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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:
2020-03-11 to 2021-05-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide
EC Number:
939-581-9
Cas Number:
1471314-81-4
Molecular formula:
Not applicable (UVCB substance)
IUPAC Name:
3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Amides, C12-C18 (even numbered), N-[3-(dimethylamino)propyl], N'-oxides
- Substance type: Clear liquid
- Lot/batch No.: BLBX017090
- Storage condition of test material: Room temperature in the dark
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Amides, C12-C18 (even numbered), N-[3-(dimethylamino) propyl], N' -oxides
- Substance type: Appearance: Almost clear colorless liquid
- Physical state: Liquid
- Analytical purity: Purity: 35.2% in water
- Purity test date: no data
- Lot/batch No.: S019341917
- Expiration date of the lot/batch: 28 March 2021
- action of test substance: no data
- correction factor: 2.85, based on purity
- purity/weighing factor: none

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Sealed container, 2 to 8°C, protected from light
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Formulations of 0.2 and 100 mg/mL (AI) were confirmed to be stable and homogenous for 15 days when stored refrigerated (2 to 8°C) or for 14 days when stored at room temperature (15 to 25°C) in Covance Study 8410552.


FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid

Test animals

Species:
rabbit
Strain:
New Zealand White
Remarks:
Hra:(NZW) SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Eighty time-mated female New Zealand White Hra:(NZW) SPF rabbits from Envigo Global Services Inc., Denver, Pennsylvania, United States of America.
- Age at start of study (day 0 of gestation): at least 20 weeks
- Weight at day 0 (after mating): at least 2.7 kg
- Fasting period before study: no data
- Housing: Animals were individually housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes (Home Office, 2014).
Suitable tray liners were provided and changed at least weekly.
- Diet (e.g. ad libitum): ad libitum, LabDiet 5322 rabbit diet (PMI Nutritional International). Each batch of diet was analyzed for specific constituents and contaminants.
Upon arrival and on some occasions when low food consumption was observed, animals were provided with moist hay or approximately 50 g of moistened diet. This was not included in any food consumption calculations. No contaminants were present in the diet at levels that might have interfered with achieving the objective of the study. Results are retained on file at Covance.
- Water (e.g. ad libitum): ad libitum, Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
No contaminants were present in the water at levels that might have interfered with achieving the objective of the study. Results are retained on file at Covance.
- Acclimation period: Acclimation was limited by mated status.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 13-23°C
- Humidity (%): not less than 45%
- Air changes (per hr): The room was air conditioned to provide a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark, with the exception of when experimental procedures dictated.

IN-LIFE DATES: From: 2020-03-16 To: 2020-05-20

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: The test article was formulated as a suspension in purified water following dispensary SOPs and the formulation method (Method 8410555_O_01D), as maintained in the study data.
- Frequency of preparation: weekly
- Storage of formulation: Refrigerated (2 to 8°C) in a sealed container protected from light.
- Test item accounting: Formulations prepared for use on GD 6 and 28 were analyzed to determine the achieved concentration.
Triplicate samples were taken from the middle of the test article formulations and were analyzed. A single sample was taken from the middle of the control formulations and was analyzed.
- Formulations were stirred continuously from at least 30 minutes prior to and throughout dosing (excluding the control group). Formulations were administered at room temperature (15 to 25 °C).

- Test item was prepared at the appropriate concentration as solution in purified water.

VEHICLE
- Justification for use and choice of vehicle: not applicable (vehicle is water)
- Concentration in vehicle: 0, 5, 12.5, 17.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations prepared for use on Gestation Day 6 and 28 of the study were analyzed to determine the achieved concentration. The mean % nominal concentration should be between 90 to 110% and with a relative standard deviation (RSD)  5.0%. Results were within these criteria. Test article was not detected in the Group 1 control samples.
The analytical procedure was validated within Covance study 8280017.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: Each female was mated with one proven male at the supplier’s laboratory.
- M/F ratio per cage: 1:1, using identified stock New Zealand White bucks
- Length of cohabitation: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: natural mating observed referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6 to 28 after mating (22 days)
Frequency of treatment:
once daily, at a similar time on each occasion.
Duration of test:
30 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
175 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20 time-mated female per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high-dose level of 175 mg/kg/day active ingredient (AI) was based on the findings from the preliminary dose range-finding study in pregnant rabbits (Covance Study 8410557). This dose level was considered the highest dose level to exhibit some toxicity (i.e., changes in body weight, food consumption, and fetal weights) without causing death. The intermediate-dose level of 125 mg/kg/day AI was selected, minimal changes in food consumption were expected, and it was anticipated to represent a no observed adverse effect level (NOAEL). The low-dose level of 50 mg/kg/day AI has been selected as the low dose, as it was expected to be well tolerated and was anticipated to represent a no observed effect level (NOEL).

- Route of administration rationale: The oral route of administration was chosen because it is an acceptable and commonly used route of exposure for regulatory studies of this type. The oral route is a potential route of accidental human exposure to be investigated in hazard and risk assessment.

- Animal species selection rationale: The New Zealand White Hra:(NZW) SPF rabbit was selected because it is a readily available non-rodent species acceptable to the regulatory authorities. Historical control data are available at the test facility for this strain of animal.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed in the home cage twice daily, at the beginning and end (nominal) of the working day for signs of ill health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination outside the home cage daily from GD 4 until necropsy. An individual record of the clinical condition of each animal was maintained.
- Animals were observed daily at 1 hour (± 30 minutes) postdose.

BODY WEIGHT: Yes
- Body weights for each animal were recorded on GD 4, 6, 7, 8, 9, 12, 15, 17, 19, 22, 25, 28, and 29.
Unscheduled body weights were recorded on GD 24, 26, and 27 for welfare reasons.

FOOD CONSUMPTION: Yes
- The amount of food was determined once daily from GD 4 to necropsy.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: on GD 29 after mating
- Females were killed by an intravenous injection (overdose) of sodium pentobarbitone.
- Females were sacrificed in replicate order.
- Macroscopic pathology: Following confirmation of death of the females that were scarified on GD 29, the major blood vessels were severed. All macroscopic lesions were dissected, freed from fat and other contiguous tissue, recorded, and retained in the relevant fixative. Animals that were sacrificed moribund or found dead prior to the scheduled sacrifice were examined macroscopically for abnormalities of the thoracic and abdominal viscera.

OTHER:
MORTALITY:
- Moribund animals were sacrificed by an intravenous injection (overdose) of sodium pentobarbitone. Following confirmation of death, the major blood vessels were severed.
Ovaries and uterine content:
- The uterus and ovaries were examined for implantations and corpora lutea, respectively. The extent of development of the implantation sites was determined as near as possible. An evaluation of the uterus was made to indicate dead or live implantations.

- The ovaries and uteri were removed and examined, and the following data were recorded.
• Pregnancy status
• Gravid uterus weight
• Terminal body weight (recorded for adjusted gravid uterus weight calculations only and have not been reported)
• Number of corpora lutea
• Number and intrauterine position of implantations subdivided into:
• Live fetuses
• Early intrauterine deaths
• Late intrauterine deaths
• Dead fetuses

- Early intrauterine deaths were classified as those that exhibited decidual or placental tissue only. Late intrauterine deaths exhibited embryonic or fetal tissue, in addition to placental tissue. Dead fetuses were classified as those that appeared to have died shortly before necropsy.
- The uterus of apparently non pregnant females was immersed in a 10% ammonium sulphide solution to reveal any evidence of implantation.
Fetal examinations:
- External examinations: Yes: [all fetuses per litter ]
- Internal examinations: Yes: [all fetuses per litter ]
- Soft tissue examinations: Yes [all viable fetuses per litter ]
- Skeletal examinations: Yes [all viable fetuses per litter ], examined in 50% glycerol and retained in glycerol/propylene glycol.
- Head examinations: Yes: [appr. one half of eviscerated fetuses per litter], fixed in Bouin's fluid. Heads were then examined by the Wilson sectioning method. Fetal head sections were retained in the relevant fixative.
- Fetal abnormalities were classified as malformations (rare and/or potentially lethal) or variations (commonly occurring non lethal abnormalities).
- Live fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose), followed by confirmation of cessation of circulation.
- Dead fetuses were examined externally and sexed, then discarded.
Statistics:
See any other information on materials and methods incl. tables.
Indices:
% Pre-Implantation Loss = Number of corpora lutea - number of implantations
Number of corpora lutea X 100
% Post-Implantation Loss = Number of implantations - number of live embryos
Number of implantations X 100
Corrected Body Weight (Carcass Weight) = Terminal body weight - uterine weight
Corrected Weight Change = Carcass weight - GD 7 body weight
Total Weight Change = GD 29 body weight - GD 7 body weight
% Male Fetuses = Number of male fetuses
Number of fetuses of determined sex X 100
Historical control data:
Historical control data will be routinely supplied for selected observations where this information is considered to assist interpretation of study data, and will normally include both fetal and litter incidences.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test article-related clinical observations were noted following administration of 125 or 175 mg/kg/day AI.
Clinical observation of increased incidence of yellow pelage around the feet, associated with increased urination, was observed for five females administered 175 mg/kg/day AI. Four females administered 175 mg/kg/day AI were also recorded with thin appearance, and decreased body tone was evident for one female (Animal B03015) which littered early, following administration of 175 mg/kg/day AI. The effects following 175 mg/kg/day AI were considered adverse, due to the mortality observed at this dose.
The increased incidence of yellow pelage around the feet was observed for five females administered 125 mg/kg/day AI was considered nonadverse.
No test article-related clinical observations were observed following 50 mg/kg/day AI and no postdose observations were noted at any dose level administered.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two test article-related unscheduled deaths occurred following administration of 175 mg/kg/day AI.
One animal administered 175 mg/kg/day AI (Animal B0319) was sacrificed on GD 26 due to body weight loss of 13% between GD 9 and GD 26, clinical observations of thin appearance, red staining on the cage tray liner, and mucoid feces. At necropsy, the animal was recorded with one early resorption and eight dead fetuses. The animal had a slightly pale and mottled liver and thin appearance. Another animal administered 175 mg/kg/day AI (Animal B0313) aborted on GD 28 and was subsequently terminated. Body weight loss was evident from GD 12 onwards. By GD 27, the animal had lost 530 g (-15% compared with the GD 12 body weight). The animal had a slightly pale and mottled liver and thin appearance at necropsy.
No further unscheduled deaths occurred as a result of test article toxicity. One animal administered 125 mg/kg/day AI (Animal B0209) died following a whole body convulsion immediately after dosing on GD 22. At necropsy, the animal was recorded with a rupture to the right lobe of the lung and red fluid in the oral and thoracic cavities. These findings were indicative of inappropriate dosing, and this was not test article related. The animal was confirmed as pregnant.
One control female (Animal B0014) was sacrificed on GD 26 following clinical observations of thin appearance and progressive body weight loss (17% between GD 12 and GD 26). The animal was confirmed as pregnant with nine live fetuses. This death was considered incidental in nature as this was a control animal.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test article-related lower mean body weights were evident following administration of 175 mg/kg/day AI, compared to controls.
Overall mean body weight gain was -37% compared with controls, from GD 6 to 29, however this value included decedent Animals B0313 and B0319 and was considered to reflect the intragroup variability in response to test article administration at 175 mg/kg/day. The effects following administration of 175 mg/kg/day AI were considered adverse, due to these mortalities. For surviving animals administered 175 mg/kg/day AI, mean body weights were 7% lower than controls on GD 29.
Animal B0315 administered 175 mg/kg/day AI showed body weight losses from GD 17 onwards. The body weight losses were considered to have resulted in the early parturition on the day of scheduled sacrifice. This was considered to be test article related.
No test article-related body weight changes were noted following administration of 50 or 125 mg/kg/day AI.
Total weight change following administration of 175 mg/kg/day AI was 25% lower than controls, and corrected weight change was 142% lower than controls. Gravid uterus weight following administration of 175 mg/kg/day AI was 10% lower than controls, although values did not attain statistical significance, and mean values were within the historical control data ranges (420.3 to 559.9 g).
Placenta weights, adjusted for litter size, were statistically significantly lower following maternal exposure of 175 mg/kg/day AI compared to controls (P < 0.05), and mean unadjusted placenta weights were also lower following administration of 175 mg/kg/day AI, compared with controls (-8%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test article-related lower mean food consumption was observed following administration of 125 or 175 mg/kg/day AI.
Marginally lower mean food consumption was evident for females administered 175 mg/kg/day AI between GD 12 and 15 (-14%), with mean values more than 20% lower than control from GD 15 onwards, with statistically significant reductions observed between GD 22 and 25 (P < 0.05) and GD 25 and 28 (P < 0.01). Overall mean food consumption from GD 6 to 29 was -16% lower than control and attained statistical significance (P < 0.05).
Marginally lower mean food consumption was observed between GD 9 and 12 ( 13%) and GD 12 to 15 ( 18%) for females administered 125 mg/kg/day AI, with changes up to -26% of control between GD 22 and 25. Overall mean food consumption from GD 6 to 29 was -16% lower than controls and attained statistical significance (P < 0.05).
The effects following administration of 175 mg/kg/day AI were considered adverse, due to mortality observed at this dose. The effects noted following administration of 125 mg/kg/day AI were considered nonadverse.
No test article-related effects were evident following administration of 50 mg/kg/day AI.
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
Endocrine 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:
effects observed, treatment-related
Description (incidence and severity):
Test article-related macroscopic findings were noted for four females administered 175 mg/kg/day AI.
At scheduled sacrifice, one female administered 175 mg/kg/day AI was recorded as thin (Animal B0320); this finding was also recorded for two decedent females administered 175 mg/kg/day AI (Animals B0313 and B0319). Two females administered 175 mg/kg/day AI were recorded with mottled livers (Animals B0315 and B0317). This was also observed for two decedent females from this group (see Section 4.2.1) and was considered test article related.
No test article-related macroscopic findings were noted for females administered 50 or 125 mg/kg/day AI.
Animal B0315 administered 175 mg/kg/day AI littered on the day of scheduled necropsy; this was considered to be test article-related, as the animal showed body weight losses from GD 17 onwards.
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:
effects observed, treatment-related
Description (incidence and severity):
Test article-related effects were evident following the cesarean section examination, for females administered 125 or 175 mg/kg/day AI.
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Test article-related effects were evident following the cesarean section examination, for females administered 125 or 175 mg/kg/day AI.
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
Test article-related effects were evident following the cesarean section examination, for females administered 125 or 175 mg/kg/day AI.
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
Test article-related effects were evident following the cesarean section examination, for females administered 125 or 175 mg/kg/day AI.
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
Test article-related effects were evident following the cesarean section examination, for females administered 125 or 175 mg/kg/day AI.
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
changes in pregnancy duration
clinical signs
dead fetuses
gross pathology
mortality
number of abortions

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Nonadverse test article-related fetal variations were observed for fetuses from females administered 125 or 175 mg/kg/day AI. Female fetal weights, adjusted for litter size were 8% lower than controls (P < 0.05) following maternal exposure of 175 mg/kg/day AI. Male fetal weights, adjusted for litter size were 5% lower than controls, although values did not attain statistical significance following maternal exposure of 175 mg/kg/day AI. Mean fetal weight values for eight litters were outside of the historical control data ranges for combined sex fetal weights (36.4 to 43 g). The magnitude of the change in fetal weight was small (≤10%), and as such, was considered nonadverse. Test article-related reductions for female fetal weights, adjusted for litter size were evident following maternal exposure of 125 mg/kg/day AI; mean values were 7% lower than controls (P < 0.05) and within the historical control data ranges (35.4 to 42.8 g). No such changes were evident for male fetal weights. Mean combined fetal weight values for eight litters were lower than the historical control data range for the combined sexes (36.4 to 43 g). Mean unadjusted placenta weights were also lower following administration of 125 mg/kg/day AI, compared with controls (-6%); although placenta weights adjusted for litter size were not affected. These changes were considered to be attributable to the effects of the test article on the maternal animal, and did not represent an adverse effect on fetal development.
No test article-related effects were noted following administration of 50 mg/kg/day AI.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.
Changes in sex ratio:
not specified
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Total weight change following administration of 175 mg/kg/day AI was 25% lower than controls, and corrected weight change was 142% lower than controls. Gravid uterus weight following administration of 175 mg/kg/day AI was 10% lower than controls, although values did not attain statistical significance, and mean values were within the historical control data ranges (420.3 to 559.9 g).
Female fetal weights, adjusted for litter size were 8% lower than controls (P < 0.05) following maternal exposure of 175 mg/kg/day AI. Male fetal weights, adjusted for litter size were 5% lower than controls, although values did not attain statistical significance following maternal exposure of 175 mg/kg/day AI. Mean fetal weight values for eight litters were outside of the historical control data ranges for combined sex fetal weights (36.4 to 43 g). The magnitude of the change in fetal weight was small (≤10%), and as such, was considered nonadverse.
Placenta weights, adjusted for litter size, were statistically significantly lower following maternal exposure of 175 mg/kg/day AI compared to controls (P < 0.05), and mean unadjusted placenta weights were also lower following administration of 175 mg/kg/day AI, compared with controls (-8%).
Test article-related reductions for female fetal weights, adjusted for litter size were evident following maternal exposure of 125 mg/kg/day AI; mean values were 7% lower than controls (P < 0.05) and within the historical control data ranges (35.4 to 42.8 g). No such changes were evident for male fetal weights. Mean combined fetal weight values for eight litters were lower than the historical control data range for the combined sexes (36.4 to 43 g). Mean unadjusted placenta weights were also lower following administration of 125 mg/kg/day AI, compared with controls (-6%); although placenta weights adjusted for litter size were not affected. These changes were considered to be attributable to the effects of the test article on the maternal animal, and did not represent an adverse effect on fetal development.
No test article-related effects were noted following administration of 50 mg/kg/day AI.
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related malformations were recorded. The malformations observed were from disparate organ systems and indicated no dose dependent trend, with similar malformations also present in litters from the control group or previously observed within the historical control data.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Nonadverse test article-related fetal variations were observed for fetuses from females administered 125 or 175 mg/kg/day AI.
The majority of the variations observed following maternal exposure of 125 or 175 mg/kg/day AI were associated with the extent of ossification of the fetal skeleton. None of the incidences are found in 100% of fetuses providing clear evidence that ossification of both the appendicular and axial skeleton was progressing and the higher incidences observed were considered delayed development associated with reduced female fetal weight and not a developmental change. A higher incidence of unossified metacarpals was noted in the litters of females administered 125 or 175 mg/kg/day AI achieving statistical significance. Peripheral bone ossification is later in development and this finding was considered to correlate with the lower fetal weights observed for females in these dose groups and associated with a delay in development. A higher percentage of litters of females administered 125 or 175 mg/kg/day AI included fetuses with unilateral ossification of the sternebra, compared with controls, however mean values at the high and low dose fell within the historical control range and in the absence of statistical significance at the high dose and no dose response, this finding was considered incidental.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Small thyroid was noted for two fetuses in a single litter maternally administered 175 mg/kg/day AI (Animal B0303 male fetus L1 and female fetus L3). This finding was not present in the concurrent control group or the historical control data. The change was marginal and only affected two fetuses; therefore, this was classified as a variation and considered nonadverse.
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Localisation:
external: limb
external: tail
skeletal: skull
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: vertebra
skeletal: hindlimb
visceral/soft tissue: hepatobiliary
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
visceral/soft tissue: central nervous system
visceral/soft tissue: endocrine system
visceral/soft tissue: respiratory system
visceral/soft tissue: eye

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Formulation analysis report:


The mean % nominal concentration should be between 90 to 110% and with a relative standard deviation (RSD) ≤ 5.0%. Results were within these criteria (see Text Table 3.1 and Text Table 3.2).


The test article was not detected in the Group 1 control samples.


Text Table 3.1:    Gestation Day 6











































Group



Concentration
mg/mL



Results as % Nominal Concentration



Mean (%)



RSD (%)



2



5.0



100



100



99



100



0.48



3



12.5



104



102



102



103



0.74



4



17.5



101



101



101



101



0.32



Mean and RSD are calculated from the data base values, not the rounded values presented above.



 


Text Table 3.2:    Gestation Day 28











































Group



Concentration
mg/mL



Results as % Nominal Concentration



Mean (%)



RSD (%)



2



5.0



104



104



105



104



0.23



3



12.5



104



103



106



105



1.27



4



17.5



107



106



108



107



0.78



Mean and RSD are calculated from the data base values, not the rounded values presented above.



 


 


 

Applicant's summary and conclusion

Conclusions:
Based on the results of this prenatal developmental toxicity study, it was concluded that the no observed adverse effect level (NOAEL) for maternal toxicity was established as 125 mg/kg/day AI.
The NOAEL for developmental toxicity was established as 125 mg/kg/day AI.