118

 

Fetal incidence

 

N (%)

 

1 (0.9)

 

1 (0.8)

 

0.0

 

0.0

 

Litter incidence

 

N (%)

 

1 (4.2)

 

1 (4.0)

 

0.0

 

0.0

Affected fetuses/litter

 

Mean%

 

2.1

 

1.0

 

0.0

 

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 7: Total fetal skeletal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

24
110

25
122

24
122

23
118

 

Fetal incidence

 

N (%)

 

109 (99)

 

116 (95)

 

120 (98)

 

109 (92)

 

Litter incidence

 

N (%)

 

24 (100)

 

25 (100)

 

24 (100)

 

23 (100)

Affected fetuses/litter

 

Mean%

 

99.2

 

95.7

 

98.3

 

91.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 8: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding

Test group 0

0 mg/kg
bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Historical control data

Mean %

(range)

Supraoccipital hole(s)

0.0

4.7*

1.9

0.0

6.5

(0.0 – 30.1)

Hole in tuberositas
deltoidea

0.0

0.0

0.0

2.6*

0.2

(0.0 – 2.3)

mg/kg bw/d = milligram per kilogram body weight per day; % = per cent* = p <= 0.05 (Wilcoxon-test [one-sided])  ** = p <= 0.01 (Wilcoxon-test [one-sided])

Table 9: Total unlassified cartilage observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

24
110

25
122

24
122

23
118

 

Fetal incidence

 

N (%)

 

62 (56)

 

62 (51)

 

70 (57)

 

62 (53)

 

Litter incidence

 

N (%)

 

22 (92)

 

23 (92)

 

22 (92)

 

22 (96)

Affected fetuses/litter

 

Mean%

 

55.1

 

49.2

 

56.8

 

51.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Applicant's summary and conclusion

Conclusions:
The substance does not cause developmental toxicity or teratogenicity in rats.
Executive summary:

In a prenatal developmental toxicity study the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle. Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 100, 300 or 1000 mg/kg bw/d test substance and controls. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.