Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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).
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).
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
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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:
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
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
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
Behaviour (functional findings):
no effects observed
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
Key result
Reproductive effects observed:
not specified

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.

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Both the reproductive screening study and teh 2-generation study in rats showed no evidence of reproductive toxicity
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 421 reproductive study (rats)

The reproduction/developmental study is compliant with OECD 421 protocol and is assigned a Klimisch 1 rating. This study represents the only repro/dev study for the test material.

The test substance was administered in graduated doses to three groups of male and female rats. The males were dosed for a period of at least six weeks (which included 2 weeks prior mating, 2 weeks during mating and 2 weeks post mating), up to and including the day before sacrifice. Females were dosed throughout the treatment period. This included two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and four days after delivery.

Treatment with the test substance 1,3 and 1,4 Cyclohexanecarboxaldehyde did not cause any test substance related effects on general health, body weights, feed consumption, mating and fertility, mean number of corpora lutea and implantations, live birth index, mean litter size, growth and development of pups. There were no test substance related changes in terminal fasting body weights and organ weights at all the doses tested. Multiple raised foci and/or diffuse thickening was observed grossly in non glandular stomach in 400 mg/kg/day dose males and females. Microscopically these observations were associated with ulcers and epithelial hyperplasia/hyperkeratosis and were considered test substance-related adverse changes. 

 

Under the conditions of this study, based on the adverse effects which were limited to the non-glandular stomach of males and females at 400 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for local effects was determined to be 150 mg/kg/day, while the NOAEL for systemic effects was 400 mg/kg/day. As there were no adverse effects on reproduction and fertility parameters up to and including 400 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was determined to be 400 mg/kg/day, the highest tested 1,3 and 1,4 Cyclohexanecarboxaldehyde dose level.

OECD 416 2 -generation reproductive toxicity study:

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 mg 1,3- 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 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.


Short description of key information:
2 Studies are available fore assessing the reproductive toxicity: and OECD 421 screening study and an OECD 416 2-generation study in rats

Justification for selection of Effect on fertility via oral route:
reliability 1, GLP OECD guideline reproductive toxicity screening study

Effects on developmental toxicity

Description of key information
A full guideline OECD 414 Developmnetal toxicity study in rats.
Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
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.

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.
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.
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.
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
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.
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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

None

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.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
225 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High: A full OCED guideline Developmental toxicity study via the oral route is available and supported by an OECD 421 reproductive/developmnetal screening study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Oral Developmental toxicity study in rats (OECD 414)

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.

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.

Given the absence of developmental toxicity in rats up to the highest dose tested, it is proposed that no further developmental toxicity testing is required (in a second species) until the next tonnage band is reached.


Justification for selection of Effect on developmental toxicity: via oral route:
The key study is an OECD guideline, GLP compliant Developmental toxicity study.

Justification for classification or non-classification

There were no adverse effects observed on reproductive and developmental endpoints and so no classification is warranted.