Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 2010 - May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
(2S,4S,5S,6R)-5-hydroxy-4-(hydroxymethyl)-11-methyl-3,7-dioxa-1,9-diazatricyclo[6.4.0.0^{2,6}]dodeca-8,11-dien-10-one
EC Number:
700-330-1
Cas Number:
433733-92-7
Molecular formula:
C10H12N2O5
IUPAC Name:
(2S,4S,5S,6R)-5-hydroxy-4-(hydroxymethyl)-11-methyl-3,7-dioxa-1,9-diazatricyclo[6.4.0.0^{2,6}]dodeca-8,11-dien-10-one
Test material form:
solid: bulk
Details on test material:
- Name of test material (as cited in study report): Anhydrothymidine
- Substance type: White powder
- Physical state: powder
- Analytical purity: 99.7%
- Purity test date: not stated
- Lot/batch No.: 073904/05
- Expiration date of the lot/batch: 11 December 2008
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals or test system and environmental conditions:
The rat (Crl:WI(Han) (outbred, SPF-Quality)) was selected as it has been recognized as appropriate for developmental toxicity studies. NOTOX B.V. has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Animal species and strain Rat: Crl:WI(Han) (outbred, SPF-Quality).
The females were nulliparous and non-pregnant at initiation of the
study. Stock male rats Crl:WI(Han) (outbred, SPF-Quality) were
used for mating with the females. After mating these males were
placed back in their stock and might be used in further studies.
Breeder/supplier Charles River, Sulzfeld, Germany.
Total numbers of animals F0-generation: 96 females.
F1-generation: 1098 fetuses
Age at start of pairing Females were approximately 12 weeks.
Acclimatization At least 5 days prior to breeding. A health inspection was
performed prior to commencement of treatment to ensure that the
animals were in a good state of health.
Breeding procedures One female was placed on a one-to-one-basis in the cage of a male
rat. Detection of mating was confirmed by evidence of sperm in
the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum. Once
mating had occurred, the female was separated from the male.
When sufficient mated females were obtained for each dose group,
the surplus females were removed from the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Ninety-six mated female Wistar Han rats were assigned to four dose groups. All groups contained twenty-four mated females. The test item was administered once daily by gavage
from Days 6 to 19 post-coitum at a dose leves of 100, 300 and 750 mg/kg/day. The rats of the control group received the vehicle, water, alone.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (23 November 2010), according to a validated method (NOTOX Project 488331). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Details on mating procedure:
One female was placed on a one-to-one-basis in the cage of a male rat. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the female was separated from the male.
When sufficient mated females were obtained for each dose group, the surplus females were removed from the study.
Duration of treatment / exposure:
From Day 6 to Day 19 post-coitum, inclusive.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Duration of test:
Delivery of Animals 10 November 2010
Experimental starting date 15 November 2010 (Start pairing)
Start treatment (Day 6 post-coitum) 22 November 2010
Necropsy (Day 20 post-coitum) 06 - 23 December 2010
Experimental completion date 23 December 2010 (end of in-life phase)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
No. of animals per sex per dose:
F0-generation: 96 females.
F1-generation: 1098 fetuses
Control animals:
yes, concurrent vehicle
Details on study design:
Group Dose level (mg/kg) Number of females
1 0 24
2 100 24
3 300 24
4 750 24

Ninety-six mated female Wistar Han rats were assigned to four dose groups. All groups contained twenty-four mated females. The test item was administered once daily by gavage
from Days 6 to 19 post-coitum at a dose leves of 100, 300 and 750 mg/kg/day. The rats of the control group received the vehicle, water, alone.
Dose levels were selected based on the results of a dose range finding study (NOTOX Project 492174) and a prenatal developmental toxicity study that was conducted as a limit test (NOTOX Project 492175). In the dose range finding study, no maternal, reproductive, or external developmental toxicity was observed up to 1000 mg/kg. The main study was
conducted as a limit test because no toxicity was observed in the pilot. However, at 1000 mg/kg, skeletal malformations characterized by bent limb bones were seen. Retrospective
examination of the skeletons from the range finding study revealed the same type of skeletal malformations at 1000 mg/kg, only. This was conducted as an independent study.
Females were checked twice daily for mortality/viability and daily for the presence of clinical signs. Food consumption was determined at periodic intervals and body weight was determined daily during treatment and at periodic intervals before the onset of treatment. On one occasion formulations were analyzed for accuracy and homogeneity.

Examinations

Maternal examinations:
All animals surviving to Day 20 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A
laparohysterectomy was performed on each female of all groups.
Ovaries and uterine content:
The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated.
Fetal examinations:
The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.
All live fetuses were euthanized. One half of the fetuses were decapitated, the heads fixed in Bouin‟s fixative, and were examined. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous alcohol and stained with Alizarin Red S for skeletal examinations; skeletal examinations were performed on one-half of the fetuses (the fetuses with heads).
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-to-one t-test) based on a pooled variance estimate was applied for the
comparison of the treated group and the control group for each sex.
- 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.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined) 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 group 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 have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Indices:
The following definitions were applicable for implantation data:
- Fetal (late) resorptions were defined as a dead fetus with external degenerative changes and presence of distinguishable features such as head or limbs.
- Embryonic (early) resorptions were defined as evidence of implantation without presence of distinguishable features such as head or limbs.
- Dead fetus was defined as a dead fetus without external degenerative changes and presence of distinguishable features such as head or limbs.
- Post-implantation loss included embryonic (early) resorptions, fetal (late) resorptions and dead fetuses.
Historical control data:
Dose levels were selected based on the results of a dose range finding study (NOTOX Project
492174) and a prenatal developmental toxicity study that was conducted as a limit test
(NOTOX Project 492175). In the dose range finding study, no maternal, reproductive, or
external developmental toxicity was observed up to 1000 mg/kg. The main study was
conducted as a limit test because no toxicity was observed in the pilot. However, at 1000
mg/kg, skeletal malformations characterized by bent limb bones were seen. Retrospective
examination of the skeletons from the range finding study revealed the same type of skeletal
malformations at 1000 mg/kg, only. This was conducted as an independent study.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs indicative of toxicity noted up to 750 mg/kg.
Alopecia, an incidental finding, was noted for 2, 2 and 2 females at 0, 100 and 750 mg/kg, respectively. No other clinical signs were seen during the treatment period.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no differences in body weights or body weight gains at any point during the treatment period up to 750 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant effects on absolute or relative food consumption up to 750 mg/kg.
A significant increase in absolute and relative food consumption was seen over Days 0-3 of the post-coitum period for animals at 750 mg/kg (Group 4). As this occurred prior to test substance administration on Day 6 of the post-coitum period, the increase was not treatment related or toxicologically relevant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related macroscopic findings seen up to 750 mg/kg.
Incidental findings including alopecia of various body regions and/or pelvic dilation of one or both kidneys was noted for individual animals in the control and treated groups. At the incidence observed and in the absence of a treatment related distribution, these were not considered to be toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
There were 23, 22, 24, and 23 animals pregnant in Groups 1, 2, 3 and 4, respectively.
The numbers of corpora lutea, implantation sites, viable and dead fetuses and early and late resorptions were similar between control and treated animals.
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea, implantation sites, viable and dead fetuses and early and late
resorptions were similar between control and treated animals.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea, implantation sites, viable and dead fetuses and early and late
resorptions were similar between control and treated animals.
Dead fetuses:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea, implantation sites, viable and dead fetuses and early and late
resorptions were similar between control and treated animals.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were 23, 22, 24, and 23 animals pregnant in Groups 1, 2, 3 and 4, respectively.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in pregnancy duration
clinical signs
dead fetuses
effects on pregnancy duration
gross pathology
maternal abnormalities
mortality
number of abortions
total litter losses by resorption

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal body weights at 750 mg/kg remained within the same range as fetuses from the control group. They were 3.5, 3.4, 3.5, and 3.5 grams for control, 100, 300 and 750 mg/kg animals, respectively.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no treatment related effect of Anhydrothymidine on litter size. The mean litter size per group was 12, 11.9, 11.4, and 12.5 fetuses for controls and animals at 100, 300 and 750 mg/kg, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No treatment-related effect on the sex ratio was noted.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no treatment related effect of Anhydrothymidine on litter size. The mean litter size per group was 12, 11.9, 11.4, and 12.5 fetuses for controls and animals at 100, 300 and 750 mg/kg, respectively.
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related effects on fetal external morphology.
No external malformations were observed in any fetus in this study. One fetus in the 300 mg/kg group (no. A064-14) had a subcutaneous hemorrhage, which was considered to be
an artefact (due to handling at necropsy). No other external developmental variations were observed.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related effects on fetal skeletal morphology.
No skeletal malformations occurred in the test substance-treated groups, whereas one control fetus (no. A001-07) had a bent humerus.
Skeletal variations that occurred in the test substance-treated groups were reduced ossification of the skull, bent ribs, unossified sternebrae nos. 5 and/or 6, 14th rudimentary ribs, ossified cervical centrum no. 1, 7th cervical ribs, 14th full ribs, 27 presacral vertebrae, unossified hyoid, slightly to moderately malaligned sternebrae, unossified sternebrae nos. 1, 2, 3 and/or 4, and reduced ossification of vertebral centra and arches. All visceral variations occurred at similar frequencies in the control group, occurred infrequently and/or in a manner that was not doserelated.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related effects on fetal visceral morphology.
Visceral malformations were observed in 0(0), 1(1), 1(1) and 5(4) fetuses (litters) in the control, 100, 300 and 750 mg/kg groups, respectively. At 750 mg/kg, there were two fetuses from one litter (nos. A092-02 and -12) that had situs inversus whereby all thoracic and abdominal organs were laterally transposed and two fetuses (nos. A079-04 and A095-13) were missing one of both thyroid glands. Although these findings were neither observed in the lower dose groups, nor in the control group, they occurred in historical control fetuses at comparable maximum incidences (the upper historical control limit was 0.7% and 0.4% per
litter for absent thyroid and situs inversus respectively, versus 0.7% and 0.6% per litter in the current 750 mg/kg group) and were therefore not considered to be related to test substancetreatment.
Multiple visceral malformations were observed in fetus A096-14 at 750 mg/kg. This fetus had a retroesophageal aortic arch, absent eye (right) and small eye (left). At 300 mg/kg, a small eye was also observed in fetus A068-02 and at 100 mg/kg, fetus no. A045-03 had a large atrium with no underlying abnormalities. The incidental occurrence of these findings did not suggest a relationship to treatment. Furthermore, the eye findings were previously seen in historical control fetuses. Therefore, these visceral malformations were considered to be spontaneous in origin.
There were no soft-tissue developmental variations attributed to the test substance. Variations noted more than once in the test substance-treated groups were small supernumerary liver lobes, small thyroids and pale spleen. All visceral variations occurred at similar frequencies in the control group, occurred infrequently and/or in a manner that was not dose-related.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Anhydrothymidine were established as 750 mg/kg body weight/day. In the former dose range finding study (NOTOX Project 492174) and the limit test (NOTOX Project 492175) there were clear skeletal malformations at 1000 mg/kg without any maternal toxicity. Thus, the test substance Anhydrothymidine is a teratogen at 1000 mg/kg. The NOAEL of 750 mg/kg determined in the present study indicates that there is a clear threshold.