Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 September 2017 to 24 November 2017
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: RccHan™;WIST rat.
- Source: Envigo RMS Limited.
- Age at study initiation: 76 - 82 days
- Weight at study initiation: 180-208 g
- Housing: individually in polycarbonate cages with a stainless steel mesh lid
- Diet: SDS VRF1 Certified pelleted diet ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days before pairing and during pairing until evidence of mating

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24ºC
- Humidity (%): 40-70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: weekly
The required amount of substance was weighed and then mixed with the vehicle (approximately 50% of the final volume). The mixture was magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.

VEHICLE: 1% w/v methylcellulose
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The first and last freshly prepared test formulations were sampled (4 × 1 mL,accurately weighed) and submitted for analysis. Duplicate samples were analyzed. The samples were dissolved using ultrasonic vibration and swirling in a suitable volume of diluent (Methanol / water 80/20 v/v). The extract was diluted using diluent (where necessary) to provide a solution containing the substance at an expected concentration within the range 2 µg/mL to 4 µg/mL.

Method:
High performance liquidchromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector.
Column: Agilent Poroshell C18-EC, 2.7 µm, 100 × 4.6 mm
Column temperature: 45ºC
Sample temperature: Ambient.
Mobile Phase: Methanol / water 85 / 15 v/v
Flow rate: 1 mL/min
Needle wash: Methanol / water 80 / 20 v/v
Detector wavelength: UV, 229 nm
Injection volume: 10 µL
Run time: 6 minutes
Approximate retention time: 4.6 minutes

Results method validation:
Calibration linearity (range 1-5 ug/mL): r > 0.999
Specificity: absence of peak for substance in control sample
Precisions calibration (CV 0.22% 1 ug/mL, 0.28% 5 ug/mL)
Accuracy: procedural recovery value of 101.6% (CV=1.13%, n=5) was obtained for 1 mg/mL and 101.0% (CV=0.13%, n=5) was obtained for 200 mg/mL.
Stability (15 days 4 ºC): 1 mg/mL 96% of intial; 200 mg/mL 97% of initial
Stability (1 day at 21 ºC): 1 mg/mL 105% of intial; 200 mg/mL 103.5% of initial
Homogeneity: CV < 5.6% at 1 mg/mL; CV < 1% at 200 mg/mL
LOQ: 2.2 ng/mL and 7.4 ng/mL

Preparation analyses: see below
Procedural recovery: no data
Accuracy: first: 97.8-99.7% of nominal for all concentrations; last: 101-104% of nominal for all concentrations
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: until detection of mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 6 to 19 after mating
Frequency of treatment:
daily (using a suitably graduated rubber-free syringe and a plastic catheter inserted via the mouth)
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
measured concentration 99.1-101% of nominal
Dose / conc.:
330 mg/kg bw/day (nominal)
Remarks:
measured concentration 99.7-101% of nominal
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
measured concentration 97.8-104% of nominal
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
Dose levels were selected based on the effects of a preliminary embryo-fetal study at this laboratory (Envigo study number CM44QG) which investigated dose levels of 330, 660 and 1000 mg/kg/day. At 1000 mg/kg/day, overall body weight gain and food consumption were marginally lower than that of Control. There was no conclusive effect of treatment on embryo-fetal survival, litter size or sex ratio at any dose level; however, mean fetal weights appeared slightly low at 660 or 1000 mg/kg/day when compared with Control.
Due to the minimal signs of maternal toxicity on the preliminary study, along with slight effects of fetal growth at 660 or 1000 mg/kg/day, dose levels of 100, 330 and 1000 mg/kg/day were selected for this study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating)

DOSING OBSERVATIONS: Pre-dose and 1-2 hours after completion of dosing of all groups (during week 1 also at the end of the working day)

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, day 3 and daily from day 6 to 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating

WATER CONSUMPTION: No

HEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: no
- How many animals: all
- Parameters checked: Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: no
- How many animals: all
- Parameters checked: Alanine amino-transferase (ALT), Aspartate amino-transferase (AST), Bilirubin (Bili), Urea, Total protein (Total Prot), Albumin (ALB) by chemical assay, Zinc (Zn)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Gross necropsy: for all animals all external features and orifices were examined visually

OTHER: residual lithium heparin samples be analyzed for Zinc on the Roche P Modular Analyzer
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
- Fetuses (live and dead): Yes
- Other: non-pregnant females: The number of uterine implantation sites were checked after staining with ammonium sulphide
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]: fixed in Bouin’s fluid
- Skeletal examinations: Yes: [half per litter]: stained with Alizarin Red
Statistics:
For body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.

For litter size and survival indices and fetal, placental and litter weight, gravid uterine weight and clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed.
Pre/post implantation loss and sex ratio were analyzed by generalized mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz 1991). Each treated group was compared to control using a Wald chi-square test. For pre implantation loss, the numerator was number of corpora lutea - number of implantations, the denominator was number of corpora lutea. For post-implantation loss, the numerator was number of implantations - number of live fetuses, the denominator was number of implantations. For sex ratio, the numerator was number of males; the denominator was number of live fetuses.

For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test (Wilcoxon 1945).

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Historical control data:
available in the report for fetal abnormalities (major and minor) and variations

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
total litter resorption in 1 female at 100 mg/kg bw appeared with discharge of blood from the vagina, piloerection of the coat and a pale skin colour across the whole body and the appearance of blood clots one day thereafter
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were lower than Controls for females receiving 1000 mg/kg/day from Gestation Days 6 to 20 (85%). It is noted that prior to treatment (Gestation Days 0 to 6), bodyweight gains for females were lower than Controls.

When adjusted for the weight of the gravid uterus the effect on body weight remained, but body weights on day 20 were within control ranges

see attached table
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption during Gestation Days 6-19 was similar to Control at 100 and 330 mg/kg/day, and slightly low at 1000 mg/kg/day (90%).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The hematological examination at termination revealed, when compared to Controls, a statistically significantly low prothrombin time for females treated at 1000 mg/kg/day (90%).

see overview table
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma at termination revealed, when compared to Control, slightly low alanine amino-transferase at 1000 mg/kg/day (86%) and slightly low aspartate amino-transferase at 330 or 1000 mg/kg/day (83% both). High urea concentrations (126%) were observed at 1000 mg/kg/day, along with slightly higher albumin/globulin ratio (115%) that were attributed to statistically significantly low total protein (88%).

see overview table
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean gravid uterine weight was similar to Control at 100 and 330 mg/kg/day, but low at 1000 mg/kg/day (86%).
Placental weights were not affected

see attached table
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no clear effects of treatment on pre/post implantation losses.

see overview table
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
total resoprtion in 1 female at 100 mg/kg bw
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no clear effects of treatment on early or late resorptions

see overview table
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no clear effects of treatment on numbers of live young

see overview table
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
1 female at 330 mg/kg bw was non-pregnant
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Corpora lutea counts were slightly low for females in treated groups, however the number of implantations for all dose groups were similar to Control

see overview table
Details on maternal toxic effects:
At 1000 mg/kg/day, body weight gain was low, and correlated with low food intake at this dose. The lower weight gain was attributed to a lower gravid uterine weight.
The hematological examination at termination revealed low prothrombin time for females treated at 1000 mg/kg/day.
The biochemical examination of the blood plasma at termination revealed slightly lower alanine amino-transferase and total protein at the high dose level, and low aspartate amino-transferase at the intermediate and high dose level. Additionally, higher urea concentrations and albumin/globulin ratio (115%) were observed at the high dose level.
Adult females were macroscopically normal.
The number of implantations, resorptions, pre and post-implantation loss, number of live young, sex ratio of males to females and placental weights were unaffected by treatment.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: observed effects were minimal

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
When compared to Control, the litter and fetal weights of animals receiving 1000 mg/kg/day was low (75% and 80%, respectively), but were similar to Controls for females receiving 100, 330 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no clear effects of treatment on numbers of live young

see overview table
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no clear effects of treatment on sex ratio

see overview table
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
In the treated groups there was a slight increased incidence of major abnormalities which included short/bent/thickened long bones. At 1000 mg/kg/day, there were three litters with major abnormalities (partially split sternum, bent scapula and short/thickened long bones) in 1-2 fetuses/litter, the incidence of which exceeded historical control data. One fetus at 330 mg/kg/day had multiple thoracic vertebral/rib abnormalities.
At 1000 mg/kg/day there was an increased incidence of medially thickened/kinked ribs; short supernumerary cervical rib; 14th supernumerary rib; 20 thoracolumbar vertebrae; unilateral shift of pelvic girdle and a generalised delay in ossification affecting, but not restricted to, cranial bones, sternebrae, vertebrae and digits compared to concurrent Control and outside of historical control data (HCD).
At 330 mg/kg/day there was an increased incidence of 14th supernumerary rib; unilateral shift of pelvic girdle and a slight delay in ossification of cranial bones compared to concurrent Control and outside of HCD.
A delay in ossification is a transient stages of fetal development and can be attributed to low fetal bodyweight.

see attached table and overview table
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
In the treated groups there was a slight increased incidence of major abnormalities which included a low incidence of heart and major vessel abnormalities. At 1000 mg/kg/day, there was one litter with major abnormalities (retosophageal right subclavian artery and malrotated heart), the incidence of which exceeded historical control data. At 100 and 330 mg/kg/day, the findings were seen in a single fetus.
At 1000 mg/kg/day there was an increased incidence of variation in lens shape; partially undescended lobe of thymus and shiny skin compared to concurrent Control and outside of historical control data (HCD).
At 330 mg/kg/day there was an increased incidence of partially undescended lobe of thymus compared to concurrent Control and outside of HCD.
The variation in lens shape and partially undescended lobe of thymus are transient stages of fetal development.

see attached table and overview table
Details on embryotoxic / teratogenic effects:
Fetal and litter weights were low at 1000 mg/kg/day compared with Control.
There was an increased incidence of major abnormalities at 1000 mg/kg/day.
Visceral and skeletal abnormalities were seen at a higher incidence at 330 and 1000 mg/kg/day. The delays in ossification, variation in lens shape and partially undescended lobe of thymus were considered to represent transient stages of fetal development. Other minor skeletal abnormalities such as short supernumerary cervical ribs and 14th supernumerary ribs seen at 1000 mg/kg/day are not attributable to the low fetal weights in this group.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
330 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: vertebra
visceral/soft tissue: cardiovascular
other: skeletal/appendicular

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects

Any other information on results incl. tables

Overview table

Dose (mg/kg bw)

0

100

330

1000

Treatment related

 

Endpoint

 

 

 

 

 

 

Mortality

0/20

0/20

0/20

0/20

 

 

Clinical signs

NTRE

 

 

Body weight (gain)

 

 

(↓ day 0-6 18%)

↓day 7 to 20 (↓ day 0-6 14%, ↓ day 7-20 15%)

yes

 

Food consumption day 0-20

 

 

 

↓ day 0-6 6%, ↓ day 7-20 10%

yes

 

Pregnancy rate

20/20

19/20

19/20

20/20*

 

 

Litter resorption

 

1/20

 

 

 

 

Gravid uterus weight

 

 

 

↓ 14% (↓ 4% adj BW)

 

 

Haematology

 

 

 

PTT ↓ 10%

 

 

Clinical biochemistry

 

 

ASAT ↓ 17%

 

ALAT ↓ 16%

ASAT ↓ 17%

Urea ↑ 26%

TP ↓ 12%

Alb ↓ 6%

A/G ↑ 15%

yes

 

Macroscopy

NTRE

 

Corpora lutea

13.3

12.0 ↓

12.1 ↓

12.1 ↓

yes

 

Implantations

11.9

10.9

11.3

11.4

 

 

Early resorptions

0.7

0.4

0.7

0.8

 

 

Late resorptions

0.1

0.1

0.0

0.1

 

 

Pre-implementation loss (%)

11.1

9.6

6.4

7.1

 

 

Post-implementation loss (%)

6.4

6.1

6.5

9.2

 

 

Live young/Litter

11.1

10.4

10.5

10.4

 

 

Sex ratio young (% males)

51.6

40.8 ↓

50.7

48.8

 

 

Placental weight

NTRE

 

Litter weight

 

 

 

↓ 25%

yes

 

Fetal weight

 

 

 

↓ 20%

yes

 

Fetal major abnormalities

Cervical/Thoracic

Skeletal

-Partially split sternum

-Multiple thoracic vertebral/rib abn

Visceral

-Retroesophageal right subclavian art

-Retroesophageal aortic arch

-Interrupted aortic arch

-Transposition of ascending aorta and pulmonary trunk

-Muscular ventricular septal defect

-Atrial septal defect

-Malrotated heart

Lumbar/Sacral/Caudal

Visceral

-Omphalocele

Appendicular

Skeletal

-Bent scapula(e)

-Short/thickened long bones

 

 

 

 

 

 

 

 

 

1/19 (1/198 fetuses)#

 

1/19 (1/198 fetuses)#

 

1/19 (1/198 fetuses)#

1/19 (1/198 fetuses)#

 

 

 

1/19 (1/198 fetuses)

 

 

 

 

 

 

 

1/19 (1/200 fetuses)

 

 

1/19 (1/200 fetuses)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1/19 (1/198 fetuses)

 

 

1/19 (2/198 fetuses)#

 

 

 

 

 

 

 

1/19 (1/198 fetuses)#

 

 

 

 

 

1/19 (1/198 fetuses)

2/19 (3/198 fetuses)

yes

 

Fetal minor abnormalities + variations

Skeletal

-Minor skeletal abnormalities

-Rib and vertebral configuration

-Thoracolumbar vertebrae 20

-Pelvic girdle (caudal/cranial shift)

-Delayed/Incomplete ossification/unossified

Visceral

 

 

 

2/20 (2/112 fetuses)

Up to 9/20 (17/112 fetuses)

 

 

 

Up to 17/20 (43/112 fetuses)

Up to 5/20 (7/110 fetuses)

 

 

 

 4/19 (4/99 fetuses)

Up to 9/19 (23/99 fetuses)

 

 

 

Up to 14/19 (31/99 fetuses)

Up to 15/19 (24/98 fetuses)

 

 

 

3/19 (3/102 fetuses)

Up to 17/19 (39/102 fetuses)

 

4/19 (4/102 fetuses)

 

Up to 17/19 (47/102 fetuses)

Up to 9/19 (12/98 fetuses)

 

 

 

11/19 (21/100 fetuses)

Up to 17/19 (54/100 fetuses)

7/19 (12/100 fetuses)

4/19 (4/100 fetuses)

 

Up to 19/19 (98/100 fetuses)

Up to 12/19 (21/98 fetuses)

Yes

 

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

*one female was not dosed during two days and was excluded from the evaluations

#effects reported in the same fetus

Applicant's summary and conclusion

Conclusions:
Based on results of this study, the substance was well tolerated and the maternal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day. Due to the occurrence of major fetal abnormalities at 1000 mg/kg/day the fetal NOAEL was considered to be 330 mg/kg/day
Executive summary:

Three groups of 20 femal rats received the substance at doses of 100, 330 and 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle,1% w/v methylcellulose at the same volume dose as treated groups and for the same duration. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

During the study clinical observations, body weight, food consumption hematology (peripheral blood) and blood chemistry investigations were undertaken. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. 

At 1000 mg/kg/day, body weight gain was low, and correlated with low food intake at this dose. The lower weight gain was attributed to a lower gravid uterine weight.   

The hematological examination at termination revealed low prothrombin time for females treated at 1000 mg/kg/day.

The biochemical examination of the blood plasma at termination revealed slightly lower alanine amino-transferase and total protein at the high dose level, and low aspartate amino-transferase at the intermediate and high dose level. Additionally, higher urea concentrations and albumin/globulin ratio (115%) were observed at the high dose level.

Adult females were macroscopically normal. 

The number of implantations, resorptions, pre and post-implantation loss, number of live young, sex ratio of males to females and placental weights were unaffected by treatment.

Fetal and litter weights were low at 1000 mg/kg/day compared with Control.

There was an increased incidence of major abnormalities at 1000 mg/kg/day.

Visceral and skeletal abnormalities were seen at a higher incidence at 330 and 1000 mg/kg/day. The delays in ossification, variation in lens shape and partially undescended lobe of thymus were considered to represent transient stages of fetal development. Other minor skeletal abnormalities such as short supernumerary cervical ribs and 14th supernumerary ribs seen at 1000 mg/kg/day are not attributable to the low fetal weights in this group.

Based on results of this study, the substance was well tolerated and the maternal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day. Due to the occurrence of major fetal abnormalities at 1000 mg/kg/day, the fetal NOAEL was considered to be 330 mg/kg/day.