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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study perofrmed according under GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Test Material Name
1,3- and 1,4-Cyclohexanedicarboxaldehyde
Chemical Name
A mixture of 1,3-Cyclohexanedicarboxaldehyde and 1,4-Cyclohexanedicarboxaldehyde
Synonyms
1,3- and 1,4-Cyclohexanecarboxaldehyde; 1,3(and 4)-Cyclohexanedicarboxaldehyde, 1,3- and 1,4-cyclohexanedicarboxaldehyde, refined; 1,3- and 1,4-CHDA
Supplier, City, State (Lot, Reference Number)
The Dow Chemical Company, Midland, Michigan (Lot# EXP-14-AI0320).
Purity/Characterization
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
Sex:
male/female
Details on test animals and environmental conditions:
Species and Sex
Rats (male and female)
Strain and Justification
Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location
Charles River (Kingston, New York)
Age at Study Start
Approximately six weeks at initiation of treatment.
Physical and Acclimation
During the acclimation period each animal was 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 animals were housed two-three per cage in stainless steel solid bottom cages with corncob bedding, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), prior to randomization. Animals were acclimated to the laboratory for at least one week prior to the start of the study.
Housing
After assignment to study, animals were housed singly in solid bottom stainless steel cages, except during breeding and during the gestation and littering phases of the study. The solid bottom cages contained ground corn cob nesting material with some heat-treated laboratory grade wood shavings for enrichment. Beginning on February 10 for the females and February 12 for the males, the use of heat-treated laboratory grade wood shavings was discontinued from this laboratory. Nylon bones were then placed in the cages for environmental enrichment except during the pairing, gestation, and lactation phases of the study. During breeding, one male and one female were placed in stainless steel cages with wire mesh floors which were suspended above catch pans in order to better visualize vaginal copulatory plugs. During gestation and littering, dams (and their litters) were housed in plastic cages containing corn cob bedding material and were provided with paper nesting material from approximately GD 0 until completion of lactation (LD 21). Cages contained a glass feed crock and a pressure activated lixit valve-type watering system.

The following environmental conditions were targeted in the animal room; however, temporary excursions from these environmental conditions may have occurred on an infrequent basis (all observed ranges were documented in the study file).
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.)

Randomization and Identification
Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Feed and Water
Feed and municipal water were provided ad libitum. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. There were no contaminants in either the feed or water at levels that would have adversely impacted the results or interpretation of this study. 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 has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities to be used for this study were DART 01, Humane Endpoints 01, and Animal ID 01. 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).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
Breeding for the P1 and P2 adults commenced after approximately ten weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until mating occurred or two weeks elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then removed from the males and returned to their home cages. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating. When available, one rat/sex/litter was randomly selected for the P2 mating to produce the F2 generation. More than one weanling was selected from the litters when necessary to achieve 25 breeding pairs/dose level for the second generation. Cohabitation of P2 male and female littermates was avoided. In cases when a mating partner had died or was otherwise not available, the animal was paired with the next available partner.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose Confirmation and Homogeneity
Dose confirmation analyses of all dose solutions were conducted at least four times (at study start, P1 mating, P1 lactation and P2 pre-mating). The homogeneity of the low-dose and the high-dose test solutions was determined concurrent with dose confirmation. Details are contained in Appendix A.
Stability
A previously conducted study (Poornachandra Shetty, 2012) has shown 1,3- and 1,4-CHDA 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.
Solubility
The test material 1,3- and 1,4-CHDA was determined to be soluble in corn oil at a concentration of 500 mg/ml.
Retainer Samples
A sample of each lot number of the bulk test material was retained. No samples of dose solutions were retained.
Duration of treatment / exposure:
approximately ten weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (three weeks) for each of two generations.
Adult rats were dosed seven days per week for two generations until necropsy. Any females in active labor at the time of dosing were not dosed on that particular day and were noted in the dosing records. Oral gavage administration to F1 offspring assigned to the P2 generation began on PND 22. F1 offspring selected for necropsy were not directly dosed via gavage.
Frequency of treatment:
Daily, 7 days per week;
Details on study schedule:
Test material administration began on December 12, 2014 and was continued up until the day prior to necropsy. The F1 weanlings were necropsied from April 6 to April 20, 2015. The P1 adults were necropsied from April 27 to April 30, 2015 (males) and April 22 to April 24, 2015 (females). The F2 weanlings were necropsied from August 17 to August 28, 2015. The P2 adults were necropsied from August 24 to August 27, 2015 (males) and August 31 to September 2, 2015 (females).
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 per sex per dose
Control animals:
yes, concurrent vehicle

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
not specified
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased relative and absolute kidney weight in females (P1 and P2) and males (P2) in the 150 mg/kg bw/day dose group
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Stomach lining of the P1 and P2 males and females in the 50 and 150 mg/kg bw dose groups
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Stomach lining of the P1 and P2 males and females in the 50 and 150 mg/kg bw dose groups
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

The only significant treatment reltated findings in the parental animals were the kidney weight changes in the top dose group (increased weight - absolute and relative) and the histopathological observations in the stomachs of male and female animals in the mid and top dose group.

Kidney effects:
increased absolute (6.8%) and relative (4.8%) kidney weights of females from the
150 mg/kg/day group. The increased absolute kidney weights were statistically identified, and the increased relative kidney weights were outside of the recent historical control range for this laboratory. Increases in kidney weights in the P0 female high-dose group were consistent with those observed in the second generation (see below). There was no histopathologic correlate for the increased kidney weights in P0 females (see Histology section).
Dose (mg/kg/day) 0 10 50 150
Final Body Weight (g) 284.3 284.5 292.9 289.8
Kidney
Absolute Weight (g) 2.054 2.049 2.083 2.194*
Kidney
Relative Weight (g/100g) 0.722 0.721 0.713 0.757


In the F1 generation at 150 mg/kg/day, absolute and relative kidney weights were increased in males (6.3 and 6.5%, respectively) and females (6.9 and 6.4%, respectively) compared to control. Relative and absolute kidney weights in both sexes at 150 mg/kg/day were outside of the recent historical control range for this laboratory. Relative and absolute kidney weight increases were statistically identified in females, and relative kidney weight increases were statistically identified in males. A histopathologic correlate for kidney weight increases was not observed in either sex.

Sex Dose (mg/kg/day) 0 10 50 150
Male Final Body Weight (g) 613.8 618.8 602.7 613.3
Kidney Absolute Weight (g) 4.045 4.004 3.933 4.301
Kidney Relative Weight (g/100g) 0.661 0.650 0.653 0.704*
Female
Final Body Weight (g) 301.9 297.2 306.3 304.3
Kidney Absolute Weight (g) 2.203 2.177 2.278 2.355*
Kidney Relative Weight (g/100g) 0.730 0.733 0.744 0.777*
*Statistically different from control mean by Dunnett’s Test, Alpha=0.05

Pathology (Gross and Histo)
Treatment-related gross pathologic observations were limited to the stomach of P0 and F1 male and female rats. The squamous mucosa at the junction of the glandular and non-glandular portions of the stomach (limiting ridge) was thickened in most P0 and F1 males and females from the 150 mg/kg/day group and in one F1 male from the 50 mg/kg/day group. This gross observation corresponded to hyperplasia and hyperkeratosis occurring at the limiting ridge of the stomach (see below, Histopathology).
All other gross observations occurred in single animals or across all dose groups and were interpreted to be incidental changes unassociated with treatment.

Treatment-related histopathological findings were limited to the stomach of P0 male and female rats from the 150 mg/kg/day group and F1 male and female rats from the 50 and 150 mg/kg/day groups. In both sexes and both generations at 150 mg/kg/day, there was very slight to slight hyperkeratosis and hyperplasia of the non-glandular gastric mucosa at the limiting ridge. In F1 males and females at 50 mg/kg/day, treatment-related very slight hyperkeratosis and very slight to slight hyperplasia of the limiting ridge was observed. Hyperkeratosis was characterized by thickening and compaction of the orthokeratotic keratin layer. In some animals, there were circular or globular forms of keratin within the laminated layers. In occasional animals with slight hyperkeratosis, the keratin of the limiting ridge contained entrapped inflammatory cells. Hyperplasia was characterized by proliferation of the basal layer of the stratified squamous epithelium and occasional hydropic degeneration of individual cells in the middle and luminal layers of the squamous epithelium. The changes in the limiting ridge of the stomach represent a reaction of the mucosa to repeated administration of an irritant material at the point of contact/entry.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
ca. 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Increased absolute and relative kidney weights female P0
Remarks on result:
other: Generation: P1, P2 (migrated information)
Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
ca. 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathological observations in the stomach of males and females at 50 and 150 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
ca. 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive effects observed in any generation in either sex
Remarks on result:
other:

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Generation:
F1
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings:
no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):
no effects observed

Effect levels (F2)

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

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

Any other information on results incl. tables

Unscheduled Deaths

Unscheduled necropsies were conducted on one female rat from the P0 generation, six males from the F1 generation, and four females from the P1 generation because the rats were either found dead or euthanized for humane reasons when observed to be moribund. The causes of death or moribundity were considered incidental and unrelated to1,3- and 1,4-CHDA treatment. 

 

The following were the animals with unscheduled necropsies and the cause of death:

1)P0 female (5934) in the 150 mg/kg/day group died on TD 84 due to accidental administration of test material into the lungs (gavage complication). At gross necropsy, test material/vehicle was present externally on the muzzle and nares and internally within the trachea and lungs.

2) F1 male (1215) from the control groupwas euthanized for humane

reasons on TD 70 due to trauma to the lumbar spine. At gross necropsy, there was a laxity within the lumbar vertebral column.

3) F1 male (1217) from the control groupwas euthanized for humane reasons on TD 94 due to accidental trauma to the muzzle resulting in a fracture of the incisors and maxilla.

4) F1 male (1249) from the 10 mg/kg/day groupwas euthanized for humane reasons on TD 120 due to accidental trauma to the tail, fracturing and exposing bone and connective tissues.

5) F1 female (1368) from the 50 mg/kg/day group diedon TD 26 due to an accidental administration of test material into the lungs (gavage complication). Histopathology of the lung demonstrated alveolar hyperplasia and chronic inflammation indicative of additional antecedent non-fatal gavage complications.

6) F1 female (1372) from the 50 mg/kg/day group diedon TD 15 due to accidental administration of test material into the lungs (gavage complication). At gross necropsy, test material/vehicle was present externally on the nares and internally within the trachea and lungs.

7) F1 female (1380) from the 50 mg/kg/day group, was euthanized on TD 97 (GD 24) for humane reasons due to dystocia. At necropsy, there were nine dead and/or resorbed fetuses within the uterus and one within the birth canal. Histologically, there was subacute, diffuse, slight inflammation of the epithelium of the uterus and the mucosa/submucosa of the vagina. 

8) F1 male (1294) from the 150 mg/kg/day group diedon TD 9 due to accidental administration of test material into the lungs (gavage complication). At gross necropsy, test material/vehicle was present externally on the muzzle and nares and internally within the trachea and lungs.

9) F1 male (1298) from the 150 mg/kg/day groupwas euthanized for humane

reasons on TD 88 due to accidental trauma to the muzzle resulting in a fracture of the incisors and maxilla.

10) F1 male (1303) from the 150 mg/kg/day groupwas euthanized for humane

reasons on TD 68 due to accidental trauma to the tail, fracturing and exposing bone and connective tissues.

11) F1 female (1384) from the 150 mg/kg/day group diedon TD 70 due to accidental administration of test material into the lungs (gavage complication). At gross necropsy, test material/vehicle was present externally on the nares and internally within the trachea and lungs.

Applicant's summary and conclusion

Conclusions:
Treatment of rats with 1,3- and 1,4-CHDA for two generations did not result in treatment-related effects on any in-life parameter or any parameter of reproductive function or offspring survival, growth or development.
Treatment-related effects on organ weight were limited to increased kidney weights in P1 females and P2 males and females administered 150 mg/kg/day. At this dose level, absolute and relative P1 female kidney weights were increased 6.8% and 4.8%, respectively, compared to control. In the P2 generation at 150 mg/kg/day, absolute and relative kidney weights were increased in males (6.3 and 6.5%, respectively) and females (6.9 and 6.4%, respectively) compared to control. There was no treatment-related effect on absolute or relative kidney weights in any parental generation at 10 or 50 mg/kg/day. There were no treatment-related effects on F1 or F2 weanling organ weights in either generation.
Treatment-related gross pathologic and histologic observations were limited to the stomach of P1 and P2 male and female rats. At 150 mg/kg/day, P1 and P2 males and females had thickened squamous mucosa at the junction of the glandular and non-glandular portions of the stomach (i.e. stomach limiting ridge). Histopathologically, this gross observation corresponded to very slight to slight hyperplasia and hyperkeratosis at the stomach limiting ridge in most P1 and P2 males and females administered 150 mg/kg/day. At 50 mg/kg/day, gross pathology was limited to one P2 male with thickening of the stomach limiting ridge, and histopathological changes were limited to very slight hyperkeratosis and very slight to slight hyperplasia of the stomach limiting ridge in P2 males and females. No treatment-related gross pathologic or histologic changes were observed in any parental generation at 10 mg/kg/day. No treatment-related gross pathologic change was observed in F1 or F2 weanlings at any dose level.
Under the conditions of the study and based on the increased P1 and P2 kidney weights at 150 mg/kg/day, the no-observed-effect level (NOEL) for systemic toxicity was
50 mg/kg/day. Based on the stomach histologic change at 50 mg/kg/day, the NOEL for point of contact toxicity was 10 mg/kg/day. Due to the lack of any treatment-related effects on reproductive performance or offspring survival, growth and development, the NOEL for reproductive toxicity was 150 mg/kg/day, the highest dose level tested.
Executive summary:

The purpose of this oral gavage two-generation reproduction toxicity study was to evaluate the potential effects of 1,3- and 1,4-cyclohexanedicarboxaldehyde (1,3- and 1,4-CHDA)on male and female reproductive function, as well as the survival, growth and development of the offspring. 

Groups of 25 male and 25 female Crl:CD(SD) rats were administered the test material seven days/week via oral gavage at dose levels of 0, 10, 50, and 150 mg1,3- and 1,4-CHDA/kg of body weight/day (mg/kg/day, mkd)for approximately ten weeks prior to breeding and continuing through breeding, gestation and lactation for two generations. In-life parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In addition, post-mortem evaluations included gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.

Treatment of rats with 1,3- and 1,4-CHDA for two generations did not resultin treatment-related effects on any in-life parameter or any parameter of reproductive function or offspring survival, growth or development. 

Treatment-related effects on organ weight were limited to increased kidney weights in P0 females and F1 males and females administered 150 mg/kg/day. At this dose level, absolute and relative P0 female kidney weights were increased 6.8% and 4.8%, respectively, compared to control. In the F1 generation at 150 mg/kg/day, absolute and relative kidney weights were increased in males (6.3 and 6.5%, respectively) and females (6.9 and 6.4%, respectively) compared to control. There was no treatment-related effect on absolute or relative kidney weights in any parental generation at 10 or 50 mg/kg/day. There were no treatment-related effects on F1 or F2 weanling organ weights in either generation.

Treatment-related gross pathologic and histologic observations were limited to the stomach of P0 and F1 male and female rats. At 150 mg/kg/day, P0 and F1 males and females had thickened squamous mucosa at the junction of the glandular and non-glandular portions of the stomach (i.e. stomach limiting ridge). Histopathologically, this gross observation corresponded to very slight to slight hyperplasia and hyperkeratosis at the stomach limiting ridge in most P0 and F1 males and females administered 150 mg/kg/day. At 50 mg/kg/day, gross pathology was limited to one F1 male with thickening of the stomach limiting ridge, and histopathological changes were limited to very slight hyperkeratosis and very slight to slight hyperplasia of the stomach limiting ridge in F1 males and females. No treatment-related gross pathologic or histologic changes were observed in any parental generation at 10 mg/kg/day. No treatment-related gross pathologic change was observed in F1 or F2 weanlings at any dose level.

Under the conditions of the study and based on the increased P0 and F1 kidney weights at 150 mg/kg/day, the no-observed-effect level (NOEL) for systemic toxicity was
50 mg/kg/day.
 Based on the stomach histologic change at 50 mg/kg/day, the NOEL for point of contact toxicity was 10 mg/kg/day. Due to thelack of any treatment-related effects on reproductive performance or offspring survival, growth and development, the NOEL for reproductive toxicity was150 mg/kg/day, the highest dose level tested.