3-cyano-3,5,5-trimethylcyclohexanone

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-07-09 to 1991-11-19

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-cyano-3,5,5-trimethylcyclohexanone

EC Number:

411-490-4

EC Name:

3-cyano-3,5,5-trimethylcyclohexanone

Cas Number:

7027-11-4

Molecular formula:

C10H15NO

IUPAC Name:

3-cyano-3,5,5-trimethylcyclohexanone

Details on test material:

Isophorne nirile from Degussa AG, purity: 99.6%, Batch No. 64 II/87/90

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Strain: Bor: WISW (SPF Cpb)
- Source: Winkelmann GmbH & Co. KG, Borchen (Germany)
- Age: males 7 weeks, females 8 weeks
- Weight at study initiation: males 186 - 225 g, females 141 - 175 g
- Housing: single housing
- Diet: ad libitum, special diet for rats, SSniff R
- Water: ad libitum, tab water
- Acclimation period: 3 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 40 - 70 % (for short periods down to 35% or up to 75 %)
- Photoperiod (hrs dark / hrs light): 12 hours artificial light, 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: aqueous Tragant(R) (1 %) suspension

Details on oral exposure:

ADMINISTRATION: 
- Preparation of test substance: daily preparation of suspension in vehicle immediately before dosing using Ultraturrax homogenizer
- Doses per time period: once daily, 7 days per week
VEHICLE
- Concentration in vehicle: 3.83, 8.25, 17.8/14.7 *mg/ml, * in test week 6 dose for group 4 were reduced because of severe clonoc convulsions
- Amount of vehicle (if gavage): 2,15 ml/kg
- Lot/batch no. (if required): Tragacanth, batch 821 V57 6905 (E. Merck)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of isophorone nitrile in aqueous tragacanth suspensions were determined by gas chromatographic method (FID). Sodium chloride
and methanol were added to the suspension and treated in an ultrasonic bath, the phases were separated using a centrifuge. Internal standard was
added to methanolic phase and the solution were analyzed using gaschromatographie
Stability test demonstrate that the isophorone nitrile remeins stable during a shipping period of approximately 24 hours at ambient temperatrures

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

once daily a.m., 7 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
8.25, 17.8, 38.3/31.6* mg/kg bw; * in test week 6 dose for group 4 were reduced because of severe clonoc convulsions
Basis:
other: in aqueous Tragant(R) (1 %) suspension

No. of animals per sex per dose:

control: 20 (including 10 recovery animals
low and mid test group: 10 animals
high testr group: 20 (including 10 recovery animals

Control animals:

other: yes, 1% tragacanth suspension

Details on study design:

- Dose selection rationale: dose finding study at doses of 17.8, 26.1, 38.3, 56.2, 68.1, 82.5 mg/kg 4 weeks treatment. At Doses below 38.3 mg/kg
no symptoms appeared, at a dose of 38.3 mg/kg convulsions were observed and doses above this level caused mortality.
- Post-exposure period: 6 weeks, Rats of main groups were sacrified at the end of the 13-week treatment period. Additional animals
(10 rats/sex/group) were allocated to the control and high-level group that served as satellite rats which were subjected to a postexposure
observation period of approx. 6 weeks.

Positive control:

no positive control

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs: were observed daily for the occurence of toxicityc symptoms and their severity and duration
- Mortality: was checked twice daily, on Saturday, Sunday and business holidays only once daily
- Body weight: once per week , starting with pretest period
- Food consumption: once a week, starting with pretest period
- Reflexes: pain, pinna and corneal refexes were tested once a week, starting with pretest period
- Hearing and Teeth: prior to first substance administration and in test weeks 6 and 13
- Ophthalmoscopic examination: in pretest week 1 and in test week 6 and 13 eyes of all animals of groups 1 and 4 were examined by biomicroscopy
with slit lamp 30 SL/M (Carl Zeiss)
- Haematology and Clinical Chemistry: weeks 6 and 12 a.m. as well as in test week 19 from the recovery animals, blood was collected from the
retro orbital venous plexus of onr eye under CO2 anesthesia :  hematocrit (Hct), hemoglobin (Hb), leukocytes (WBC), erythrocytes (RBC) ,mean 
corpuscular volume (MCV),  mean corpuscular hemoglobin concentration (MCHC),  reticulocytes,  Leukocyze differential count,
aspartate  aminotransferase (optimized, ASAT), alanine aminotransferase(optimized, ALAT), glutamate dehydrogenase (GLDH), gamma-
Glutamylaminotransferaase, alkaline phosphatase (AKP),  albumin, total bilirubin, blood glucose, total protein, triglycerides,  cholesterol, creatinine,
blood urea, sodium, potassium
- Urinalysis: in test week 13 with test strips COMBUR9 and photometer determination of  pH, protein, glucose, hemoglobin/erythrocytes, bilirubin, 
urobilinogen, ketones, nitrite, osmolality and  leukocytes. Microscopical examinations of the urine sediment were done in animals whose urine
state showed pathological changes in hemoglobin/erythrocytes or protein

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Sacrifice: at the end of the treatment or recovery period the survived animals were anesthetized with CO2 ans sacrificed by exsanguination
- Organ weights: Adrenals, brain, heart, kidneys, liver, ovaries, pituitary, prostate/seminal vesicles in toto, spleen, testes, thymus.
Organs weights were expressed as absolute and relative to body weight after exsanguination
- Gross Necropsy: all animals were subjected to full cross necroscopy which included examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contens.
Following organs or tissues were preserved: all gross lesions, adrenal glands, aorta, axillary lymph node, bone (femur, tibia, sternum), bone
marrow, bone marrow smear, brain, caecum, cervix, colon, duodenum, epididymides, eyes with optic nerve, Harderian  glands,  heart, jejunum, 
ileum, kidneys, knee joint, liver, lungs, mammary gland, mesenteric lymph nodes, oesophagus,  ovaries, pancreas, parathyroid glands,
peripheral nerve, pituitary gland, prostate, rectum, salivary  glands, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes,
thymus,  thyroid glands, tongue, trachea, urinary bladder, uterus, vagina
- Microscopical: all slides prepared with exception of bone marrow smears (no respective changes in peripheral blood parameterers) were
examined microscoically

Other examinations:

no other examinations

Statistics:

Mean values of all parameters and - where indicated- standard deviations were calculated separately for each group and sex.
For statistical evaluation of food consumption, body weights, and organ weights the DUNNETT-Test was used. For values of hematological and clinical chemistry examinations the DUNETT-Test was used in case of normal distribution, otherwise the STEEL-test was employed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality: no substance related mortality
Food consumtion/Body weight: neither food consumption nor body weight were affected. 
Clinical signs /Neurobehaviour: dose related, temporary and reversible changes in the behaviour (hyperkinesia, hyperactivity, clonic/tonic convulsion, salivation, tremor). No changes in reflexes, teeth and hearing  
Macroscopicaly: no substance related findings
Microscopicaly/Histopathology a dose related partial disappearance of glycogen deposits in liver cells of males, after 6-week recovery period the finding was 
no longer detected.
Organ weights: slight increases in absolute organ weights in high dose group in males and females in week 14

Effect levels

Dose descriptor:

NOAEL

Effect level:

8.25 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

no further remarks

Applicant's summary and conclusion

Conclusions:

In a 13 week oral subcronic toxicity study, conducted with rats, the test item Isophorone nitrile showed effects on central and/or motoric nervous system ( hyperkinesia, hyperactivity, clonic convulsion, salivation) as well as histopathological alterations in the liver of males. The non-toxic dose (NOAEL) of the test item Isophorone nitrile is determined to be 8.25 mg/kg bw under the conditions of this study.

Executive summary:

The toxicity of Isophorone nitrile after repeated oral administration was examined in a 13 week subchronic toxicity study conducted in rats. Obvious changes in the behaviour of treated animals were the temporary and fully reversible increase of locomotor activity (hyperkinesia, hyperactivity), the occurence of clonic and tonic convulsions and tremor. These findings occured in a dose releated pattern and may be judged as expression of a stimulation of the central and/or the motoric nervous system. Regarding the observed salivation, this effect can be attributed to a stimulation of vegetative functions, so that different parts of the central nervous system and the vegetative nervous system must be considered as target regions for Isophorone nitrile.

In the microscopic examination of the preserved tissues the liver is noticed as an affected organ. The partial disappearance of glycogen deposits in liver cells is dose and in so far treatment related. As there was no functional correlate, e.g. reduction of serum glucose levels and degenerative changes were absent, this finding is considered to have an adaptive nature. After the 6 -week recovery period the finding was no longer detected. A final evaluation of this phenomen on the basis of this study is not feasible.

Because only two low dose animals showed slight hyperkinesia occasionally in week 4 and no other effects were noted in the low dose group the non toxic dose of isophorone nitrile is still considered to be 8.25 mg/kg bw.