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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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
A mixture of 1,3-Cyclohexanedicarboxaldehyde and 1,4-Cyclohexanedicarboxaldehyde
Supplier, City, State (Lot, Reference Number): The Dow Chemical Company, Midland, Michigan (Lot# EXP-14-AI0320).
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 95.5% area (corrected for water) by gas chromatography with identification by nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Supplier: Charles River (Raleigh, North Carolina)

Age and Weight at Study Start: Sexually mature adult weighing approximately 200-250 g.
Physical and Acclimation
During the acclimation period each animal will be evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals will be housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least four days prior to the start of dosing.
Housing
After assignment, animals will be housed one per cage in stainless steel cages. Cages will have solid floors with corncob bedding. Cages will contain a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions will be maintained in the animal room.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 50% with a range of 30-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Temporary excursions from these ranges for temperature and humidity may occur on an infrequent basis; all observed ranges will be documented in the study file.
Enrichment
Enrichment for rats includes the use of ground corn cob bedding, paper nesting material, and open areas on the cage sides for visualization of other rats.
Randomization and Identification
Animals will be stratified by GD 0 body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform mean group weights and standard deviations at the start of the study. Animals that are placed on study will be uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that are correlated to unique alphanumeric identification numbers. If a transponder stops functioning or is lost, it will be replaced with a new transponder that will be correlated with the unique animal number.
Feed and Water
Feed and municipal water will be provided ad libitum. Animals will be provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Analyses of the feed will be performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source is periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants are conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained in the study file.
Animal Welfare
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC determined that the proposed Activities are in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities used for this study are DART 02, Humane Endpoints 01, Subchronic/Chronic 01, and Animal ID 01.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration verification of all dose solutions and homogeneity of the low- and high-dose solutions was determined pre-exposure. The method used for analyzing the test material in corn oil was gas chromatography with flame ionization detection (GC/FID).
Stability
A previously conducted study (Poornachandra Shetty, 2012) has shown 1,3- and 1,4-Cyclohexanecarboxaldehyde to be stable for at least eight days in corn oil at concentrations of 1 and 250 mg/ml. Dose solutions for this study were used within the established concentration range and stability duration; therefore, additional stability analyses were not conducted.
Details on mating procedure:
Not applicable - animals arrive pregnant.
Duration of treatment / exposure:
Seven days/week on gestation days (GD) 6-20
Frequency of treatment:
Daily
Duration of test:
15 days
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of an oral gavage developmental toxicity probe study in Crl:CD(SD) rats - Groups of five time-mated female Cr1:CD(SD) rats were administered 0, 250, 500, or 750 mg/kg/day 1,3- and 1,4-Cyclohexanedicarboxaldehyde in corn oil by oral gavage at a dose volume of 4 ml/kg on gestation day (GD) 6 through 20. In-life parameters evaluated for all groups included clinical observations, body weight, body weight gain, and feed
consumption. On GD 21 all surviving dams were euthanized and examined for gross pathologic alterations. Liver and kidney weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead fetuses. Histopathological examination of the stomach was conducted on all control and 250 mg/kg/day animals.
Treatment-related noisy respiration was observed in one dam in each of the 500 and 750 mg/kg/day groups. There were no treatment-related clinical observations noted on animals in the 250 mg/kg/day group.
In the 500 and 750 mg/kg/day groups, treatment-related decreases in body weight were present at GD 18-21. For both of these dose groups, there was a treatment-related decrease in maternal body weight gain and feed consumption beginning at the GD 9-12 interval and continuing through the GD 18-21 interval. Body weight gain of animals in the 250 mg/kg/day group showed a treatment-related decrease at the GD 9-12 interval; however, body weight gains were similar to control for all other measured intervals, and body weight was similar to controls throughout the study.
There was a primary treatment-related increase in absolute and relative liver weights in the 750 mg/kg/day group. In addition, a treatment-related increase in relative kidney weights was observed in the 750 mg/kg/day group that was deemed secondary to decreased body weight. There were no treatment-related liver or kidney weight effects in the 250 or 500 mg/kg/day groups.
At all dose levels, point of contact irritation of the stomach was observed. In the 500 and 750 mg/kg/day groups, stomach gross pathology findings in all animals included multifocal or focally extensive thickening of the non-glandular mucosa. Additional gross findings in some animals given 500 or 750 mg/kg/day included glandular and nonglandular mucosal ulceration, glandular mucosal hyperemia, and gastric wall abscesses. A single animal in the 250 mg/kg/day group had a gross focal thickening of the non-glandular stomach. Microscopically, this focal observation was associated with epithelial ulceration, subacute inflammation, hyperkeratosis, and hyperplasia. Histological findings (without a gross pathology correlate) of very slight gastric epithelial hyperkeratosis and hyperplasia at the limiting ridge were present in four and three of five animals, respectively in the 250 mg/kg/day group.
Oral gavage administration of 1,3- and 1,4-Cyclohexanedicarboxaldehyde to time-mated Crl:CD(SD) rats resulted in maternal toxicity at all dose levels tested but no indication of embryo/fetal lethality at any dose level tested. Based upon the treatment-related increase in stomach epithelial ulceration observed at 250 mg/kg/day in this study, 1,3- and 1,4-Cyclohexanedicarboxaldehyde oral gavage dose levels less than 250 mg/kg/day are appropriate for a full prenatal developmental toxicity study in Crl:CD(SD) rats.

Examinations

Maternal examinations:
Daily Observations
A cage-side examination will be conducted twice daily, preferably at the same time each day. This examination is typically performed with the animals in their cages and is designed to detect significant clinical abnormalities that are clearly visible upon a limited examination and to monitor the general health of the animals. The animals are not hand-held for these observations unless deemed necessary. Significant abnormalities that could be observed include, but are not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. Any animals found dead will be necropsied as soon as practical. In addition, all animals will be observed for morbidity, mortality, and the availability of feed and water at least twice daily. For animals showing indications of premature delivery, the delivered fetuses will be counted and examined to the extent possible. Dosing will be discontinued, and animals will be euthanized and necropsied as soon as practical.

Clinical Observations
Clinical observations will be conducted on all animals at least once daily. Animals will be observed approximately one hour after dosing, or at the anticipated time of peak effects, if known. Clinical observations include a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

Body Weights/Body Weight Gains
Statistical analysis of body weights will be performed using data collected on GD 0, 6, 9, 12, 15, 18, and 21. Statistical analysis of body weight gains will be conducted for the following intervals: GD 0-6, 6-9, 9-12, 12-15, 15-18, 18-21, 6-21, and 0-21.

Feed Consumption
Feed consumption will be recorded and statistically analyzed for all animals every three days from GD 3-21 by weighing feed containers at the start and end of a measurement cycle.

On GD 21, all surviving females (not fasted) will be sedated with a mixture of isoflurane vapors and medical oxygen, euthanized by carbon dioxide inhalation and a limited gross pathologic examination (necropsy) will be performed. The sequence of the maternal necropsies will be counterbalanced across groups (e.g., control, high, middle, low) to control for potential confounding influences of timing on fetal growth and skeletal ossification.
The maternal necropsy will include an examination of the external tissues and all orifices. The skin will be reflected from the carcass, the thoracic and abdominal cavities will be opened and the viscera will be examined. The stomach, liver, and kidneys will be dissected from the carcass and will be incised. Any obvious gross pathologic alterations will be recorded, and the weight of the liver, kidneys, and gravid uterus will be recorded. The ratios of liver and kidney weights to terminal body weight will be calculated. Representative portions of liver, kidneys, stomach and gross lesions will be preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination will of the lived, kidneys, and gross lesions will not be conducted unless deemed necessary to interpret other observations made during the study. The stomach will be processed and examined as per the histopathology section below. Transponders will be removed and placed in with the preserved tissues.

Histopathology
In order to help interpret histological stomach effects that were seen in the probe study, the stomach from all pregnant animals that survive to necropsy will be processed by standard histologic procedures. The stomach of animals that die or are sacrificed in a moribund condition will not be processed unless deemed necessary to interpret other observations made during the study. Paraffin embedded tissues will be sectioned approximately 6 µm thick and batch stained with hematoxylin and eosin. Stomachs from the control and high-dose group animals will be examined by a veterinary pathologist using a light microscope. Stomachs from the low- and intermediate-dose group animals will be microscopically examined if determined to be a target organ by histology.
Selected histopathologic findings will be graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades will be used for conditions that are altered from the normal textbook appearance of an organ/tissue, but are of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change would neither be expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade will be used for conditions that are of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue may be adversely affected but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but, generally. lesions graded as moderate would not be life threatening. A severe grade will be used for conditions that are extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may be life threatening.

Ovaries and uterine content:
A detailed examination of the reproductive tract will be performed, and the number and position of implantations, viable fetuses, dead fetuses, and resorptions will be recorded. Resorptions will be classified as either “early” or “late” based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form, while a “dead fetus” indicates a very recent death as evidenced by a lack of external degenerative changes. For females with one or more viable fetuses, the number of ovarian corpora lutea will be counted. The uteri of females lacking visible implantations will be stained with a 10% aqueous solution of sodium sulfide based on (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.
Fetal examinations:
The sex and body weight of all viable fetuses will be recorded. All fetuses will be given an external examination that will include observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses will be euthanized by sublingual oral administration of sodium pentobarbital solution. Approximately one half of all the fetuses in each litter will be chosen randomly via computer for visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974; Stuckhardt and Poppe, 1984). The visceral examination will include observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. The heads of these fetuses will be removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue (Wilson, 1965). The remaining fetuses not selected for visceral examination will then be skinned, eviscerated, preserved in alcohol and double stained with Alcian Blue and Alizarin Red S for cartilage and bone according to methods based on Trueman et al. (1999) and Zablotny (2002). A thorough evaluation of the fetal skeleton will be conducted on the remaining fetuses not selected for visceral examination. However, a fetus may be intentionally changed from one selected for visceral examination to one processed for skeletal examination (and vice versa) if it is deemed that such examination will provide more meaningful data about a suspected abnormality.
All fetal alterations will be classified as a variation or malformation. A variation is defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation is defined as a permanent structural change that may adversely affect survival, development or function and/or which occurs at a relatively low incidence in the specific species/strain.
Statistics:
Maternal body weights, maternal body weight gains, gravid uterine weights, fetal body weights, and feed consumption will be evaluated by Bartlett’s test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) will be performed. If the ANOVA is significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with Bonferroni's correction (Miller, 1966) will be performed, respectively. Feed consumption values will be excluded from analysis if the feed is spilled or scratched.
Frequency of pre- and post-implantation loss (calculations shown below), and fetal alterations will be analyzed using a censored Wilcoxon test (Haseman and Hoel, 1974) with Bonferroni’s correction applied when the incidence is greater than 5%. The number of corpora lutea, implantations and litter size will be evaluated using a non-parametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction. Pregnancy rates will be analyzed using the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni’s correction. Fetal sex ratios will be analyzed using a binomial distribution test. Females lacking visible implantations or totally resorbed litters at the scheduled necropsy will be excluded from the appropriate analyses. Statistical outliers will be identified, using a sequential method (alpha = 0.02; Grubbs, 1969) and, if excluded, will be excluded for sound scientific reasons. Both Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control group to keep the experiment-wise alpha at 0.05. Both will be reported at the experiment-wise alpha level.
Indices:
Calculation of Pre- and Post-Implantation Loss
• Pre-implantation loss* = (No. corpora lutea-implantations) x 100
No. corpora lutea
• Post-implantation loss* = (No. implantations – viable fetuses) x 100
No. implantations
* Note: Percent pre- and post-implantation loss will be determined for each litter, followed by calculation of the mean of these litter values.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Gavage administration of 1,3- and 1,4-cyclohexanedicarboxaldehyde resulted in no treatment-related effects on clinical observations, body weight, body weight gain, feed consumption, organ weights, or gross pathology in dams in any treated groups. Histopathological examination of dams revealed treatment-related very slight or slight hyperkeratosis and hyperplasia of the nonglandular mucosa of the stomach at the limiting ridge with associated chronic active inflammation of the underlying submucosa at dose levels of 75 and 225 mg/kg/day. In addition to hyperkeratosis and hyperplasia, a single animal given 225 mg/kg/day 1,3- and 1,4-cyclohexanedicarboxaldehyde had focal moderate ulceration of the nonglandular mucosa at the limiting ridge and moderate focal chronic-active inflammation within associated submucosal tissues, all these changes are typical of point of contact effects.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
225 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: based on overall effects
Key result
Dose descriptor:
NOEL
Remarks:
point of contact
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity: based on point of contact irritation resulting in hyperkeratosis and hyperplasia in the stomach at dose levels ≥ 75 mg/kg/day
Key result
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
225 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: based on the absence of systemic effects

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Administration of 1,3- and 1,4-cyclohexanedicarboxaldehyde via gavage at dose levels up to and including 225 mg/kg/day produced no indications of embryo/fetal toxicity or teratogenicity.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the no-observed-effect level (NOEL) for maternal toxicity was 25 mg/kg/day, due to point of contact irritation (hyperkeratosis and hyperplasia in the stomach) noted at dose levels ≥ 75 mg/kg/day. Based on the absence of systemic effects up to and including 225 mg/kg/day, the NOEL for systemic maternal toxicity was 225 mg/kg/day. The embryo/fetal no-observed-effect level (NOEL) was 225 mg/kg/day, the highest dose level tested.
Executive summary:

The purpose of this study was to evaluate the maternal and developmental toxicity of 1,3- and 1,4-cyclohexanedicarboxaldehyde in Crl:CD(SD) rats following repeated gavage administration.Groups of 24 time-mated female rats were administered 1,3- and 1,4 -cyclohexanedicarboxaldehyde by gavage in corn oil on gestation day (GD) 6 through 20 at dose levels of 0, 25, 75 and 225 mg/kg/day. These dose levels were selected based upon a treatment-related ulceration of the stomach in a single dam at 250 mg/kg/day along withgastric hyperkeratosis and hyperplasia in three of five damsin a preceeding developmental toxicity probe study. The stomach ulceration observed at 250 mg/kg/day in the developmental toxicity probe study showed that this dose level exceeded an acceptable high dose level for the full developmental toxicity study. In-life maternal study parameters included clinical observations, body weight, body weight gain and feed consumption. On GD 21, all rats were euthanized and examined for gross pathologic alterations. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions and live/dead fetuses. Histopathological examination of the stomachs from all pregnant dams was conducted. All fetuses were weighed, sexed and examined for external alterations. Approximately one half of the fetuses were examined for visceral alterations while skeletal examinations were conducted on the remaining fetuses.

Gavage administration of1,3- and 1,4-cyclohexanedicarboxaldehyderesulted in no treatment-related effects on clinical observations, body weight, body weight gain, feed consumption, organ weights, or gross pathology in dams in any treated groups.

Histopathological examination of dams revealed treatment-related very slight or slight hyperkeratosis and hyperplasia of the non-glandular mucosa of the stomach at the limiting ridge with associated chronic active inflammation of the underlying sub-mucosa at dose levels of 75 and 225 mg/kg/day. In addition to hyperkeratosis and hyperplasia, a single animal given 225 mg/kg/day, 1,3- and 1,4 –cyclohexanedicarboxaldehydehad focal moderate ulceration of the non-glandular mucosa at the limiting ridge and moderate focal chronic-active inflammation within associated sub-mucosal tissues.

Administration of 1,3- and 1,4-cyclohexanedicarboxaldehyde via gavage at dose levels up to and including 225 mg/kg/day produced no indications of embryo/fetal toxicity or teratogenicity.

Due to point of contact irritation resulting in hyperkeratosis and hyperplasia in the stomach at dose levels ≥75 mg/kg/day, the no-observed-effect level (NOEL) for maternal toxicity was 25 mg/kg/day. Based on the absence of systemic effects up to and including 225 mg/kg/day, the NOEL for systemic maternal toxicity was 225 mg/kg/day. The embryo/fetal no-observed-effect level (NOEL) was 225 mg/kg/day, the highest dose level tested.