Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Following ECHA decision (CCH-D-2114361700-57-01/F) on3-p-cumenyl-2-methylpropionaldehyde, EC No 203-161-7, it was requested to conduct additional toxicological studies:
1. In vitro cytogenicity study in mammalian cells (Annex VIII, Section 8.4.2., test method: OECD TG 473) or in vitro micronucleus study (Annex VIII, Section 8.4.2, test method: OECD TG 487
2. In vitro gene mutation study in mammalian cells (Annex VIII, Section 8.4.3.; test method: OECD TG 476 or TG 490) with the registered substance provided that the study requested under 1. has negative results;
3. Sub-chronic toxicity study (90-day), oral route (Annex IX, Section 8.6.2.; test method: EU B.26./OECD TG 408) in rats with the registered substance;
4. Pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: EU B.31./OECD TG 414) in a first species (rat or rabbit), oral route with the registered substance;
5. Extended one-generation reproductive toxicity study (Annex IX, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance after providing the OECD 408 outcome.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Remarks:
Exceptions to GLPs: characterization of the test substance performed by the Sponsor according to established SOPs, controls, and approved test methodologies to ensure integrity and validity of the results generated
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-p-cumenyl-2-methylpropionaldehyde
EC Number:
203-161-7
EC Name:
3-p-cumenyl-2-methylpropionaldehyde
Cas Number:
103-95-7
Molecular formula:
C13H18O
IUPAC Name:
2-methyl-3-[4-(propan-2-yl)phenyl]propanal
Test material form:
liquid
Remarks:
colorless to pale-yellow liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han
Details on test animals or test system and environmental conditions:
Test animals: time-mated female Crl:WI(Han) rats were received from the lab on Gestation Day 1, 2, 3, or 4. The animals were approximately 12–13 weeks old and weighed between 186 and 240 g at the initiation of dosing on Gestation Day 6.

Housing: single/individual housing in solid-bottom cages containing appropriate bedding material. Cages were arranged on the racks in group order. Where
possible, control group animals were housed on a separate rack from the test substance-treated animals.

Food:
Type: Meal
Frequency: Ad libitum.
Analysis: Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Testing Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Type: Municipal tap water, treated by reverse osmosis and ultraviolet irradiation.
Frequency: Ad libitum, via an automatic watering system. Water bottles were provided, if required.
Analysis: Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

Acclimation period: After receipt at the Testing Facility, the Crl:WI(Han) rats were acclimated prior to the initiation of dosing.

Environmental conditions:
Temperature: 68°F to 77°F (20°C to 25°C)
Humidity: 30% to 70%
Light Cycle: 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Dose formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion.
Vehicle was used as received and test substance was prepared daily. For both, the storage conditions were set to maintain 18°C to 24°C, protected from light, purged with nitrogen.
Dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The prepared formulations were not adjusted for purity.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis.

Concentration analyses:
Concentrations: all groups
Stratum: middle
Number of samples per concentration: 4 collected samples, 2 analyzed samples, 2 backup samples
Intervals: first and last preparation
Storage conditions: temperature set to maintain 18°C to 24°C.
Acceptance criteria: mean sample concentration within 100% ± 10% of theoretical concentration. Individual sample concentration of ± 15%.

Analyses were performed by a high performance liquid chromatography method with ultraviolet absorbance detection using a validated analytical procedure.
Details on mating procedure:
Time-mated rats were received by the lab on Gestation Day 1, 2, 3 and 4. The day on which confirmation of mating is made will be designated Gestation Day 0.
Duration of treatment / exposure:
Gestation Days 6–20
Frequency of treatment:
Once daily
Duration of test:
Gestation Days 0-21
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
No. of animals per sex per dose:
22 females/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The oral route of exposure was selected because this a possible route of human exposure. The dose levels were selected based on information provided by the Sponsor. In a previous
1 generation reproduction toxicity study, gestating rats given 150 mg/kg/day of cyclamen aldehyde were observed with reduced mean body weight and body weight gains compared to the control group generally throughout gestation. Based on these results, 150 mg/kg/day was selected as the high dose for the current study. It was expected that this high dose would induce maternal toxicity while not resulting in death or severe suffering. Lower doses were selected to assess dose response.

Examinations

Maternal examinations:
Mortality: checked at least twice daily (morning and afternoon), beginning upon arrival through termination. Animals were observed within their cage unless necessary for identification or confirmation of possible findings.

Clinical observations: checked once daily, beginning with the day of animal arrival and continuing through (and including) the day of euthanasia. Animals were removed from the cage. Mortality and all signs of overt toxicity were recorded on the day observed. The observations included, but were not limited to, evaluations for changes in appearance of skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous systems, somatomotor activity, and behavior. On days of scheduled dosing, the observations were collected prior to dosing.

Postdose observations (with animal handling): checked approximately 1 hour postdose. Animals were removed from the cage. Animals were observed for findings that were potentially related to treatment or that might change before the next scheduled observation.

Individual Body Weights: checked at Gestation Days 0 (by supplier) and 5–21 (daily). Gestation Day 0 bodyweight collected under Non-GLP conditions. Not collected from animals found dead.

Food Consumption: checked at Gestation Days 5–21 (daily). Quantitatively measured. Reported as g/animal/day for each corresponding body weight interval
during gestation.

Examinations after necropsy: liver and thyroid gland (organ weights, histology, histopathology)
Ovaries and uterine content:
The uterus of each dam was excised, and its adnexa trimmed. Corpora lutea were also counted and recorded. Gravid uterine weights were obtained and recorded. The uterus of each dam was opened, and the number of viable and nonviable fetuses, early and late resorptions, and total number of implantation sites were recorded, and the placentae were examined. The individual uterine distribution was documented using the following procedure: all implantation sites, including
early and late resorptions, were numbered in consecutive fashion beginning with the left distal uterine horn, noting the position of the cervix and continuing from the proximal to the distal right uterine horn. Uteri which appear nongravid by macroscopic examination were opened and placed in a 10% ammonium sulfide solution for detection of early implantation loss. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible.
Blood sampling:
Thyroid Hormone Analyses:
Time: Gestation Day 21.
Special requirements: Collected prior to noon on each day of collection, around the same time each day, and within a 2-hour window on each collection day, to reduce variability due to normal diurnal variations in physiological levels of thyroid hormones. Blood collections were performed in an animal ante room, the necropsy laboratory, or as far away from live animals as possible to minimize stress-induced hormone fluctuations.
Method: Venipuncture from a jugular vein using the hand-held restraint method.
Target volume: approximately 1.0 mL/time point collected.
Anticoagulant: none
Processing: serum
Number of aliquots: 2
Analyses: for total T3 and T4 analyses, hormone samples were analyzed using validated ultra-high performance liquid chromatography with dual mass spectroscopy (UHPLC/MS/MS) assays. Analysis of serum samples to determine TSH concentrations was conducted using a validated Luminex Bead Based (TSH) assay.
Fetal examinations:
Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal), malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.).

External: Each viable fetus was examined in detail, sexed, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. Following euthanasia, anogenital distance was measured for all viable fetuses. The absolute and relative values (to the cube root of fetal body weight) were reported. 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.

Internal (visceral): The sex of all fetuses was confirmed by internal examination. Approximately one-half of the fetuses in each litter were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The heads from these fetuses were removed and placed in Harrison’s fixative for subsequent processing and soft-tissue examination.

Skeletal: The remaining fetuses (approximately one-half from each litter, excluding any carcasses without heads) were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, fetuses were stained with Alizarin Red S and Alcian Blue. The skeletal examination was made following this procedure.
Statistics:
Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Indices:
The following parental indices and litter calculations were included, where applicable:
Pre-Implantation Loss = (No. of corpora lutea – no. of implants) x 100/No. of corpora lutea
Post-Implantation Loss = (No. of implants – no. of live fetuses) x 100/No. of implants
Sex Ratio (% males) = No. male fetuses x 100/Total no. of fetuses
Litter % of Fetuses with Abnormalities = No. of fetuses in litter with a given finding x 100/No. of fetuses in litter examined
Historical control data:
Historical control data were available from the testing laboratory.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related clinical observations noted at any dose level at the daily examinations or approximately 1 hour postdose.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females in the control, 25, 75, and 150 mg/kg/day groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 150 mg/kg/day group, a slight mean body weight loss with correspondingly lower mean food consumption were noted following the initiation of dosing (Gestation Days 6–7) compared to the control group. Mean body weight gains and food consumption were generally comparable to the control group for the remainder of the study and when the entire treatment period (Gestation Days 6–21) was evaluated and the mean absolute body weights in this group were unaffected. Therefore, the initial decrements in the 150 mg/kg/day group were considered test substance-related, but nonadverse.
Mean body weights and body weight gains in the 25 and 75 mg/kg/day groups and mean corrected body weights, corrected body weight gains in the 25, 75, and 150 mg/kg/day groups were unaffected by test substance administration.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 150 mg/kg/day group, a slight mean body weight loss with correspondingly lower mean food consumption were noted following the initiation of dosing (Gestation Days 6–7) compared to the control group. Mean body weight gains and food consumption were generally comparable to the control group for the remainder of the study and when the entire treatment period (Gestation Days 6–21) was evaluated and the mean absolute body weights in this group were unaffected. Therefore, the initial decrements in the 150 mg/kg/day group were considered test substance-related, but nonadverse.
Food consumption in the 25 and 75 mg/kg/day groups was unaffected by test substance administration.
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:
effects observed, treatment-related
Description (incidence and severity):
Lower mean total T3 and T4 levels were observed in the 75 (-34.0% and -23.4% respectively) and 150 (-44.3% and -45.3% respectively) mg/kg/day groups compared to the control group; differences were statistically significant. Mean total T3 and T4 levels in the 25 mg/kg/day group were comparable to the control group mean.
Mean TSH levels in the 25, 75 and 150 mg/kg/day groups were higher than the control group mean; differences were non-dose responsive and statistically significant only for the 25 and 75 mg/kg/day groups. In addition, TSH mean values across all treated groups were within the range of the lab historical control data for Sprague Dawley rats and the control group mean was atypically low, and outside the range of the historical control data; when evaluated on an individual animal basis, more than 50% of the animals in the control group had TSH levels that fell outside of the range of the historical control data.
There were no differences in thyroid gland weights or any corresponding changes in thyroid histopathology in any of the treated groups when compared to the control group. However, test article-related higher mean (absolute) liver weights were observed in the 75 and 150 mg/kg/day group females.
Based on these data, the lower T3 and T4 levels noted in the 75 and 150 mg/kg/day groups were likely attributable to increased hormone metabolism in the liver, rather than a direct effect on the thyroid gland, and hence considered nonadverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test article-related higher mean (absolute) liver weights were observed in the 75 and 150 mg/kg/day group females. Higher mean liver weights were not dose-responsive, but multiple individual absolute liver values in the 75 and 150 mg/kg/day group females were out of the concurrent control group range. There were no microscopic correlates.
No test substance-related organ weight changes were noted in the thyroid gland.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related gross pathology findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of Cyclamen Aldehyde.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, microscopic liver changes were present in the 25, 75, and 150 mg/kg/day group females and consisted of minimal hepatocellular single-cell necrosis. Single-cell necrosis was perivascular and/or random, and was characterized as individual eosinophilic rounded hepatocytes with pyknotic nuclei. Single-cell necrosis was dose-responsive in incidence rate.
Of note, a marginal increased incidence of focal or multifocal hepatocellular necrosis (n=4), characterized as random groups (vs. single-cell/individual) of necrotic hepatocytes with associated inflammatory infiltrates, was also observed in the 150 mg/kg/day group females, but was considered incidental given the observation of this change in the concurrent control group females (n=2), and frequent occurrence of this change in both the lab historical control database and in untreated rats used in toxicity studies (Thoolen et al., 2010; McInnes, 2011).
There were no test substance-related microscopic changes in the thyroid gland. Remaining changes were considered incidental, of the nature commonly observed in this strain and age of
rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of Cyclamen Aldehyde.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
There were no adverse effects on maternal parameters observed at any dosage level.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Intrauterine survival was unaffected by test substance administration at dose levels of 25, 75 and 150 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss,
mean number of live fetuses, and fetal sex ratios.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Intrauterine survival was unaffected by test substance administration at dose levels of 25, 75 and 150 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss,
mean number of live fetuses, and fetal sex ratios.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Intrauterine survival was unaffected by test substance administration at dose levels of 25, 75 and 150 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss,
mean number of live fetuses, and fetal sex ratios.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Intrauterine survival was unaffected by test substance administration at dose levels of 25, 75 and 150 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss,
mean number of live fetuses, and fetal sex ratios.
Dead fetuses:
no effects observed
Description (incidence and severity):
Intrauterine survival was unaffected by test substance administration at dose levels of 25, 75 and 150 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss,
mean number of live fetuses, and fetal sex ratios.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
There were no adverse effects on maternal parameters observed at any dosage level.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
dead fetuses
early or late resorptions
endocrine findings
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean fetal body weights (males, females, and combined) in the 75 and 150 mg/kg/day groups were lower than the concurrent control group (3.978%–5.913% and 8.539%–9.499%, respectively) and below the mean values in the lab historical control data. The lower mean fetal body weights in these groups were considered test substance-related and adverse at 150 mg/kg/day based on the magnitude of change from the control group, but nonadverse at 75 mg/kg/day due to the low magnitude of change from the control group and lack of any fetal morphological effects.
Intrauterine growth at 25 mg/kg/day was unaffected by test substance administration.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Mean anogenital distances (absolute and relative to the cube root of the fetal weight) were unaffected by test substance administration at all dose levels. Differences from the control group were slight and not statistically significant.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external developmental malformations were observed for fetuses in the test substance-treated groups. In the control group, Fetus No. 1505-01 was noted with a thread-like tail which consisted
skeletally of absent sacral and caudal vertebrae. No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related skeletal developmental malformations were noted. In the 150 mg/kg/day group, a malformation of bent humerus was observed for a single fetus (Fetus No. 4501-12); variations of bent scapula, wavy ribs, thoracolumbar supernumerary ribs, and incomplete ossification of the frontal, parietal, squamosal, and supraoccipital bones of the skull were also observed for this fetus. As the bent limb bone was only observed for a single fetus, it was not considered to be test substance-related.
No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the lab historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
No visceral developmental malformations were observed in fetuses in this study.
No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the lab historical control data.
A discolored liver lobe was noted for Fetus No. 2503-04 in the 25 mg/kg/day group and was not classified as either a malformation or variation. This finding was considered incidental and was not considered to be test substance-related because it occurred infrequently and/or in a manner hat was not dose-related.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
In the 75 and 150 mg/kg/day groups, test substance-related lower (up to 5.913% and 9.499%, respectively) mean fetal body weights (males, females, and combined) were noted compared to the control group. Based on the magnitude of change versus the control group, the effects on mean fetal body weights were considered nonadverse at 75 mg/kg/day but adverse at 150 mg/kg/day. There were no test substance-related effects on intrauterine growth at 25 mg/kg/day or intrauterine survival and fetal morphology (external, visceral, and skeletal) at 25, 75, and 150 mg/kg/day.
The numbers of fetuses (litters) available for morphological evaluation were 177(20), 186(19), 202(20), and 209(22) in the control, 25, 75, and 150 mg/kg/day groups, respectively.
Malformations were observed in 1 fetus each in the control and 150 mg/kg/day groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the lab historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
In the 150 mg/kg/day group, a malformation of bent humerus was observed for a single fetus (Fetus No. 4501-12); variations of bent scapula, wavy ribs, thoracolumbar supernumerary ribs, and incomplete ossification of the frontal, parietal, squamosal, and supraoccipital bones of the skull were also observed for this fetus. As the bent limb bone was only observed for a single fetus, it was not considered to be test substance-related.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, there were no adverse effects on maternal parameters observed at any dosage level but a slight decrease in maternal body weight during gestation was reported at the high dose. Therefore, a dose level of 150 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL for maternal toxicity. Based on lower mean fetal weights at 150 mg/kg/day, a dose level of 75 mg/kg/day was considered to be the NOAEL for developmental toxicity when cyclamen aldehyde was administered orally by gavage to time-mated Crl:WI(Han) rats. These low mean fetal weights occurred in the presence of a slight decrease in maternal body weight during gestation at the high dose.
Executive summary:

The objectives of this study were to determine the potential of Cyclamen Aldehyde to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a NOAEL (no-observed-adverse-effect level) for maternal and developmental toxicity.

Animals were dosed with Cyclamen Aldehyde at doses of 25, 75 and 150 mg/kg bw/d via oral gavage once daily during Gestation Days 6–20.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormones, gross necropsy findings, organ weights, intrauterine growth and survival, and fetal morphology.

In the maternal animals, test substance-related effects included a slight mean body weight loss with correspondingly lower mean food consumption in the 150 mg/kg/day group immediately following the initiation of dosing (Gestation Days 6–7). Mean body weight gains and food consumption in this group were generally comparable to the control group for the remainder of the study and the initial decrements did not impact the mean absolute body weights, and therefore were considered nonadverse. Test substance-related lower total T3 and T4 levels were observed in the 75 and 150 mg/kg/day groups. These differences were considered nonadverse based on lack of corresponding changes on thyroid gland weights or thyroid histopathology in these groups. Test substance-related higher mean liver weights were observed at 75 and 150 mg/kg/day with no microscopic correlate. Based on these data, the lower T3 and T4 levels observed at 75 and 150 mg/kg/day groups were likely attributable to increased hormone metabolism in the liver, rather than a direct effect on the thyroid gland, and hence considered nonadverse. Additionally, no test substance-related effect on TSH levels were noted at any dose level. Although higher TSH levels were noted in the 25, 75, and 150 mg/kg/day groups, the differences from the control group were non-dose responsive, statistically significant for only the 25 and 75 mg/kg/day groups, and within the range of the lab historical control data for Sprague Dawley rats. Additionally, minimal single-cell necrosis was observed dose-responsively in all test substance groups but considered nonadverse given the minimal severity grade and absence of an associated inflammatory response.

In the 75 and 150 mg/kg/day groups, test substance-related lower (up to 5.9% and 9.5%, respectively) mean fetal body weights (males, females, and combined) were noted compared to the control group. Based on the magnitude of change versus the control group, the effects on mean fetal body weights were considered nonadverse at 75 mg/kg/day but adverse at 150 mg/kg/day. There were no test substance-related effects on intrauterine growth at 25 mg/kg/day or intrauterine survival and fetal morphology (external, visceral, and skeletal) at 25, 75, and 150 mg/kg/day.

In conclusion, there were no adverse effects on maternal parameters observed at any dosage level but a slight decrease in maternal body weight during gestation was reported at the high dose. Therefore, a dose level of 150 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL for maternal toxicity. Based on lower mean fetal weights at 150 mg/kg/day, a dose level of 75 mg/kg/day was considered to be the NOAEL for developmental toxicity when cyclamen aldehyde was administered orally by gavage to time-mated Crl:WI(Han) rats. These low mean fetal weights occurred in the presence of a slight decrease in maternal body weight during gestation at the high dose.