Registration Dossier

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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Effects on fertility

Description of key information

Two reproductive and developmental toxicity studies were performed. In first study, at 1000 mg/kg no maternal toxicity was observed, but a significant increase in the incidence of skeletal malformations. In second study, there were no treatment-related effects on fetal external, visceral or skeletal morphology, nor were there any effects on viability, litter size, sex ratio or fetal body weights at any dose level. The NOAEL for this study was considered to be 750 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Qualifier:
according to guideline
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

There were no treatment related effects on fetal viability, litter size, sex ratio, fetal body weights or external, visceral or skeletal morphology up to 750 mg/kg.

At 1000 mg/kg an increased incidence in the mean litter proportion of fetuses with skeletal malformations, characterized by bent limb bones including the humerus and scapula was seen. No toxicologically significant findings were noted for external or visceral malformations or

variations.

Fetuses from the control and 1000 mg/kg groups had mean litter proportions of external malformations at 0.0% and 0.8%, respectively. No external variations were seen. Visceral malformations were observed in 2.3% and 0.4% of fetuses per litter, and visceral variations were

observed in 8.9% and 7.5% of fetuses per litter in the control and 1000 mg/kg groups, respectively. Skeletal examination revealed 0.0% of controls and 2.3% of fetuses per litter (Four fetuses from two litters) in the 1000 mg/kg group with malformations and 41.5% and 43.5% of

fetuses per litter with variations in the same respective groups.

There were no treatment related effects on fetal viability, litter size, sex ratio, or fetal body weights.

Link to relevant study records

Referenceopen allclose all

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
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
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.
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)
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.
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.
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
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
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
Key result
Abnormalities:
no effects observed
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
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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day
Treatment related:
no
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.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar 2010 - Oct 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals or test system and environmental conditions:
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.
Total numbers of animals F0-generation: 48 females.
F1-generation: 559 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 and/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 had been obtained for each dose group, the surplus females were removed from the study.
Randomization Upon detection of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.
Identification By tattoo.
Room temperature 19.5 to 21.0°C.
Room relative humidity 40 to 66%
Ventilation Approximately 15 air changes per hour.
Lighting cycle Fluorescent light for a 12-hour light/12-hour dark cycle
Accommodation
Pre-mating During acclimatization, females were housed in groups of 5 animals/cage in Macrolon cages (MIV type, height 18 cm). The acclimatization period was at least 5 days
prior to pairing under laboratory conditions.
Mating Females were caged together with males on a one-toone-basis in Macrolon cages (MIII type, height 18 cm). During the weekend, mating procedures were suspended and the females were housed in groups of maximum 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Post-coitum Females were individually housed in Macrolon cages (MIII type, height 18 cm).
General Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cageenrichment (Enviro-dri, Wm. Lillico & Son (Wonham
Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. Analysis of the diet did not reveal any findings that were considered to have affected study integrity.
Diet Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Analysis of diet Results of analyses for nutrients and contaminants were examined and archived. Analysis of diet did not reveal any findings that were considered to have affected study integrity.
Water Free access to tap-water.
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration analyzed in the Group 2 formulation was in agreement with the target concentration (i.e. mean accuracies between 90% and 110%). In the Group 1 formulation, a small response at the retention time of the test substance was observed. It was not considered to derive from the formulation since the response was not observed in the duplicate sample.
The formulation of Group 2 was homogenous (i.e. coefficient of variation ≤ 10%).
Details on mating procedure:
Females were caged together with males on a one-toone-basis in Macrolon cages (MIII type, height 18 cm).
During the weekend, mating procedures were suspended and the females were housed in groups of maximum 5 animals/sex/cage in Macrolon cages (MIV type, height
18 cm).
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 and/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 had been 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 28 April 2010
Start pairing 04 May 2010
Start treatment (Day 6 post-coitum) 11 May 2010
Necropsy (Day 20 post-coitum) 25 – 28 May 2010 and
31 May – 03 June 2010
Experimental completion date 03 June 2010 (end of in-life phase)
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
Forty-eight mated female Wistar Han rats were assigned to two dose groups. Groups 1 and 2 each contained twenty-four mated females. The test item was administered once daily by gavage from Days 6 to 19 post-coitum at a dose of 1000 mg/kg/day (Group 2). The rats of the control group received the vehicle, water, alone.
Control animals:
yes, concurrent vehicle
Details on study design:
Forty-eight mated female Wistar Han rats were assigned to two dose groups. Groups 1 and 2 each
contained twenty-four mated females. The test item was administered once daily by gavage from
Days 6 to 19 post-coitum at a dose of 1000 mg/kg/day (Group 2). The rats of the control group
received the vehicle, water, alone.
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.
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 the two groups. 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. 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 and the heads were fixed in Bouin‟s fixative and 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).
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 the two 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 and the heads were fixed in Bouin‟s fixative and 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 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 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 might 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 did include embryonic (early) resorptions, fetal (late) resorptions and dead fetuses.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs indicative of toxicity up to 1000 mg/kg.
Alopecia, an incidental finding, was noted for two females at 1000 mg/kg. 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 between animals at 1000 mg/kg and controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Absolute and relative food consumption was unaffected up to 1000 mg/kg.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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:
not specified
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related macroscopic findings seen up to 1000 mg/kg.
A watery-clear cyst on the right oviduct was noted for a single control animal. As this animal only received the vehicle, it was not related to treatment with Anhydrothymidine.
Two animals at 1000 mg/kg were noted as having alopecia on the forelegs or inguinal region at macroscopic examination; these findings were incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
There were 24 and 23 animals pregnant in the control and 1000 mg/kg groups, 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:
no effects observed
Description (incidence and severity):
There were 24 and 23 animals pregnant in the control and 1000 mg/kg groups, respectively. The numbers of corpora lutea, implantation sites, viable and dead fetuses and early and late resorptions were similar between control and treated animals.
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were 24 and 23 animals pregnant in the control and 1000 mg/kg groups, respectively. 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):
There were 24 and 23 animals pregnant in the control and 1000 mg/kg groups, respectively. 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
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical signs
dead fetuses
early or late resorptions
effects on pregnancy duration
food consumption and compound intake
gross pathology
maternal abnormalities
mortality
number of abortions
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal body weights at 1000 mg/kg remained within the same range as fetuses from the control group. They were 3.4 and 3.5 grams for control and 1000 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 11.8 and 12 fetuses for controls and animals at 1000 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 11.8 and 12 fetuses for controls and animals at 1000 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.
External malformations were seen in 0 and 0.8% of fetuses per litter in Groups 1 and 2, respectively. No external variations were seen for any animal. In this study, two fetuses in the 1000 mg/kg group showed external malformations. Umbilical herniation of the intestine was observed for fetus A025-05 and this finding went together with sternoschisis that was found skeletally. The other fetus, no A039-01, had craniorachischisis (the cranial vault and vertebral column were open through the cervical region) and maxillary micrognathia accompanied by an open and protruding eye (right), and small and malpositioned pinna (right). At visceral
examination the left eye of this fetus appeared to be small. Skeletal findings of this fetus confirmed malformations that were observed externally and viscerally and in addition a bent limb bone (right clavicle) was observed. Because these findings occurred coherently in single fetuses and/or were seen in NOTOX historical controls (craniorachischisis and maxillary micrognathia), they were not considered to be related to the test substance.
No external developmental malformations or variations were observed in any other fetuses in this study.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There was a statistically significant increase in the mean litter proportion of fetuses with total skeletal malformations in the 1000 mg/kg group (2.3% per litter) compared to the control group (0.0% per litter) due to the occurrence of bent limb bones and sternoschisis. Bent limb bones (clavicle, scapula and/or humerus) were observed in 5 fetuses out of 3 litters, while no cases were evident in the concurrent control group. The mean litter proportion of bent limb bones at 1000 mg/kg was 2.0% per litter. Bending of limb bones for fetus no. A039-01 was limited to one clavicle and occurred in conjunction with other malformations of skeletal origin which were not attributed to treatment with the test substance. As such, the bent clavicle was also not considered to be related to the test substance. In 4 fetuses out of 2 litters (nos. A027-01, 03, 14 and A037-13) the scapula and/or humerus were affected; these fetuses also showed bent ribs, reduced ossification of the skull bones and vertebral arches with slight to severe gradations. Recalculation of the mean litter proportion of bent limb bones, excluding bent clavicle, results in a mean litter percentage of 1.5% per litter for bent scapula and/or humerus which is above the historical control data range of 0.0-0.9% per litter. Therefore, a relation between the occurrence of bent scapula and/or humerus and treatment with the test substance could not be excluded.
Sternoschisis was observed for a fetus (no. A025-05) that also had umbilical herniation of intestine and these two coherently occurring malformations in a single fetus were not considered test substance-related.
At 1000 mg/kg, the mean litter proportion of reduced ossification of the skull (19.3% per litter) was significantly increased compared to the control group (10.2% per litter), but remained within the NOTOX historical control data range (5.8-20.1% per litter). In contrast, other major ossification parameters such as cervical centrum no. 1 and sternebrae nos. 5 and 6 were comparable between the control and 1000 mg/kg groups as was the case with mean fetal body weights (3.4 and 3.5 grams in the control and 1000 mg/kg groups, respectively). Therefore, the finding of reduced ossification of the skull at 1000 mg/kg was considered to have arisen by chance, and was not a result of treatment with the test substance.
Other skeletal developmental variations observed in 1000 mg/kg group were 14th rudimentary ribs, bent ribs, reduced ossification of vertebral arches, 7th cervical ribs, 14th full ribs, unossified sternebrae nos. 1, 2, 3 and/or 4, reduced ossification of ribs, 27 presacral vertebrae, slightly to moderately malaligned sternebrae and unco-ossified vertebral centra. All these variations occurred at similar frequencies in the control group, occurred infrequently and/or in a manner that was not dose-related.
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 noted for 2.3 and 0.4, and soft tissue variations were noted for 8.9 and 7.5% of fetuses per litter in the control and 1000 mg/kg groups, respectively. In the 1000 mg/kg group, a small eye was observed for the fetus with craniorachischisis, no. A039-01 (also see paragraph 6.4.1) which was not considered to be test substance related due to the single occurrence and its relation with craniorachischisis.
Four control fetuses in this study had visceral malformations. Fetus no. A020-09 had abnormal lobation of the liver and lung and fetus no. A022-04 showed situs inversus (all thoracic and abdominal organs were laterally transposed). Cephalic examination revealed two fetuses (nos. A010-04 and -06) in one litter with external hydrocephaly (with an increased space between the brain and skull). No other visceral malformations were observed in this study.
Small supernumerary liver lobes were noted at 1000 mg/kg at a frequency similar to that in the control group; thus, this finding was not regarded as a test substance effect. Other soft tissue developmental variations that were noted in this study were pale spleen, extra papillary muscle and an accessory blood vessel. These variations occurred infrequently and no relation with treatment with the test substance could be established.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: clavicle
skeletal: scapule
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: vertebra
skeletal: hindlimb
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Developmental findings

At 1000 mg/kg an increased incidence in the mean litter proportion of fetuses with skeletal malformations, characterized by bent limb bones including the humerus and scapula was seen. No toxicologically significant findings were noted for external or visceral malformations or variations.

Fetuses from the control and 1000 mg/kg groups had mean litter proportions of external malformations at 0.0% and 0.8%, respectively. No external variations were seen. Visceral malformations were observed in 2.3% and 0.4% of fetuses per litter, and visceral variations were observed in 8.9% and 7.5% of fetuses per litter in the control and 1000 mg/kg groups, respectively. Skeletal examination revealed 0.0% of controls and 2.3% of fetuses per litter (Four fetuses from two litters) in the 1000 mg/kg group with malformations and 41.5% and 43.5% of fetuses per litter with variations in the same respective groups.

There were no treatment related effects on fetal viability, litter size, sex ratio, or fetal body weights.

Conclusions:
Based on the results in this prenatal developmental toxicity study the maternal No. Observed Adverse Effect Level (NOAEL) for Anhydrothymidine was established as being 1000
mg/kg body weight/day and the developmental NOAEL could not be determined based on the skeletal malformations (bent limb bones) seen at 1000 mg/kg.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results in prenatal developmental toxicity study no 495576 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.

Additional information