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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5,5-trimethylhexanoic acid
EC Number:
221-975-0
EC Name:
3,5,5-trimethylhexanoic acid
Cas Number:
3302-10-1
Molecular formula:
C9H18O2
IUPAC Name:
3,5,5-trimethylhexanoic acid
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): i-Nonanoic Acid
- Physical state: liquid, colorless, clear
- Analytical purity: 99.9 area-% (AT-1000) or 98.6 area-% (DB-1)
- Lot/batch No.: B52 19.09.2012
- Expiration date of the lot/batch: 19.09.2014
- Stability under test conditions: Stability guaranteed by sponsor
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10-13 weeks
- Weight at study initiation:
- Housing: 1 rat per cage
- Diet: Ground Kliba maintenance diet ad libitum
- Water : filtered tap water ad libitum
- Acclimation period: From GD 0 (day of supply) to the beginning of administration (GD6), the animals were accustomed to the environmental conditions and to the diet

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 airchanges per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
4 mL/kg body weight; for test substance preparation, the specific amount of test substance will be weighed, topped up with corn oil in a calibrated beaker and intensely mixed with a magnetic stirrer; during administration, the preparations were kept homogenous with a magnetic stirrer; preparations are stored at room temperature
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
Animals paired by the breeder (time-mated animals) were supplied at noon on the day of evidence of mating; this day is referred to as GD0
Duration of treatment / exposure:
Animals are treated once daily from GD6-GD19
Frequency of treatment:
Once daily
Duration of test:
Dams are sacrificed on day 20
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, abnormalities and changes are documented

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Bw is recorded on day 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consuption is recoreded from GD0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19, 19-20

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined:

Ovaries and uterine content:
The ovaries and uterine content was examined after termination:
Examinations included:
- Gross-pathological examination
- Weight of the unopened uterus
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Site of implantations in the uterus
Fetal examinations:
After removal of fetuses from the uterus, the following examinations have been made:
- weight of each fetus
- sex
- weight of placentas
- gross pathological examination of fetuses after dissection from uterus
- half of the fetuses of each dam is skined, fixed in ethyl alcohol and the skeleton and cartilage stained (method by Kimmel and Trammell)
- the other half of the fetuses is fixed in Harrisons fluid and soft tissues examined
Statistics:
Dunnett's test, Fishers exact test and Wilcoxon test
Historical control data:
Results were compared to historical control data for Wistar rats that were available in the test facility

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
200 mg/kg bw/day group: 2 dams died shortly before term
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
200 mg/kg bw/day group: Decreased net body weights (8% below control) at the end of gestation, distinctly reduced net body weight gain during treatment (47% below control)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
200 mg/kg bw/day group: Decreased food consumption towards the end of gestation (13-23% below control)
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
200 mg/kg bw/day group: Increased red blood cell (RBC) counts, hemoglobin and hematocrit values, decreased relative reticulocyte counts, increased absolute and relative monocyte and large unstained cell (LUC) counts
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increased urea levels, decreased total protein, albumin, globulin, cholesterol, triglyceride and calcium levels
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
200 mg/kg bw/day group: Increase in liver weight (absolute +13%, relative +23%)
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
mortality
organ weights and organ / body weight ratios

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
200 mg/kg bw/day group: Reduced fetal weights (14% below control), at 60 mg/kg bw/day: only minimal weight reduction (5% below control), not considered as adverse: there was no no effects on the degree of fetal maturity and the reduction was within the historical control range
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
200 mg/kg bw/day group: Morphological changes indicating a delay or disturbance of development, such as increased
incidences of supernumerary ribs (73.9 % affected fetuses per litter) and wavy
ribs (14.4%), incompletely ossified Skulls 43.7%, unossified sternebra (31.5%), incomplete
ossification of metacarpal (7.3%), incomplete ossification of pubis (4.7%), incomplete
ossification of ischium (3.3%)
Increased incidence of severely altered rib cages (multiple rib findings like wavy, bent or
knobby) in 9.4% fetuses per litter, resulting in a skeletal malformation rate of 11.1% affected
fetuses per litter

60 mg/kg bw/day group: skull ossification was decreased in mid-dose animals; however, these decreases were still within historical control data
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A yellow discoloration of fetal livers was noted in almost all high-dose litters (200 mg/kg bw/day) and approximately half of the mid-dose litters (60 mg/kg bw/day). The rate of affected fetuses per litter was about 60 and 20%, respectively. The dose-relationship suggests an association to the treatment, but the cause of this discoloration is not known. The shape and size of those livers were completely unchanged. As there is no frank morphological change observable, the level of concern for this finding is rather low. It is not considered as evidence for an adverse effect.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Test Group 3 (200 mg/kg/d)
• Reduced fetal weights (14% below control)
• Morphological changes indicating a delay or disturbance of development, such as increased incidences of supernumerary ribs (73.9 % affected fetuses per litter) and wavy ribs (14.4%), incompletely ossified Skulls 43.7%, unossified sternebra (31.5%), incomplete ossification of metacarpal (7.3%), incomplete ossification of pubis (4.7%), incomplete ossification of ischium (3.3%)
• Increased incidence of severely altered rib cages (multiple rib findings like wavy, bent or knobby) in 9.4% fetuses per litter, resulting in a skeletal malformation rate of 11.1% af-fected fetuses per litter

Test Group 2 (60 mg/kg/d)
• No test substance-related adverse effects on fetuses

Test Group 1 (20 mg/kg/d)
• No test substance-related adverse effects on fetuses

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
other: developmental effects at maternal toxic doses

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: sternum
skeletal: rib
skeletal: supernumerary rib
skeletal: pelvic girdle
Description (incidence and severity):
supernumary and wavy ribs, altered rib cages, other localisations: incomplete ossification

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day
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:
yes

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of I-Nonanoic acid to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity at a dose of 200 mg/kg bw/d, such as mortality, reduced body weights/weight gain, hematological alterations, dysregulated liver cell metabolism and dose-relatedly increased liver and adrenal weights. An increase in liver weight in the mid dose group was without histopathological correlate and hence not considered adverse. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 60 mg/kg bw/d.
Pups of the high-dose group showed signs of developmental defecets as depicted by reduced body weights, severely altered rib cages (eading to an increased malformation rate), wavy ribs, decreased ossification of skulls, sternebrae, sacral arch, metacarpal, pubis and ischium. Considering strong systemic toxicity observed in dams, these effects are most likely due to maternal toxicity and not a direct effect of the substance on development. In the mid dose-group (60 mg/kg/d), fetal body weights were statistically significantly reduced (5%). Since there were no effects on the degree of fetal maturity at 60 mg/kg bw/d, the slight weight reduction in this group is not considered to be an adverse, toxicologically relevant effect of the test substance. Moreover, the decrease in fetal body weight was within the historical control range. Accordingly, skull ossification was decreased in mid-dose animals; however, these decreases were still within historical control data, indicating that this effects is not considered adverse. A yellow discoloration of fetal livers was noted in almost all high-dose litters (200 mg/kg bw/d) and approximately half of the mid-dose litters (60 mg/kg bw/d). The rate of affected fetuses per litter was about 60 and 20%, respectively. The dose-relationship suggests an association to the treatment, but the cause of this discoloration is not known. The shape and size of those livers were completely unchanged. As there is no frank morphological change observable, the level of concern for this finding is rather low. It is not considered as evidence for an
adverse effect.
Since no effects have been noted in low-dose animals and effects observed in mid-dose rats are not considered to be an adverse, toxicologically relevant effect of the test substance, the NOAEL for developmental toxicity is set at the mid dose, i.e. 60 mg/kg/d.
Since morphological changes in offspring were only observed at 200 mg/kg bw/d, a dose level which caused distinct maternal toxicity, including mortality isononanic acid does not need to be classified with respect to teratogenic effects.
Executive summary:

i-Nonanoic acid was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an emulsion in corn oil to groups of 25 time-mated female Wistar rats by gavage at doses of 20, 60, and 200 mg/kg body weight/day (mg/kg bw/day) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group.

The following test substance-related adverse effects/findings were noted:

Test group 3 (200 mg/kg bw/day):

Dams

􀁸 Mortality in 2 dams shortly before term

􀁸 Decreased food consumption towards the end of gestation (13-23% below control)

􀁸 Decreased net body weights (8% below control) at the end of gestation, distinctly reduced net body weight gain during treatment (47% below control)

􀁸 Increased red blood cell (RBC) counts, hemoglobin and hematocrit values

􀁸 Decreased relative reticulocyte counts

􀁸 Increased absolute and relative monocyte and large unstained cell (LUC) counts

􀁸 Increased urea levels

􀁸 Decreased total protein, albumin, globulin, cholesterol, triglyceride and calcium levels

􀁸 Increase in liver weight of absolute +13% and relative +23%

Fetuses

􀁸 Reduced fetal weights (14% below control)

􀁸 Morphological changes indicating a delay or disturbance of development, such as increased incidences of supernumerary ribs (73.9 % affected fetuses per litter) and wavy ribs (14.4%), incompletely ossified Skulls 43.7%, unossified sternebra (31.5%), incomplete ossification of metacarpal (7.3%), incomplete ossification of pubis (4.7%), incomplete ossification of ischium (3.3%)

􀁸 Increased incidence of severely altered rib cages (multiple rib findings like wavy, bent or knobby) in 9.4% fetuses per litter, resulting in a skeletal malformation rate of 11.1% affected fetuses per litter

Test group 2 (60 mg/kg bw/day):

􀁸 No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 1 (20 mg/kg bw/day):

􀁸 No test substance-related adverse effects on dams, gestational parameters or fetuses

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 60 mg/kg bw/day.

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 60 mg/kg bw/day, based on reduced fetal weights, as well as a delay of development and a slightly increased malformation rate at 200 mg/kg bw/day.