Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January - March 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

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)
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
429-320-2
EC Name:
-
Cas Number:
24748-23-0
Molecular formula:
C12H24O6
IUPAC Name:
3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexaoxonane
Constituent 2
Reference substance name:
Trigonox 301
IUPAC Name:
Trigonox 301
Details on test material:
Batch no.: DVT 0706516079 (see CoA attached)
Storage: at room T in the dark
Expiry date: 15 August 2008

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered
to be approximately 15 air changes per hour, a temperature of 21 ± 3°C (actual range: 20.0-
21.8°C), a relative humidity of 30 - 70% (actual range: 39 - 64%) and 12 hours artificial light and
12 hours darkness per day.
Accommodation
Females were individually housed in labelled cages with perforated floors (Techniplast, Italy,
dimensions 75 x 70 x 45 cm, or Ebeco, Germany, dimensions 67 x 62 x 55 cm).
Diet
Free access to pelleted diet for rabbits (K-H from SSNIFF® Spezialdiaten GmbH, Soest,
Germany). Results of analyses for nutrients and contaminants of each batch were examined
and archived. Copies of the certificates are enclosed in Appendix 10 of this report.
In addition, hay (Tecnilab-BMI BV, Someren,the Netherlands) was provided three times a week.
Water
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and
archived.
Analysis of diet and water did not reveal any findings that were considered to have affected
study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Oral gavage, using a plastic catheter attached to a plastic
disposable syringe.
Formulations were placed on a magnetic stirrer during dosing.
Once daily for 7 days per week, approximately the same time each
day with a maximum of 4 hours difference between the earliest and
latest dose.
Female 50 was not dosed on Day 21 post-coitum as this animal
showed rapid breathing on this day. Females 76 and 77 were not
dosed on Day 26 post-coitum due to bad health. Female 48 was
not dosed on Day 22 post-coitum as this animal showed rapid
breathing.
1 mL/kg body weight. Actual dose volumes were calculated
according to the latest body weight.
From day 7 to day 28 post-coitum, inclusive.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC-MS
Details on mating procedure:
Artificial insemination (number of donor bucks not indicated)
Duration of treatment / exposure:
Day 7 up to and including Day 28 post coitum (day of insemination = Day 0)
Frequency of treatment:
Daily
Duration of test:
Animals were killed on Day 29
No. of animals per sex per dose:
12 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
Four groups of twenty-three (control group) or twenty-four (treatment groups) New Zealand White rabbits were inseminated (Day 0 post-coitum) and exposed by oral gavage to 0, 30, 100 and 300 mg/kg TRIGONOX 301 from Day 7 to 28 post-coitum. Groups 1, 2, 3 and 4 consisted of 17,19,22 and 18 pregnant animals, respectively. The numbers of litters available for morphological evaluation were 16, 19, 20 and 12 in the control, 30, 100 and 300 mg/kg/day, groups respectively. Females were checked daily for the presence of clinical signs. Body weight and food consumption of females was determined at periodic intervals and from February 15th onwards, water consumption was measured daily. Formulation analysis was performed on prepared dosing solutions. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The ovaries and uterine horns were dissected and examined for the number of corpora lutea, the weight of the gravid uterus, the number and distribution of live/dead fetuses and embryo-fetal deaths, the weight of each fetus, fetal sex and externally visible fetal macroscopic abnormalities. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin' fixative and subsequently sliced, 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. Analyzes demonstrated accuracy, homogeneity and stability of dose preparations.

Examinations

Maternal examinations:
Mortality I Viability: At least twice daily. Animals showing pain, distress or discomfort,
which was considered not transient in nature or was likely to
become more severe, were sacrificed for humane reasons based
on OECD guidance document on humane endpoints
(ENV/JM/MONOI (2000)7). The time of death was recorded as
precisely as possible.

Clinical signs: At least once daily from day 0 post-coitum onwards. The time of
onset, degree and duration were 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
A different project number (NOTOX Project 486690) was used for
recording of clinical signs.
Cage debris was examined to detect abortion or premature birth.

Body weights: Days 0, 4 and 7-29 (daily) post-coitum.

Food consumption: Days 0-4, 4-7,7-10, 10-13, 13-16, 16-20,20-23,23-26 and 26-29
post-coitum.

Water consumption: From study initiation, water consumption was visually assessed.
From 15 February 2008 onwards, water consumption was
determined for all animals due to a possible treatment related
effect on water consumption.

Necropsy: All animals were subjected to an examination post-mortem. All animals surviving to day 29 postcoitum,
all moribund animals and all animals showing abortion or premature delivery were
euthanised by intravenous injection of pentobarbital (approx. 1 ml/kg Euthesate®; Ceva Sante
Animale BV, Maassluis, The Netherlands) immediately prior to necropsy.

Organ weights: The weight of the liver, kidneys and terminal body weight were recorded from all surviving
females on the scheduled day of necropsy. A different project number (NOTOX Project 486690)
was used for recording of organ weights.

Histopathology: The following slides were examined by a pathologist:
The liver and kidneys of the first ten pregnant females of Groups 1 and 4.
The liver of Female 58 (Group 3), as this female showed accentuated lobular pattern of the
liver at macroscopic examination (this finding was also noted for five animals of the high
dose group).
Due to treatment related changes in the liver at the high dose group, the liver of the first ten
pregnant females of Groups 2 and 3 were examined.
All abnormalities.
All abnormalities were described and included in the report. An attempt was made to correlate
gross observations with microscoRic findings.

Ovaries and uterine content:
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 (ovaries in situ)
- The weight of the gravid uterus
- The number and distribution of live and dead foetuses
- The number and distribution of embryo-foetal deaths
- The number of former implantation sites
- The weight of each live foetus
- The sex of each foetus (during further foetal examination)
- Externally visible macroscopic foetal abnormalities.
Fetal examinations:
External, visceral and skeletal foetal findings were recorded as developmental variations or
malformations. See Appendix 3 for the details of all foetal examination methods.
External:
Each viable foetus was examined in detail and weighed. All live foetuses were euthanised by
subcutaneous injection of 0.1 ml pentobarbital (Euthesate®; Ceva Sante Animale BV,
Maassluis, The Netherlands) in the area between the scapulas. The crown-rump length of late
resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a
gross external examination performed (if possible) and the tissue was discarded.
Visceral (Internal):
Foetuses were 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 foetuses was determined by internal examination.
The heads were removed from approximately one-half of the foetuses in each litter and placed
in Bouin's solution (Klinipath, Duiven, The Netherlands) for subsequent processing and softtissue
examination using the Wilson sectioning technique (Wilson, 1965). After examination, the
tissues were stored in neutral phosphate buffered 4% formaldehyde solution (Klinipath, Duiven,
The Netherlands). The heads from the remaining one-half of the foetuses in each litter were
examined by a mid-coronal slice.
All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in
identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for
subsequent examination of skeletons.
Skeletal:
Each eviscerated foetus, following fixation in alcohol, was macerated in potassium hydroxide
(Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The
Netherlands) by a method similar tothat described by Dawson (Dawson, 1926). The skeletal
examination was made following this procedure. 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.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of
significance.

The following interpretation was performed for fetal pathology data:
All statistical tests were performed using appropriate computing device programs. Analyses
were conducted using two-tailed tests (except as noted otherwise) for minimum significance
levels of 1 % and 5%, comparing each compound-treated group to the control group. Each mean
was presented with the standard deviation (S.D.) and the number of animals (N) used to
calculate the mean. Due to the different rounding conventions inherent in the types of software
used, the means and standard deviations on the summary and individual tables may differ by ±
1 in the last significant figure. Where applicable, the litter was used as the experimental unit.
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.
Indices:
Pre-implantation loss (Number of corpora lutea - number of implantation sites) x 100
Number of corpora lutea
Post-implantation loss (Number of implantation sites - number of live foetuses) x 100
Number of implantation sites
The following calculations were performed for fetal developmental findings:
The fetal developmental findings were summarized by: 1) presenting the incidence of a given
finding both as the number of fetuses and the number of litters available for examination in the
group; and 2) considering the litter as the basic unit for comparison and calculating the number
of affected fetuses in a litter on a proportional basis

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At the high dose group (300 mg/kg body weight/day), severe toxicity was noted. Eight rabbits were killed in extremis before planned necropsy. Six of these animals showed an early delivery. All females showed clinical signs, severe body weight loss, and reduced food consumption and water consumption. One additional female of the high dose group showed an early delivery on the day of planned necropsy. Clinical signs observed at the high dose group consisted of lethargy, hunched posture, slow breathing, pale appearance, piloerection, brown discolouration of the urine, pale faeces, reduced faeces production, lean appearance and hypothermia. Animals treated at 300 mg/kg showed severe body weight loss, severely reduced food consumption, and reduced water consumption.
Treatment related macroscopic findings for the liver and gallbladder were observed at 300 mg/kg. Six animals were emaciated at necropsy. Liver findings consisted of accentuated lobular pattern, enlargement, hardening and black-brown discoiouration. Gallbladder findings consisted of black discoloured contents and a gelatinous gallbladder. Increased liver weights (absolute and relative) and kidneys weights (relative) were noted at the high dose group. Histopathological examination revealed severely affected livers with hypertrophic hepatocytic changes and proliferative bile channel changes.
At the intermediate dose group (100 mg/kg body weight/day), similar but less severe effects on the liver were noted. These consisted of accentuated lobular pattern for one animal and minimal centriacinar hepatocytic hypertrophy for three animals.
No maternal toxicity was observed at the low dose group (30 mg/kg);

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
30 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:
No effects on pre- and post implantation loss, litter size and sex ratio were noted. Significantly reduced body weights of fetuses were noted at the high dose group (300 mg/kg). This was considered to be related to the decreased maternal body weight at this dose level. An increase in the mean litter proportion of fetuses with unossified metacarpais (statistically significant) and unossified sternebrae nos. 5 and/or 6 were noted at a dose level of 300 mg/kg/day. This delayed ossification, however, was considered secondary to the fetal body weight reduction noted at this dose level.
There were no test substance related effects on fetal morphology in the 30 and 100 mg/kg/day group.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

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 TRIGONOX 301 was established as being 30 mg/kg body weight/day. The NOAEL for embryo/fetal developmental toxicity, which was considered to be secondary to the documented maternal toxicity, was 100 mg/kg body weight/day.
Executive summary:

Four groups of twenty-three (control group) or twenty-four (treatment groups) New Zealand White rabbits were inseminated (Day 0 post-coitum) and exposed by oral gavage to 0, 30, 100 and 300 mg/kg TRIGONOX 301 from Day 7 to 28 post-coitum. Groups 1, 2, 3 and 4 consisted of 17,19,22 and 18 pregnant animals, respectively. The numbers of litters available for morphological evaluation were 16, 19, 20 and 12 in the control, 30, 100 and 300 mg/kg/day, groups respectively. Females were checked daily for the presence of clinical signs. Body weight and food consumption of females was determined at periodic intervals and from February 15th onwards, water consumption was measured daily. Formulation analysis was performed on prepared dosing solutions. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The ovaries and uterine horns were dissected and examined for the number of corpora lutea, the weight of the gravid uterus, the number and distribution of live/dead fetuses and embryo-fetal deaths, the weight of each fetus, fetal sex and externally visible fetal macroscopic abnormalities. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin' fixative and subsequently sliced, 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. Analyzes demonstrated accuracy, homogeneity and stability of dose preparations.

Maternal findings At the high dose group (300 mg/kg body weight/day), severe toxicity was noted. Eight rabbits were killed in extremis before planned necropsy. Six of these animals showed an early delivery. All females showed clinical signs, severe body weight loss, and reduced food consumption and water consumption. One additional female of the high dose group showed an early delivery on the day of planned necropsy. Clinical signs observed at the high dose group consisted of lethargy, hunched posture, slow breathing, pale appearance, piloerection, brown discolouration of the urine, pale faeces, reduced faeces production, lean appearance and hypothermia. Animals treated at 300 mg/kg showed severe body weight loss, severely reduced food consumption, and reduced water consumption. Treatment related macroscopic findings for the liver and gallbladder were observed at 300 mg/kg. Six animals were emaciated at necropsy. Liver findings consisted of accentuated lobular pattern, enlargement, hardening and black-brown discolouration. Gallbladder findings consisted of black discoloured contents and a gelatinous gallbladder. Increased liver weights (absolute and relative) and kidneys weights (relative) were noted at the high dose group. Histopathological examination revealed severely affected livers with hypertrophic hepatocytic changes and proliferative bile channel changes. At the intermediate dose group (100 mg/kg body weight/day), similar but less severe effects on the liver were noted. These consisted of accentuated lobular pattern for one animal and minimal centriacinar hepatocytic hypertrophy for three animals . ................... No maternal toxicity was observed at the low dose group (30 mg/kg). Developmental findings No effects on pre- and post implantation loss, litter size and sex ratio were noted. Significantly reduced body weights of fetuses were noted at the high dose group (300 mg/kg). This was considered to be related to the decreased maternal body weight at this dose level.

An increase in the mean litter proportion of fetuses with unossified metacarpals (statistically significant) and unossified sternebrae nos. 5 and/or 6 was noted at a dose level of 300 mg/kg/day. This delayed ossification, however, were considered secondary to the fetal body weight reduction noted at this dose level. There were no test substance related effects on fetal morphology in the 30 and 100 mg/kg/day group.