4-(1,1-dimethylethyl)cyclohexyl acrylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(1,1-dimethylethyl)cyclohexyl acrylate

EC Number:

282-104-8

EC Name:

4-(1,1-dimethylethyl)cyclohexyl acrylate

Cas Number:

84100-23-2

Molecular formula:

C13H22O2

IUPAC Name:

4-tert-butylcyclohexyl prop-2-enoate

Test animals

Species:

rat

Strain:

Wistar

Remarks:

CRL: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 141-187g (males), 108-142g (females)
- Housing: groups of up to 5 animals
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21 °C
- Humidity (%): 44-74% (The values that were outside the targeted mean humidity range of 40-70% occurred occasionally and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.)
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle: solubility
- Concentration in vehicle: 0 -100 mg/mL
- Amount of vehicle: 5mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All analyzed formulation of all dose groups (day 1 and in weeks 6 and 12) were within 90 to 110% of the target concentration. No test material was detected in the control formulations. All formulations were homogeneous, i.e., coefficient variation <=10%)

Duration of treatment / exposure:

at least 90 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on previous OECD 422

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, 0-1h post dose

CHECK FOR MORTALITY: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

ARENA OBSERVATION
- before first administration and weekly during treatment

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: regular basis via visual inspection of water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once prior to dosing (all groups) and during week 13 (group 1 and 4, remaining animals only, if differences noted)


HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: WBC, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, RBC, reticulocytes, hemoglobin, hematocrit, MCV, MCH, MCHC, platelets, red blood cell distribution width

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters examined: prothrombin time, APTT, ALT, AST, ALP, total protein, albumin, bilirubin, urea, creatinine, glucose, cholesterol, triglycerides, HDL and LDL cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, T3, T4, TSH

URINALYSIS: Yes
- Time schedule for collection of urine: end of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume, specific gravity, clarity, color, pH, blood, leukocyte esterase, bilirubin, urobilinogen protein, ketones, glucose, nitrite

NEUROBEHAVIOURAL EXAMINATION (FOB): Yes
- Time schedule for examinations: once during weeks 12-13
- Dose groups that were examined: 5 animals per group
- Battery of functions tested: sensory activity, grip strength, motor activity, general appearance/reactions to handling

ESTROUS STAGE
- Time schedule: day of necropsy

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (for low and mid dose only gross lesions and target tissues unless differences observed between high dose and control)

Optional endpoint(s):

staining for alpha-2µ in high dose and control males

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg: Slight, transient tremors of the forelimbs, head and shoulder muscles, and closed eyes were observed on Days 86-88 in all males (prior to functional observations on Day 86) and females.

Salivation was observed in all dose groups, which is considered a consequence of local irritation and/or bad taste of the test substance due to the temporary and short appearance right after dosing.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1 control animal was euthanized on Day 46 due to complication with the gavage procedure

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500mg/kg, females body weights were higher from day 57 onward, resulting in a 12% increase by day 91. In absence of corroborating findings, this change was considered not adverse.

In addition, male body weights were 5% lower on day 91 as a consequence of lower body weight gain after day 50. The difference was not statistically significant and because of the low magnitude of the change likely represents normal variation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

at 500mg/kg: higher food consumption in females starting on day 50 (+10% over entire treatment period). This finding was not considered adverse.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, females showed a higher white blood cell count (1.43x of control), consisting of higher neutrophil (1.51x, not statistically significant), lymphocyte (1.41x), monocyte (1.70x) and eosinophil (1.42x) count. In addition, higher large unstained cell (2.22x) and reticulocyte count (1.20x) were noted in females. Also, a non-significant shorter activated partial thromboplastin time was observed (0.89x of control). In the absence of corroborating findings, these changes were considered not adverse.

Females at 500 mg/kg bw/day also showed a higher reticulocyte count, which correlated with an increased incidence and/or severity of extramedullary hematopoiesis and pigment in the spleen. The minor increase in extramedullary hematopoiesis and pigment were considered non-adverse due to their adaptive nature.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, lower albumin concentration at 50, 150 and 500 mg/kg bw/day (0.97, 0.95 and 0.95x of control, respectively), and lower total protein concentrations at 150 and 500 mg/kg bw/day (both 0.95x) were noted. In addition, higher urea (1.16x) and inorganic phosphate (1.13x) concentrations, and lower glucose concentrations (0.80x) were observed at 500 mg/kg bw/day.
In females, higher cholesterol (1.28x; not statistically significant), HDL cholesterol (1.26x; not statistically significant), and potassium (1.11x) concentrations were observed at 500 mg/kg bw/day.
The changes were generally minor, there were no corroborating findings, and might largely be related to enhanced liver metabolism. They are not considered adverse.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

In females at 500 mg/kg bw/day, higher T4 concentrations were observed (1.80x of control). The T4 value for 7/10 females at 500mg/kg remained within the normal range historically observed in rats under similar study conditions. In parallel studies, higher control values matching those of the high dose group were observed.
T3 and TSH concentrations were not affected, there were no histopathological findings.
There was no effect in males.
Overall, the change is not considered adverse and relation to treatment is unlikely.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher kidney weights in males (relative at 150mg/kg, absolute and relative at 500mg/kg)
Higher liver weights (relative in males at 500mg/kg; absolute and relative in females at 150 and 500mg/kg). This change was considered adaptive and non-adverse.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pale discoloration of kidneys in 2 high dose males (500mg/kg), as a consequence of α2µ-globulin nephropathy

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the kidney of males, microscopic findings were noted starting at 50 mg/kg/day. These consisted of an increased incidence and severity of hyaline droplet accumulation (minimal to marked), granular casts (minimal to marked), tubular degeneration (minimal to mild) and an increased incidence and severity of tubular basophilia (minimal to marked). All these changes were observed to be in a dose-related manner. Immunohistochemistry for alpha 2u-globulin was performed on the kidney of all control and treated males. Hyaline droplets as well as the granular casts stained positive for alpha 2u-globulin in all control (hyaline droplets only) and treated males.

In the liver, hepatocellular hypertrophy (minimal) was noted in 2/10 males and females at 500 mg/kg bw/day. In the thyroid gland, an increased incidence of follicular cell hypertrophy (minimal) was noted in males at 500 mg/kg bw/day. Because of the low grade of these findings they were not considered adverse.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

> 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

2

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: a2µ nephropathy

Remarks on result:

other: male rat specific finding, not relevant for human risk assessment

Target system / organ toxicity

Key result

Critical effects observed:

no