Registration Dossier

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:
18 February 2015 - 30 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Storage condition of test material: At room temperature protected from light

Test animals

Species:
rabbit
Strain:
other: Charles River, Chatillon sur Chalaronne, France
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Age at delivery: 17-19 weeks
- Weight at study initiation (Day 0 post coitum): 2995 to 4574 gram
- Fasting period before study: no
- Housing: Females were individually housed in labelled cages with perforated floors (Ebeco, Germany) and shelters (Ebeco, Germany).
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Italy). In addition, pressed hay (Tecnilab-BMI bv, The Netherlands) and wooden sticks (Swedish aspen wood, The Netherla nds) were provided during the study period.
- Water: Free access to tap-water - Acclimation period: At least 5 days prior to pairing

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

IN-LIFE DATES:
From: 27 March 2015 To: 02 May 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% Aqueous carboxymethyl cellulose with 0.1% Tween-80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visuall y acceptable level. Adjustment was made for specific gravity of the test substance. No correction was made for the purity/composition of the test substance.

VEHICLE - Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch. 1% Aqueous carboxymethyl cellulose (carboxymethyl cellulose: BUFA, The Netherlands; water: Elix, Millipore S.A.S., France) with 0.1% Tween-80 (Merck-Schuchardt, Germany). - Amount of vehicle (if gavage): 2 mL/kg bw/day


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The delegated phase was performed by the Principal Investigator for Formulation Analysis (ABL). Samp les for analyses were taken in Study weeks 1 and 4. The samples were dispatched on dry ice to ABL w here they were analyzed to assess accuracy of preparation (all groups) and homogeneity (Group 2, 4 and 7). In addition, stability in vehicle over 5 hours at room temperature protected from light was determined on samples taken on 08 April 2015 (Group 2 and Group 4) and taken on 05 April 2016 (Group 7).
Details on mating procedure:
- Impregnation procedure: cohoused One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was est ablished by visual observation of mating. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
From Days 6 to 28 post-coitum, inclusive
Frequency of treatment:
Once daily for 7 days/week, approximately the same time each day with a maximum of 6 hours differe nce between the earliest and latest dose.
Duration of test:
Until Day 29 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
130 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
No. of animals per sex per dose 22 females/dose in groups 1, 2, 3, 6 and 7 (0, 10, 30, 130 and 0 mg/kg bw/day)

23 females/dose in group 4 (100 mg/kg b/wday). One female from this group died on the first treatment day due to an oral gavage accident. To compensate for her loss, a spare female was mater and added to group 4).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Groups 1-4 (0, 10, 30 and 100 mg/kg bw/day) Dose levels were based on a range finding study in which mated rabbits were exposed by oral gavage from Day 6 to Day 28 post-coitum, inclusive, at dose levels of 0, 50, 100 and 200 mg/kg bw/day. Clear toxicity was observed at 200 mg/kg bw/day. One female had to be euthanized on Day 16 and another on Day 23 post-coitum due to severe toxicity. Additional toxicologically relevant findings in this group co nsisted of clinical signs (lethargy, pale and lean appearance, piloerection and/or hunched posture), signifi cant body weight loss and reduced food consumption. No macroscopic abnormalities were noted at necr opsy. At 100 mg/kg bw/day, there was only a single female with signs of toxicity. These included a calm and lean appearance on Day 27 post-coitum, severely reduced food consumption from Days 13-16 and 23-26 post-coitum, and body weight loss (-11.3% after correction for gravid uterine weight). At necropsy, no macroscopic abnormalities were noted for the 100 mg/kg bw/day group. There was no toxicity at 50 mg/kg bw/day.
There were 2-5-6-5 pregnant females in the control, 50, 100, and 200 mg/kg bw/day groups, respectivel y. Since 2 females had to be euthanized preterm in the high dose group, only 3 litters were available for evaluation in this group. The only finding was a trend towards a lower mean fetal body weight (-17% as compared to the concurrent control group). There were no other developmental findings at either dose level. No external developmental malformations or variations were noted.

Groups 6 and 7 (0 and 130 mg/kg bw/day) Dose levels were based on the results from the additional group added to the dose range finding study. At 150 mg/kg bw/day, faeces abnormalities, body weight loss, reduced food and/or water consumption were noted. In addition, one female had an early delivery. Based on these results, a dose of 150 mg/kg bw/day was judged too high. Therefore, it was decided in consultation with the Sponsor to select a dose of 130 mg/kg bw/day for the additional Group 7.

NB. Please note that for computer technical reasons, the two additional groups had to be planned into the computer as Groups 6 and 7. No Group 5 was included in this study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
-Time schedule for examination: Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 postcoitum.

WATER CONSUMPTION : Yes
Subjective appraisal was maintained during the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: liver sampling for groups 130 mg/kg bw/day and concurrent control group on Day 29 post-coitum

OTHER: Yes
-Blood sampling for groups 130 mg/kg bw/day and concurrent control group on Day 28 post-coitum (last day of dosing).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter from dose groups 100 and 130 mg/kg bw/day and control groups
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test)
based on a pooled variance estimate was applied for the comparison of the treated groups and the co
ntrol group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal
distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of
viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and
sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations
(external, visceral and skeletal), and each particular external, visceral and skeletal malformation or vari
ation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differe
nces. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used
to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead
fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
For each litter the following calculations were performed:
Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites) / number of corpora lutea) x 100
Post-implantation loss (%) = ((number of implantation sites - number of live fetuses) / number of implantation sites) x 100
Viable fetuses affected/litter (%) = (number of viable fetuses affected/litter / number of viable fetuses/litter) x 100
Historical control data:
Historical control data included for Study: date range 2011-2015

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Description (incidence and severity)
None of the clinical signs seen for surviving animals in this study were considered to be adverse.
Reduced faeces production, varying in duration and severity, was observed in females of both treated and
control groups. The incidence was slightly higher at 100 and 130 mg/kg bw/day. The number of females
with reduced faeces production was 13 and 14 females in the two control groups, 13 females at 10 mg/
kg bw/day, 14 females at 30 mg/kg bw/day, 18 females at 100 mg/kg bw/day and 19 females at 130 mg/
kg bw/day. For females with a more severely reduced faeces production (moderate to severe) also often
reduced intake of food and water was noted.
Incidental findings noted for control and/or treated animals included pale feces, diarrhea, thickened area
of the throat region, broken teeth, a lean or pale appearance, piloerection, a hunched posture, alopecia,
scars, swelling, scabbing or a wound on various body areas.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were 5 unscheduled deaths, though none of these were attributable to treatment related toxicity.
One control female was euthanized on Day 18 post-coitum due to a poor condition. She was noted with
lethargy, pale and lean appearance, piloerection and reduced feces production prior to euthanasia. From
start of treatment (Day 6 post-coitum) she had lost 18% of her weight, which was reflected in the finding
of emaciation at the macroscopic examination. As this was a control female, this death was not treatment
related.
Three females died due to complications during dosing: one female from dose group 30 mg/kg bw/day
was found dead on Day 27 post-coitum, and two females from dose group 100 mg/kg bw/day were found
dead on Days 6 and 22 post-coitum, respectively. Two of these animals had labored respiration during or
immediately after the dosing procedure. When found dead, one female was noted with blood around her
mouth and nose, and had reduced feces production from Day 19 post-coitum onwards. At necropsy, the t
wo females from dose group 100 mg/kg bw/day were found with hemorrhagic/clotted blood and foamy co
ntents in the trachea. They also had grey-white fluid in the lungs and reddish and/or grey-white discolorati
on of the lungs. There were no macroscopic findings seen for animal from dose group 30 mg/kg bw/day.
As one female (100 mg/kg bw/day) died very early in the study (Day 6 post-coitum) she could be
replaced by a reserve animal. However, this latter female was found dead on the morning of Day 21 postcoitum.
Most likely she aborted during the night and died from associated complications. Her cage was
covered with blood, but no uterine contents or fetuses were found. She had 13 fetuses in her uterus who
all were dead. There were no further necropsy findings. This female did not consume any food on Day
20 post-coitum. Throughout treatment, however, she had no clinical signs and her body weights, weight
gain, and food consumption were normal. Taken together, her death was considered to be secondary to
complications during the abortion and was not directly attributable to treatment with the test substance
itself. At this single occurrence it was considered a chance finding and thus not related to treatment with
TMPTA.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 130 mg/kg/day, slightly reduced body weight gains were noted from Days 16-29 post-coitum onwards
compared to the concurrent control group (not statistically significant on Day 29 post-coitum). Body
weight gain corrected for weight of the gravid uterus was slightly lower in this group compared to the con
current controls (-6.8% versus -6.2%; not reaching statistical significance). Because of the slight and nonsignificant
difference, this finding was not considered as adverse.
There were no toxicologically relevant effects on body weights or weight gains with treatment up to and
including 100 mg/kg bw/day.
A trend towards slightly lower body weight gains was noted for females at 100 mg/kg bw/day from Day
16 post-coitum onwards, though the difference from controls was not statistically significant. Females at
30 and 10 mg/kg bw/day also had lower body weight gains from Day 20 post-coitum onwards, though the
differences from controls were slight and not statistically significant. Terminal body weight corrected for
(gravid) uterine weight was unaffected by treatment up to and including 100 mg/kg bw/day.
A slightly, but statistically significantly lower body weight gain was recorded for the 10 mg/kg/day group
as compared to its concurrent control (Group 1) on Day 0 post-coitum. This was by no mean related to
the test item, since treatment did not start before Day 6 post-coitum .
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 130 mg/kg bw/day, there was a trend towards slightly lower food consumption (absolute and relative
to body weight) from Days 13-29 post-coitum onwards compared to the concurrent control group, but not
reaching statistical significance. This finding was considered treatment-related, but not adverse as the
differences from controls were slight (reaching no statistical significance) and transient.
There were no toxicologically relevant effects on food consumption noted with treatment up to 100 mg/kg/
day.
At 100 mg/kg bw/day, there was a trend towards lower food consumption (absolute and relative to body
weight) from Days 13-23 post-coitum. Differences from controls were relatively slight, reaching statistical
significance for relative food consumption from Days 16-20 post-coitum only. In addition, statistically
significantly reduced food consumption was noted at 10 mg/kg bw/day from Days 3-9 post-coitum
(absolute and relative to body weight). As the differences from controls were transient and/or occurred in
the absence of a dose-related trend, these were not considered to be toxicologically relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
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
Description (incidence and severity):
There were no toxicologically relevant macroscopic findings seen at the scheduled necropsy with trea
tment up to and including 130 mg/kg bw/day. One female at this dose level was noted emaciated. However,
when correcting her terminal body weight for gravid uterus weight it was almost the same (- 0.2%) as
at start of treatment (Day 6 post-coitum). Moreover, also one control female in Group 1 was noted
emaciated at necropsy. Taken together, this necropsy finding was not considered to be related to treatment.
Incidental macroscopic findings noted for control and/or treated animals were within the background
range of findings that are encountered among rabbits of this age and strain, were observed for individual
animals only and did not show a dose-related trend. These necropsy findings were therefore considered
to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One 100 mg/kg bw/day treated female was found dead on Day 21 post-coitum most likely due to complications caused by abortion.
Thirteen dead fetuses were found in her uterus.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A significant increase in pre-implantation loss was found in the 130 mg/kg bw/day group compared to its
concurrent control. As treatment is started after implantation, this is in no way related to treatment. In the
same group, the slightly higher post-implantation loss compared to the concurrent control (9.2 vs 3.6%
per litter, respectively) could be contributed to one female in this group with 100% of late resorptions
only. This female had only 3 implantation sites. When excluding her, the post-implantation loss per litter
was within the normal range (i.e. 4.9% per litter).
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
One female in the 130 mg/kg bw/day showed 100% of late resorptions, which is considered to be incide
ntal (within the range of normal biological variation).
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
At 130 mg/kg bw/day, one female had 3 late resorptions
Dead fetuses:
no effects observed
Description (incidence and severity):
One dead fetus was found in Group 1. As this group was treated with the vehicle alone, this finding was
by no means related to treatment with TMPTA.
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:
effects observed, non-treatment-related
Description (incidence and severity):
There was one non-pregnant female in the first control group and the 30 and 100 mg/kg bw/day groups.
In the second control group (concurrent control for dose group at 130 mg/kg bw/day), 4 females were
found non-pregnant. At 130 mg/kg bw/day, one female had 3 late resorptions only. All remaining females
were pregnant with viable fetuses at Day 29 post-coitum, but one female from the first control group
was euthanized, and one female at 30 mg/kg bw/day and three females at 100 mg/kg bw/day (including
the one with abortion mentioned above) were found dead before scheduled necropsy on Day 29 post-coit
um. Consequently, the number of litters with viable fetuses was 20, 22, 20 and 19 in the first control group
and the 10, 30 and 100 mg/kg bw/day groups, respectively; and 21 and 18 litters in the 130 mg/kg bw/ day
group and its concurrent control, respectively.
Other effects:
not examined
Details on maternal toxic effects:
No relevant maternal toxicity was observed up to and including 130 mg/kg bw/day. There was no test-i
tem related mortality following treatment up to and including 130 mg/kg bw/day.
Reduced faeces production, varying in duration and severity, was observed in females of both treated and
control groups. The incidence was slightly higher at 100 and 130 mg/kg bw/day.
There was a trend towards slightly lower food consumption (absolute and relative to body weight) from
Days 13-23 post-coitum in the 100 mg/kg bw/day group and from Days 13-29 post-coitum in the 130 mg/
kg bw/day, which resulted in slightly reduced body weight gains from Days 16-23 post-coitum and Days
16-29 post-coitum, respectively, compared to the concurrent control group. As these changes in food
consumption and body weight were relatively slight (not always reaching statistical significance), they
were considered as non-adverse.
No test-item related macroscopic abnormalities were noted following treatment up to and including 130
mg/kg bw/day.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 130 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 130 mg/kg bw/day, a trend towards slightly lower mean fetal body weights (male, female and combin
ed) was noted when compared to its concurrent control group. Mean combined fetal body weights were
37.8 and 39.3 grams, respectively. This change, however, did not reach statistical significance and
although low, values remained within the available historical control range .
There were also no toxicologically relevant changes in fetal body weights at the lower dose levels tested
(10, 30 and 100 mg/kg bw/day). Mean fetal body weights in females, but not males were slightly lower
at 100 mg/kg/day as compared to controls (not statistically significant). Mean fetal body weights (both
sexes combined) were 41.0, 40.0, 40.1 and 40.1 grams in the first control, 10, 30 and 100 mg/kg bw/day
group, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 130 mg/kg bw/day, a trend towards slightly lower mean fetal body weights (male, female and combin
ed) was noted when compared to its concurrent control group. Mean combined fetal body weights were
37.8 and 39.3 grams, respectively. This change, however, did not reach statistical significance and
although low, values remained within the available historical control range .
There were also no toxicologically relevant changes in fetal body weights at the lower dose levels tested
(10, 30 and 100 mg/kg bw/day). Mean fetal body weights in females, but not males were slightly lower
at 100 mg/kg/day as compared to controls (not statistically significant). Mean fetal body weights (both
sexes combined) were 41.0, 40.0, 40.1 and 40.1 grams in the first control, 10, 30 and 100 mg/kg bw/day
group, respectively.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Litter sizes were not affected by treatment up to and including 130 mg/kg bw/day. Mean litter sizes were
8.7, 7.3, 8.5 and 8.1 for the first control, 10, 30 and 100 mg/kg bw/day group, respectively. The mean
litter size in the 130 mg/kg bw/day group was significantly lower compared to its concurrent control group
(8.0 versus 9.4 fetuses/litter), but remained within the normal range of biological variation.
One dead fetus was found in the control group. As this group was treated with the vehicle alone, this
finding was by no means related to treatment with TMPTA.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no treatment related effects on the sex ratio between control and treated animals.
Mean sex ratios (% males : % females) were 52:48, 54:46, 52:48 and 49:51 for respectively the first
control, 10 30 and 100 mg/kg bw/day groups, and 48:52 each for the 130 mg/kg bw/day group and its
concurrent control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter sizes were not affected by treatment up to and including 130 mg/kg bw/day. Mean litter sizes were
8.7, 7.3, 8.5 and 8.1 for the first control, 10, 30 and 100 mg/kg bw/day group, respectively. The mean
litter size in the 130 mg/kg bw/day group was significantly lower compared to its concurrent control group
(8.0 versus 9.4 fetuses/litter), but remained within the normal range of biological variation.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
There were no external developmental malformations or variations for fetuses up to and including 130
mg/kg bw/day that survived until planned necropsy.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology up to and including 130 mg/kg bw/day.
The only skeletal malformation seen at 130 mg/kg bw/day was a caudal vertebral anomaly observed in
one fetus. As this was an isolated finding which was also observed in historical controls (3 fetuses in 3
litters), it was considered to be a chance finding.
Fusion between sternebrae occurred in one control, one control from additional control group and two 100
mg/kg bw/day fetuses (litter proportions were 0.4%, 0.5% and 1.8% per litter in these respective dose gr
oups). However, this malformation is the most commonly one observed in historical controls (upper range
value is 2.4% per litter) and was therefore not considered to be toxicologically relevant.
At 100 mg/kg bw/day, sternum with supernumerary ossification site was observed in three fetuses out
of three litters, resulting in a mean litter proportion of 3.8% per litter, while a sternal supernumerary os
sification site was not observed in any of the control fetuses. Although the incidence of 3.8% is above
the upper historical control limit (1.6% per litter), this variation was not observed at 130 mg/kg bw/day.
Furthermore, for a related finding, namely supernumerary skull site, no dose-related increase in the mea
n litter proportion was observed. Additionally, general ossification parameters like unossified sternebra no
s. 5 and/or 6, unossified metacarpals and/or metatarsals, unossified tarsals, unossified hyoid body and/
or arches, and unossified skull bone line were unaffected by treatment. Taken together, the increased
incidence of sternum with supernumerary ossification site in the 100 mg/kg/day group was considered to
be of no toxicological relevance.
Remaining skeletal variations were not considered treatment-related as they occurred infrequently,
occurred at frequencies that were within the range of available historical control data or were observed in
control fetuses only.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to and including
130 mg/kg bw/day.
Treatment at 100 mg/kg bw/day resulted in a higher litter proportion of visceral malformations due to mal
formations of the eye, liver edema and ascites. The total percent per litter with visceral malformations was
1.3%, 0.6%, 1.7% and 4.3% per litter in the control, 10, 30 and 100 mg/kg bw/day group, respectively.
Three fetuses in the 100 mg/kg bw/day group had an eye malformation, while none was noted at lower
dose levels. A small eye occurred in two fetuses out of two litters and cataract was observed in one
fetus. The mean litter proportion for absent and/or small eyes in the high dose group was higher than
the maximum historical control value (1.5 versus 0.5% per litter) and cataract was not seen previously
among actual historical controls. Because small eyes and cataracts are anomalies of embryologically dist
inct tissues, they are not considered developmentally linked findings. It is highly unlikely that these ocular
findings reported at 100 mg/kg bw/day in the definitive study of TMPTA are related to treatment.
In two fetuses of 1 litter in the 100 mg/kg bw/day group unusual malformations was observed. Edema of
all liver lobes and ascites (accumulation of fluid in the abdomen) were noted for both fetuses. Of these
related malformations (portal hypertension causes ascites), only ascites was seen previously in a single
historical control fetus.
To investigate if the above findings were spontaneous in origin, an extra dose group of 130 mg/kg bw/day
was added to the study. The total percent per litter with visceral malformations was 1.5% and 1.2% per
litter in the concurrent control and 130 mg/kg bw/day group, respectively. Among the fetuses of the 130
mg/kg bw/day group there were none with an eye malformation, and no cases of liver edema or ascites
were observed. Therefore, it was considered that the eye and liver/abdomen malformations observed at
100 mg/kg bw/day had occurred by chance and were not related to treatment.
Any remaining visceral malformations and variations were not considered treatment related as they
occurred infrequently, did not follow a dose-related trend, occurred at frequencies that were within the
range of available historical control data or were observed in control fetuses only.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
At 130 mg/kg bw/day, a trend towards slightly lower mean fetal body weights (male, female and combined)
was noted when compared to its concurrent control group. Mean combined fetal body weights were
37.8 and 39.3 grams, respectively. This change occurred secondary to the slightly reduced food consumption
and slightly lower body weight gains observed for dams in this highest dose group as compared
to the concurrent control group. However, as examination of the fetal skeletons did not reveal any signs
for growth delay and considering the fact that fetal body weights remained within the available historical
range, it was regarded as non-adverse.
There were no effects on litter size, sex ratio, and no increase in fetal abnormalities (external, visceral and
skeletal malformations and variations) was seen following treatment at 130 mg/kg bw/day.
No developmental toxicity was observed at 10, 30 and 100 mg/kg/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 130 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
130 mg/kg bw/day (actual dose received)

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study the No Observed Adverse Effect Levels
(NOAELs) for maternal and developmental toxicity were at least 130 mg/kg bw/day. Data from a previous
dose range finding study revealed insufficient tolerance (including an early delivery) at 150 mg/kg bw/day
and unscheduled deaths at 200 mg/kg bw/day.
Executive summary:

TMPTA was investigated for prenatal developmental toxicity in rabbits in an study performed according to

OECD 414 guideline and in accordance with GLP principles. Dose levels were based on a range finding

study in which mated rabbits were exposed by oral gavage from Day 6 to Day 28 post-coitum, inclusive, at

dose levels of 0, 50, 100 and 200 mg/kg bw/day. Clear toxicity was observed at 200 mg/kg bw/day, while

at 100 mg/kg bw/day, there was only a single female with signs of toxicity. In the first part of the study,

eighty-nine mated female New Zealand White rabbits were assigned to four groups. Groups 1, 2 and 3

consisted of 22 females, and Group 4 of 23 females. The test item, TMPTA, was administered once daily

by oral gavage from Days 6 to 28 post-coitum at doses of 10, 30 and 100 mg/kg bw/day (Groups 2, 3 and 4,

respectively). Rabbits of the control group received the vehicle, 1% aqueous carboxymethyl cellulosewith

0.1% Tween-80, only.

In the first part of this study (Groups 1-4), possible treatment-related developmental findings were observed

at the high dose of 100 mg/kg bw/day. To investigate this further, it was deemed necessary to add an extra

group. To test the feasibility of a higher dose level for this extra group, an extra group of 6 mated females

was added to the dose range finding study to test a dose of 150 mg/kg bw/day. Animals were treated and

observed under the same conditions as for the previous groups. At 150 mg/kg bw/day, faeces abnormalities,

body weight loss, reduced food and/or water consumption were noted. In addition, one female had an early

delivery. Based on these results,a dose of 150 mg/kg bw/day was judged too high. Therefore, it was decided

to select a dose of 130 mg/kg bw/day for the additional group in the definitive study.

For the second part of the study, forty-four mated female New Zealand White rabbits were assigned to

two groups (Groups 6 and 7) of 22 animals each. The test item, TMPTA, was administered once daily

by oral gavage from Days 6 to 28 post-coitum at 130 mg/kg bw/day (Group 7). Rabbits of the concurrent

control group (Group 6) received the vehicle, 1% aqueous carboxymethyl cellulosewith 0.1% Tween-80,

only. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

No relevant maternal toxicity was observed up to and including 130 mg/kg bw/day. There was no test-item

related mortality following treatment up to and including 130 mg/kg bw/day. Reduced faeces production,

varying in duration and severity, was observed in females of both treated and control groups. The incidence

was slightly higher at 100 and 130 mg/kg bw/day.

There was a trend towards slightly lower food consumption (absolute and relative to body weight) from Days

13-23 post-coitum in the 100 mg/kg bw/day group and from Days 13-29 post-coitum in the 130 mg/kg bw/

day, which resulted in slightly reduced body weight gains from Days 16-23 post-coitum and Days 16-29

post-coitum, respectively, compared to the concurrent control group. As these changes in food consumption

and body weight were relatively slight (not always reaching statistical significance), they were considered

as non-adverse. No test-item related macroscopic abnormalities were noted following treatment up to and

including 130 mg/kg bw/day.

At 130 mg/kg bw/day, a trend towards slightly lower mean fetal body weights (male, female and combined)

was noted when compared to its concurrent control group. Mean combined fetal body weights were 37.8 and

39.3 grams, respectively. This change occurred secondary to the slightly reduced food consumption and

slightly lower body weight gains observed for dams in this highest dose group as compared to the concurrent

control group. However, as examination of the fetal skeletons did not reveal any signs for growth delay and

considering the fact that fetal body weights remained within the available historical range, it was regarded as

non-adverse. There were no effects on litter size, sex ratio, and no increase in fetal abnormalities (external,

visceral and skeletal malformations and variations) was seen following treatment at 130 mg/kg bw/day. No

developmental toxicity was observed at 10, 30 and 100 mg/kg bw/day.

Based on the results in this prenatal developmental toxicity study the No Observed Adverse Effect Levels

(NOAELs) for maternal and developmental toxicity were at least 130 mg/kg bw/day. Data from a previous

dose range finding study revealed insufficient tolerance (including an early delivery) at 150 mg/kg bw/day

and unscheduled deaths at 200 mg/kg bw/day.