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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 January 2009 - 13 February 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
Details on test material:
Identification: XTJ-568 dihydrochloride
Molecular formule: C10H26Cl2N2O2 (component A) C14H34Cl2N2O3 (component B)
Molecular weight: 277.23 (component A) 349.34 (component B)
Description: Clear yellow liquid (determined at NOTOX)
Batch: 8666-080-C
Purity: ~ 100%
Composition: This product consists essentially of two components, derived from the reaction of ethylene glycol and butylene oxide in ratios of 2/1 and 3/1. The 2/1 adduct (component A) is approximately 80% of the mixture and the 3/1 adduct (component B) is approximately 20%.
The concentration of the salt in this solution is 70.5%.
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 15 June 2009
Specific details on test material used for the study:
Identification: XTJ-568 dihydrochloride
Molecular formule: C10H26Cl2N2O2 (component A) C14H34Cl2N2O3 (component B)
Molecular weight: 277.23 (component A) 349.34 (component B)
Description: Clear yellow liquid (determined at NOTOX)
Batch: 8666-080-C
Purity: ~ 100%
Composition: This product consists essentially of two components, derived from the reaction of ethylene glycol and butylene oxide in ratios of 2/1 and 3/1. The 2/1 adduct (component A) is approximately 80% of the mixture and the 3/1 adduct (component B) is approximately 20%.
The concentration of the salt in this solution is 70.5%.
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 15 June 2009

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Test System: Rat Crl:WI(Han) (outbred, SPF-Quality).
Source: Charles River, Sulzfeld, Germany.

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.

Age at start of pairing: approximately 12 weeks.

Conditions
A controlled environment was maintained in the room with optimal conditions of approximately 15 air changes per hour, a temperature of 21±3ºC (actual range: 18.6-22.8ºC ), a relative humidity of 30-70% (actual range: 22-66%) and a 12 hour light/12 hour dark cycle. Based on laboratory historical data these fluctuations were considered not to affect the study integrity.

Accommodation
Pre-mating
During acclimatization, females were housed in groups of 5 animals/cage in Macrolon cages (MIV type, height 18 cm). Acclimatization period was at least 5 days prior to pairing under laboratory conditions.

Mating
Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm). During the weekend, mating procedures were suspended and the animals 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).

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.

General
Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied. Certificates of analysis were examined and then retained in the NOTOX archives.

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.

Water
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.

Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Vehicle:
Water (Elix, Millipore S.A.S., Molsheim, France)
Rationale for vehicle: Based on information provided by the sponsor and trial formulations performed at NOTOX.
Stability of test substance in vehicle: At least 8 days, if stored in the refrigerator (determined as part of NOTOX Project 489273).
Method of formulation: Formulations (w/w) were prepared weekly and were homogenised to a visually acceptable level. Adjustment was made for density of the test substance (1.106 g/mL).
Storage conditions: In the refrigerator.

Dose levels:
0, 150, 450, 1000 mg/kg/day

Dose volume:
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Concentrations of formulations:
0, 30, 90 and 200 mg/mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were done on one day during the treatment period according to a validated method (NOTOX Project 489272) as specified below.

Group Analysis (type of sample)

1 acc (M)
2 acc + hom (TMB)
3 acc (M)
4 acc + hom (TMB)

Duplicate samples were analyzed
acc=accuracy, hom=homogeneity, T=top, M=middle, B=bottom position of container

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was 10%.
Results:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation. The maximum contribution based on area to the Group 2 samples was 1.2%. The source for the response in Group 1 is probably due to carry-over of the analytical method, as no analytical column is used. Due to the nature of the test substance, this could not be circumvented.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation 10%).
Details on mating procedure:
Age at start of pairing: approximately 12 weeks.
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 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:
20 days
No. of animals per sex per dose:
24 females/sex/dose
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Mortality / Viability
At least twice daily.

Clinical signs
At least once daily from Day 0 post-coitum onwards. The time of onset, degree and duration was recorded. All symptoms were graded according to fixed scales: Maximum grade 1: grade 0 = absent, grade 1 = present.
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females.

Body weights
Days 0, 3 and 6-20 (daily) post-coitum.

Food consumption
Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

Water consumption
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
Ovaries and uterine content:
All animals were subjected to an examination post-mortem. All animals surviving to Day 20 post-coitum were sacrificed using an oxygen/carbon dioxide procedure. External, thoracic and abdominal examinations were performed for the detection of macroscopic abnormalities. All abnormalities were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the gravid uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy)
and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.

In case a female was not pregnant, the uterus was stained using the Salewski technique (Salewski, 1964) in order to determine any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

The female genital tract including the placentas was preserved in 10% buffered formalin for possible histopathological examination.

Fetal examinations:
External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.

External:
Each viable fetus was examined in detail, sexed and weighed. All live fetuses were euthanized by subcutaneous injection of 0.01 mL pentobarbital (Euthesate®; Ceva Sante Animale B.V., Maassluis, The Netherlands for necropsies before 01 February 2009; Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands for necropsies from 01 February 2009 onwards) in the area between the scapulas. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined, crown-rump length measured, weighed and sexed. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed and the tissue was discarded.

Subsequently, each litter was divided into two groups:
- Approximately one half of the fetuses in each litter was examined for visceral anomalies (for details see below).
- The remaining half of the fetuses in each litter was used for skeletal examination (for details see below).

Visceral (Internal):
Approximately one-half of the fetuses in each litter was examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Stuckhardt, 1984). This examination included the heart and major vessels. The sex of all fetuses was confirmed by internal examination.

The heads of these fetuses were removed and placed in Bouin's solution (Klinipath, Duiven, The Netherlands) for subsequent processing and soft-tissue examination using the Wilson sectioning technique (Wilson, 1965). After examination, the tissues were stored in 10% buffered formaline.

The carcasses were eviscerated and stored in identified containers containing 96% aqueous alcohol (Klinipath, Duiven, The Netherlands) without further examinations.

Skeletal:
Following confirmation of the sex by internal examination, the carcasses of the remaining half of the fetuses in each litter were eviscerated and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.

Following fixation in alcohol, all fetuses were macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson (Dawson, 1926). For the selected fetuses, the skeletal examination was performed. The specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Merck, Darmstadt, Germany) as preservative.
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 (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) 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 (Kruskal and Wallis 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn¿s test (Dunn 1964) 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.
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.
- Post-implantation loss included embryonic (early) resorptions, fetal (late) resorptions and dead fetuses.

Historical control data:
Yes, available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, slight body weight loss occurred during the first two days of treatment. Lower body weights and body weight gain as compared to controls were noted from the second day of treatment onwards. In addition, (for uterus weight) corrected body weight gain was statistically significantly decreased for the high dose group as compared to control animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, a lower food intake (before and after allowance for body weight) as compared to the control group was recorded during the whole treatment period (not always statistically significant). Effects were most pronounced in the first week of treatment.
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 examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
450 mg/kg bw/day
Basis for effect level:
body weight and weight gain
other: based on reduced body weights and body weight gain of the high dose animals at 1000 mg/kg/day
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
> 1 000 mg/kg bw/day
Basis for effect level:
other: In the absence of treatment-related effects on reproductive parameters, fetal body weight and fetal morphology, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for developmental toxicity.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
not specified

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks:
embryotoxicity
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study, the maternal No Observed Adverse Effect Level (NOAEL) for the test substance was established as being 450 mg/kg/day, based on reduced body weights and body weight gain of the high dose animals at 1000 mg/kg/day. In the absence of treatment-related effects on reproductive parameters, fetal body weight and fetal morphology, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for developmental toxicity.