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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:
Oct 2017 - 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, 55116 Mainz
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF, B756
- Expiration date of the lot/batch: 01 Feb 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: guaranteed by sponsor
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle used

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no purification step before application to animals

FORM AS APPLIED IN THE TEST: pure, unformulated substance

Test animals

Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl:KBL(NZW)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Breeder: Charles River Laboratories, France)
- Age at study initiation: 15-17 weeks
- Weight at study initiation: pregnant females between 3054 – 4070 g
- Fasting period before study: not applicable
- Housing: during acclimatization housed singly in Type 4X03B700CP cages (TECNIPLAST Deutschland GmbH, Hohenpeißen-berg, Germany; floor space 4264 mm², internal height 450 mm)
- Diet: ad libitum; pelleted Kliba maintenance diet (Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland)
- Water: ad libitum, potable tap water in water bottles
- Acclimation period: 5 days before artificial insemination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±2
- Humidity (%): 45-65
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12/12 (6.00 h to 18.00 h / 18.00 h to 6.00 h)

IN-LIFE DATES: From: 2017-11-13 To: 2017-12-21

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: applied as pure substance without being formulated with a vehicle

DIET PREPARATION
- no diet applied

VEHICLE
- no vehicle used
Analytical verification of doses or concentrations:
no
Remarks:
no stability, homogeneity and concentration control analyses of test item formulations were conducted due to pure substance application
Details on mating procedure:
- Impregnation procedure: artificial insemination
- Day of insemination referred to as day 0 of pregnancy
Duration of treatment / exposure:
implantation to one day prior to the expected day of parturition (GD 6-28)
Frequency of treatment:
daily
Duration of test:
30d
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on dose-range finding studies with non-pregnant (300 and 1,000 mg/kg bw/d for 21 d) and pregnant (300 and 600 mg/kg bw/d for GD 6 through GD 28) female New Zealand White rabbits the doses were selected. In the first study at 1,000 mg/kg bw/d, one out of three female animals were found dead on study day 12 with similar necropsy findings in all three animals. At 300 mg/kg bw/d no adverse effects were observed. In the second study at 600 mg/kg bw/d, one out of five female animals were killed moribund on study day 21 after showing reduced nutritional condition. At 300 mg/kg bw/d no adverse effects were observed.
- Rationale for animal assignment: There are historical control data available from the test facility for New Zealand White rabbits and the specific strain has been proven to be sensitive to substances with a teratogenic potential.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity; animals examined several times daily (GD 0-29) if signs occured
- during administration period (GD 6-28): all animals checked daily for abnormal clinical signs before administration as well as within 5 hours after

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29

FOOD CONSUMPTION: Yes
- Time schedule for examination: daily during GD 0-29

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uterus
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: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter (and the heads of any fetus which revealed severe findings during the external examina-tion, e.g. anophthalmia, microphthalmia or hydrocephalus)
Statistics:
DUNNETT-test (two-sided) for the hypothesis of equal means:
Food consumption ("mean of means" = values that allow a rough estimation of the total food consumption during different time intervals [pretreatment, treatment and entire study]), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight

FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions:
Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

WILCOXON-test (one-sided) for the hypothe-sis of equal medians:
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter

For all:
* for p < 0.05; ** for p < 0.01
Indices:
The conception rate (in %):
(number of pregnant animals / number of fertilized animals) * 100


The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice:
([number of corpora lutea – number of implantations] / number of corpora lutea) * 100


The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice:
([number of implantations – number of live fetuses] / number of implantations) * 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- control: one female had blood in bedding before and after treatment on GD 16, reduced defecation was observed in four females
- 100 mg/kg bw/d: reduced defecation in one animal observed
- 250 mg/kg bw/d: reduced defecation in one animal observed
- 500 mg/kg bw/d: reduced defecation in six females observed, no defecation observed in three animals

- adverse effect is indicated by higher incidence of both findings in the high-dose group
Mortality:
mortality observed, treatment-related
Description (incidence):
- 500 mg/kg bw/d: one female were found dead on the day of scheduled sacrifice (GD 29), two females were sacrificed after abortion ahead of schedule (GD 29 and GD 28)
- simultaneous abortion/death of 3 high-dose does immediately before term points to maternal toxicity
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- no significant treatment related effect on body weights/body weight gain
- particularly no effect on carcass weights or corrected (net) body weight gain at all dose levels
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- the mean food consumption of the does in the 500 mg/kg bw/d group was reduced from GD 9 onwards
- this effect was statistical significant on GD 9-10 and GD 13-15
- afterwards (around GD 17) the effect recovered, being comparable to or exceeding the control values

- overall, the high-dose does consumed 7% less food than the control animals during the treatment period (GD 6-28)
- food consumption of the low- and mid-dose rabbits (100 and 250 mg/kg bw/d) was comparable to thecontrol
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- gravid uterus weights were not significantly different from controls, differences between all groups were assessed to be without biological relevance and no dose-dependence was observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- spontaneous findings: noted in individual females of all groups, i.e. watery feces in one control and one high-dose doe, a blind ending uterine horn (left) in one high-dose doe, absence of uterine horn (left), absent kidney (left) and absent ureter (left) in one high-dose doe
- additional fiindings associated with unscheduled maternal death or sacrifice: findings after gavage error (thoracic cavity filled with blood) in one doe, watery feces in one doe (sacrificed after abortion on GD 29), an empty stomach in one doe No. 78 (died on GD 29), watery feces, stomach filled to distension with feed, very dry, hard feces in rectum in one doe

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
- no test substance-related and/or biologically relevant differences between the different test groups
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
- no test substance-related and/or biologically relevant differences between the different test groups
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
- no test substance-related and/or biologically relevant differences between the different test groups
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
- one dead fetus found at cesarean section of one 500 mg/kg bw/d doe
- it is considered a rare finding but may occur spontaneously in this rabbit strain
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
- conception rate: 88% in 100 mg/kg bw/d group , 96% in 250 and 500 mg/kg bw/d groups and 100% in the control group (0 mg/kg bw/d)
Details on maternal toxic effects:
All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
food consumption and compound intake
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
food consumption and compound intake

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
- mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance, no biologically relevant differences in comparison to the control group
Changes in sex ratio:
no effects observed
Description (incidence and severity):
- distribution of the fetuses in test groups 100, 250 and 500 mg/kg bw/d was comparable to the control fetuses
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- occurred in test groups 100 and 250 mg/kg bw/d), as listed in Tab. 1
- male fetus No. 31-02 had multiple soft tissue and skeletal malformations additionally
- male fetus No. 70-04 had an associated soft tissue malformation

The distribution of external malformations about the dose groups does not indicate an association to the treatment, no statistically significant differences between the groups were noted (Tab. 2).
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
- detected in single fetuses of test groups 100 and 500 mg/kg bw/d, as shown in Tab. 7
- male 100 mg/kg bw/d fetus No. 31-02 had multiple skeletal malformations affecting the vertebral column, ribs, sternum and pelvic girdle, furthermore, additional external and multiple visceral malformations, considered to be spontaneous in origin and not treatment-related
- in the 500 mg/kg bw/d group the incidences for all individual malformations as well the significantly increased litter incidence and mean% affected fetuses per litter with skeletal malformations (Tab. 8) were covered by the historical control ranges
- exception: the litter and affected fetuses/litter incidences for severely fused sternebrae (bony plate) exceeded their historical control ranges at 500 mg/kg bw/d
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
- occurred in fetuses of the test groups 100, 250 or 500 mg/kg bw/d, as listed in Tab. 4
- male low-dose fetus No. 31-02 had multiple visceral malformations, i.e. absent kidney, absent ureter, absent adrenals and a right-sided aortic arch, furthermore, additional external and multiple skeletal malformations
- male mid-dose fetus No. 70-04 had an additional external malformation
- three fetuses (out of two litters) of the 500 mg/kg bw/d group multiple visceral malformations affecting the urogenital tract were recorded

- no statistically significant differences in the overall incidences between the groups were noted (Tab. 5)
- the 500 mg/kg bw/d group litter incidence (9.5%; historical range 0 – 4.8%) as well as affected fetuses/litter incidence (mean% 1.5; historical range 0 – 0.6) for fetuses with multiple visceral malformations were above the historical control
Details on embryotoxic / teratogenic effects:
Fetal external variations:
- one external variation, i.e. paw hyperflexion, was recorded in six fetuses of four 500 mg/kg bw/d litters - resulted in an increased litter incidence (19% vs. 0 in control) and mean% of affected fetuses per litter (4.6 vs. 0 in control)
- the increased overall incidences of external variations are given in Tab. 3

Fetal soft tissue variations:
- broad variety of soft tissue variations, i.e. cystic dilatation of the brain, malpositioned carotid branches, short innominate, absent lung lobe (Lobus inferior medialis) and dilated renal pelvis in individual fetuses of test groups 0, 100, 250 or 500 mg/kg bw/d
- incidences were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant (Tab. 6)

Fetal skeletal variations:
- skeletal variations of different bone structures were observed in all test groups, with or without effects on corresponding cartilages
- observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing
- overall affected fetuses/litter incidences of skeletal variations were statistically significantly increased in all substance-treated groups (Tab. 9)
- this effects were above the historical control range (HCD: mean% 93.9 [83.2 - 100.0])
- all skeletal variations with statistically significant differences between the control and the treated groups are given in table 10
- two of the findings – supernumerary thoracic vertebra and supernumerary rib (13th, cartilage present) had higher incidences than the controls in all treated groups
- this increase was not statistically significant and hovered around the upper limit of the historical range in the low-dose group and not dose-related for the supernumerary rib (13th, cartilage present).

Fetal skeletal unclassified cartilage observations:
- the results are given table 11
- incidence ‘knobby rib cartilage’ was statistically significantly increased in test group 250 mg/kg bw/d (affected fetuses/litter, mean%: 0.0/1.9/1.2*/1.1 [p≤0.05])
- the vaue was within the historical control range (mean% 0.4 [0.0 - 1.3]) and there is no dose-response - no association to the treatment was assumed
- incidence ‘branched rib cartilage’ was statistically significantly increased in test group 500 mg/kg bw/d (affected fetuses/litter, mean%: 0.0/0.7/0.8/3.1* [p≤0.05])
- litter and affected fetus-es/litter incidences were outside the historical control range (mean% 0.0 [0.0 - 0.5])

Fetal external unclassified observations (table 12):
- two unclassified external observations, i.e. placentae discolored and placentae necrobiotic, were recorded in one fetus of test groups 250 or 500 mg/kg bw/d
- the effects were not considered to be related to treatment

Fetal soft tissue unclassified observations: (table 13)
- in one control fetus, three low-dose, seven mid-dose and three high-dose fetuses: a blood coagulum around urinary bladder observed
- this finding is not considered to be treatment-related

Effect levels (fetuses)

open allclose all
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
visceral malformations
other: significantly increased incidences of external, skeletal and cartilage variations above the historical control range
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: rib
skeletal: vertebra
skeletal: pelvic girdle
visceral/soft tissue: urinary

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
not specified

Any other information on results incl. tables

Table 1: Individual fetal external malformations

Test group

Doe No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

none

 

1 (100 mg/kg bw/d)

31-02 Ma)b)

acaudate

 

46-01 F

umbilical hernia

2 (250 mg/kg bw/d)

70-04 Ma)

umbilical hernia

3 (500 mg/kg bw/d)

none

 

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a)fetus with additional soft tissue malformation (see Tab. 4)

b)fetus with additional skeletal malformation (see Tab. 7)

Table 2: Total external malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

250 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter
Fetuses

N
N

25
226

22
192

24
196

21
160

Fetal incidence

 

N (%)

 

0.0

 

2 (1.0)

 

1 (0.5)

 

0.0

Litter incidence

 

N (%)

 

0.0

 

2 (9.1)

 

1 (4.2)

 

0.0

Affected fetuses/litter

 

Mean%

 

0.0

 

1.2

 

0.6

 

0.0

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

Table 3: Total external variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

250 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter
Fetuses

N
N

25
226

22
192

24
196

21
160

Fetal incidence

 

N (%)

 

0.0

 

0.0

 

0.0

 

6 (3.8)

Litter incidence

 

N (%)

 

0.0

 

0.0

 

0.0

 

4 (19)*Fi

Affected fetuses/litter

 

Mean%

 

0.0

 

0.0

 

0.0

 

4.6*Wi

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

*Fi= p ≤ 0.05 (Fisher’s exact test [one-sided])   *Wi= p ≤ 0.05 (Wilcoxon-test [one-sided])

Table 4: Individual fetal soft tissue malformations 

Test group

Doe No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

none

 

1 (100 mg/kg bw/d)

31-02 Ma)b)

multiple visceral malformations

2 (250 mg/kg bw/d)

70-04 Ma)

malpositioned kidney

3 (500 mg/kg bw/d)

82-02 F, 82-04 F

multiple visceral malformations

 

83-04 F

multiple visceral malformations

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a)fetus with additional external malformation (see Tab. 2)

b)fetus with additional skeletal malformation (see Tab. 7)

Table 5: Total soft tissue malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

250 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter
Fetuses

N
N

25
226

22
192

24
196

21
160

Fetal incidence

 

N (%)

 

0.0

 

1 (0.5)

 

1 (0.5)

 

3 (1.9)

Litter incidence

 

N (%)

 

0.0

 

1 (4.5)

 

1 (4.2)

 

2 (9.5)

Affected fetuses/litter

 

Mean%

 

0.0

 

0.6

 

0.6

 

1.5

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

 

Table 6: Total soft tissue variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

250 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter
Fetuses

N
N

25
226

22
192

24
196

21
160

Fetal incidence

 

N (%)

 

10 (4.4)

 

3 (1.6)

 

5 (2.6)

 

8 (5.0)

Litter incidence

 

N (%)

 

7 (28)

 

3 (14)

 

4 (17)

 

3 (14)

Affected fetuses/litter

 

Mean%

 

4.9

 

1.8

 

3.2

 

4.5

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

Table 7: Individual fetal skeletal malformations

Test group

Doe No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

none

 

1 (100 mg/kg bw/d)

31-02 Ma)

multiple skeletal malformations

 

37-06 M

misshapen thoracic vertebra

2 (250 mg/kg bw/d)

none

 

3 (500 mg/kg bw/d)

80-12 F

sternebrae severely fused (bony plate)

 

87-09 F

severely malformed vertebral column and/or ribs

 

88-03 M

misshapen lumbar vertebra

 

92-09 F

thoracic hemivertebra, sternebrae severely fused (bony plate)

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a)fetus with additional external and soft tissue malformations (see Tabs. 1 and 4)

Table 8: Total skeletal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

250 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter
Fetuses

N
N

25
226

22
192

24
196

21
160

Fetal incidence

 

N (%)

 

0.0

 

2 (1.0)

 

0.0

 

4 (2.5)

Litter incidence

 

N (%)

 

0.0

 

2 (9.1)

 

0.0

 

4 (19)*Fi

Affected fetuses/litter

 

Mean%

 

0.0

 

1.0

 

0.0

 

2.2*Wi

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

*Fi= p ≤ 0.05 (Fisher’s exact test [one-sided])   *Wi= p ≤ 0.05 (Wilcoxon-test [one-sided])

 

Table 9: Total fetal skeletal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

250 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter
Fetuses

N
N

25
226

22
192

24
196

21
160

Fetal incidence

 

N (%)

 

210 (93)

 

189 (98)

 

194 (99)

 

160 (100)

Litter incidence

 

N (%)

 

25 (100)

 

22 (100)

 

24 (100)

 

21 (100)

Affected fetuses/litter

 

Mean%

 

92.1

 

97.3*

 

99.1**

 

100.0**

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

* = p ≤ 0.05 (Wilcoxon-test [one-sided])           ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 10: 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

250 mg/kg
bw/d

Test group 3

500 mg/kg bw/d

HCD

Mean %

(range)

Supernumerary thoracic vertebra

19.4

27.6

39.9**

46.9**

17.9

(7.8 - 25.6)

Supernumerary lumbar
vertebra

0.7

0.6

1.5

7.2*

0.3

(0.0 - 2.4)

Unossified sternebra;
unchanged cartilage

11.6

18.7*

21.9**

45.4**

15.4

(7.7 - 27.5)

Misshapen sternebra;
unchanged cartilage

6.4

3.6

6.3

18.2**

9.8

(3.1 - 17.7)

Supernumerary rib (13th); cartilage present

56.8

69.9

77.0**

70.7**

60.4

(50.0 - 70.9)

Incomplete ossification of talus; cartilage present

1.5

0.6

2.8

14.2**

2.1

(0.0 - 4.8)

Unossified talus;
cartilage present

0.4

1.0

2.3

16.7**

0.9

(0.0 - 2.6)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p ≤ 0.05 (Wilcoxon-test [one-sided])            ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 11: Total unclassified cartilage observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

250 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter
Fetuses

N
N

25
226

22
192

24
196

21
160

Fetal incidence

 

N (%)

 

62 (27)

 

35 (18)

 

37 (19)

 

59 (37)

Litter incidence

 

N (%)

 

23 (92)

 

16 (73)

 

16 (67)

 

19 (90)

Affected fetuses/litter

 

Mean%

 

30.5

 

18.7

 

17.1

 

34.3

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

 

Table 12: Total external unclassified observations 

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

250 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter
Fetuses

N
N

25
226

22
192

24
196

21
160

Fetal incidence

 

N (%)

 

0.0

 

0.0

 

1 (0.5)

 

1 (0.6)

Litter incidence

 

N (%)

 

0.0

 

0.0

 

1 (4.2)

 

1 (4.8)

Affected fetuses/litter

 

Mean%

 

0.0

 

0.0

 

0.8

 

0.7

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

Table 13: Total soft tissue unclassified observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

250 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter
Fetuses

N
N

25
226

22
192

24
196

21
160

Fetal incidence

 

N (%)

 

1 (0.4)

 

3 (1.6)

 

7 (3.6)

 

3 (1.9)

Litter incidence

 

N (%)

 

1 (4.0)

 

2 (9.1)

 

2 (8.3)

 

2 (9.5)

Affected fetuses/litter

 

Mean%

 

0.3

 

1.7

 

2.8

 

1.4

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

 

Applicant's summary and conclusion

Conclusions:
The oral administration of B-TEGME to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of maternal toxicity at the high-dose level of 500 mg/kg bw/d, such as abortions/mortality and reduced food consumption. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 250 mg/kg bw/d.

Since there was evidence for treatment-related adverse effects of the test substance on fetal morphology at the high-dose of 500 mg/kg bw/d, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 250 mg/kg bw/d.
Executive summary:

B-TEGME was tested for its prenatal developmental toxicity in New Zealand White rabbits according to OECD 414.

The test substance was administered as pure substance without being formulated with a vehicle to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 100, 250 and 500 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The control group, consisting of 25 females, was dosed with drinking water (same quantity as the high-dose animals) in parallel. At terminal sacrifice on GD 29, 21-25 females per group had implantation sites.

Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

The following test substance-related adverse effects/findings were noted:

Test group 3 (500 mg/kg bw/d):

Dams

- Death or abortion in 3 females immediately before term (GD 28/29), corroborative necropsy findings indicating enteropathy

- Lower food consumption (7% below control)

Fetuses

- Increased incidence of fetuses showing visceral (urogenital tract) or skeletal (axial skeleton) malformations, some above historical range

- Increased incidences of external, skeletal and cartilage variations, all above historical control range

- Increased incidences of total malformations and variations

Test group 2 (250 mg/kg bw/d):

- No test substance-related adverse effects on does, gestational parameters or fetuses.

Test group 1 (100 mg/kg bw/d):       

- No test substance-related adverse effects on does, gestational parameters or fetuses.

Under the conditions of this prenatal developmental toxicity study, the oral administration of B-TEGME to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6 -28) caused evidence of maternal toxicity at the high-dose level of 500 mg/kg bw/d, such as abortions/mortality and reduced food consumption. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 250 mg/kg bw/d.

Since there was evidence for treatment-related adverse effects of the test substance on fetal morphology at the high-dose of 500 mg/kg bw/d, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 250 mg/kg bw/d.