Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2014-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, for read-across justification refer to IUCLID section 13.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
refer to confidential details on test material

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Venray, The Netherlands
- Age at study initiation: Age at delivery: Females were approximately 10 to 14 weeks.
- Weight at study initiation: 17.6 - 22.2 g (group means)
- Fasting period before study: no
- Housing: individually housed in Macrolon plastic cages (MIII type)
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24 °C
- Humidity (%): 40 - 70%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 25 December 2014 To: 14 January 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and
were homogenized to a visually acceptable level

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations
Analytical verification of doses or concentrations:
yes
Details on mating procedure:

Untreated females were mated at the supplier, and were at Day 0, 1 or 2
post-coitum on arrival at the test facility (Day 0 post-coitum was the day
of successful mating; confirmed by vaginal plug).
Duration of treatment / exposure:
From Day 6 to Day 20 post-coitum, inclusive
Frequency of treatment:
once daily
Duration of test:
treatment from Day 6 to Day 20 post-coitum, inclusive; necropsy on day 21 post-coitum
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 60, 200, 600 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
22 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: prestudy
- Rationale for animal assignment: random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:Days 3, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
Days 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: organ weights: (gravid) uterus, liver, spleen, necrospy: Gross lesions, ovary and uterine horn, spleen, liver, Identification marks

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: number and distribution of live and dead fetuses, weight of each fetus, sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination), weight of each placenta.
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
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-toone
t-test) based on a pooled variance estimate was applied for the comparison of the treated
groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow
a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the
number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation
loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations
(external, visceral and skeletal), and each particular external, visceral and skeletal malformation or
variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine
intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance,
Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data
(scores) in the summary tables. Test statistics were calculated on the basis of exact values for means
and pooled variances. Individual values, means and standard deviations might be rounded off before
printing. Therefore, two groups might display the same printed means for a given parameter, yet
display different test statistics values.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of
dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
-increased absolute and/or relative liver weights at 200 and 600 mg/kg bw/d
- lower total protein and increased urea at 600 mg/kg bw/d

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At 600 mg/kg bw/day, reduced fetal body weights were noted (approximately 8% lower than control levels, combined for both sexes) along with a correspondingly higher incidence of reduced ossification parameters. An increase in unossified metacarpal(s) and/or metatarsal(s) was the most pronounced effect and an increase in reduced ossification of the skull also occurred, but to a lesser degree.
An increase in skeletal variations at 600 mg/kg bw/day that were not body weight dependent included increased incidence of 14th rudimentary rib(s), 14th full rib(s), 7th cervical rudimentary rib(s), and caudal shift of the pelvic girdle.
These effects indicate a generalized delay in ossification, a well documented effect often associated with maternal toxicity of various different mechanisms and/or affected homeostasis. In the present study, increased maternal liver weights and changes in clinical chemistry parameters indicate an adversely disturbed liver homeostasis. In addition, a generalized delay in ossification is expected to be readily repairable during early postnatal development and is therefore not considered as specific developmental toxicity.
There were no treatment-related effects on fetal external, visceral or skeletal malformations, on
external and visceral variations and on litter size, male:female ratios and placenta weights up to and including 600mg/kg bw/day. No developmental toxicity was observed in the 60 and 200 mg/kg bw/day groups.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

At 600 mg/kg bw/day, reduced fetal body weights were noted (approximately 8% lower than control levels, combined for both sexes) along with a correspondingly higher incidence of reduced ossification parameters. An increase in unossified metacarpal(s) and/or metatarsal(s) was the most pronounced effect and an increase in reduced ossification of the skull also occurred, but to a lesser degree. An increase in skeletal variations at 600 mg/kg bw/day that were not body weight dependent included increased incidence of 14th rudimentary rib(s), 14th full rib(s), 7th cervical rudimentary rib(s), and caudal shift of the pelvic girdle. These effects indicate a generalized delay in ossification, a well documented effect often associated with maternal toxicity of various different mechanisms and/or affected homeostasis. In the present study, increased maternal liver weights and changes in clinical chemistry parameters indicate an adversely disturbed liver homeostasis. In addition, a generalized delay in ossification is expected to be readily repairable during early postnatal development and is therefore not considered as specific developmental toxicity. There were no treatment-related effects on fetal external, visceral or skeletal malformations, on external and visceral variations and on litter size, male:female ratios and placenta weights up to and including 600mg/kg bw/day. No developmental toxicity was observed in the 60 and 200 mg/kg bw/day groups.

Applicant's summary and conclusion