4-methylcyclohexanone

Endpoint summary

Description of key information

Oral route

A 28-day repeated dose toxicity study in the rat via the oral route was conducted under GLP according to guideline OECD TG 407. Groups of 5 male and 5 female Han Wistar Crl:WI (Han) rats received the test substance at dose levels of 50, 150, and 500 mg/kg/day along with a concurrent control. The animals were observed for general health/mortality and moribundity, as well as clinical signs and functional observations. Body weights, food consumption and motor activities were measured and recorded at pre-determined intervals. Blood samples were collected from all animalsat necropsy to evaluate haematology, coagulation and clinical chemistry parameters. Urine was collected from animals during Week 4 and urinalysis parameters were evaluated. All animals were terminated after completion of 28 days of treatment and underwent a detailed necropsy examination with selected organs weighed. Tissues from the control and high dose groups were subjected to a comprehensive histological examination with the liver being examined from all four dose groups.

 

There were no premature decedents. Decreased activity, partial closing of the eyes and irregular respiration were noted in all males and females receiving 500 mg/kg/day from approximately Day 1 until Day 10 of the study; however, the duration of the observation decreased over time suggesting a tolerance had developed. Erect fur was also noted in all animals receiving 500 mg/kg/day. These findings correlated with increased ease of handling in all animals and the hunched posture. They also correlated with the piloerection/erect fur and fur staining noted in some males and females receiving 500 mg/kg/day during the functional observation battery. In addition, ploughing and salivation were noted in all animals receiving 150 or 500 mg/kg/day.Overall body weight gain and food consumption was higher in females, but not males, receiving 500 mg/kg/day compared with controls. There were no differences in qualitative or quantitative functional observations or motor activity assessments noted during the neurotoxicity assessment.

 

Haematology and coagulation parameters and gross pathology findings in males and females were unaffected by administration of the test substance. Alanine aminotransferase (40%) and alkaline phosphatase (50%) were higher in males receiving 150 or 500 mg/kg/day compared with controls. Alanine aminotransferase was up to 140% higher in females receiving 50, 150 or 500 mg/kg/day compared with controls. Clinical chemistry changes were associated with dose-dependent increased liver weights recorded in all treated males and females and centrilobular hepatocyte hypertrophy present in males and females at 500 mg/kg/day. Liver weights were statistically significantly higher for males and females at 150 mg/kg/day and above. Clinical chemistry and liver weight changes in animals at 150 mg/kg/day and below, are not associated with the pathology changes, so considered to be adaptive and non-adverse.

 

Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) following 28 days of dosing with 4 -methylcyclohexanone was 150 mg/kg/day.  The NOAEL is based on increased body weight in females and decreased activity, irregular respiration and convulsions in males and females at 500 mg/kg/day. In addition centrilobular hepatocyte hypertrophy of the liver with increased liver weights and liver enzymes activity was noted in males and females at 500 mg/kg/day. The study was conducted under GLP according to a guideline method, and was thus assigned a Klimisch reliability rating 1. In accordance with criteria set out in REACH, Annex VIII, 8.6.1, column 2, no further testing is required at this tonnage band.

Inhalation route

The repeated dose toxicity via the inhalation route was studied in rabbits in a non-standard, pre-OECD guideline experiment (Treon et al. 1943), which is considered as supporting information due to the methodological deficiencies. Groups of four healthy, young rabbits were exposed whole body to nominal concentrations of 0.82, 2.31, 5.12 or 8.19 mg/L for 6 hours per day, 5 days per week. The test duration was 10 weeks for the two lower concentrations and 3 weeks for the two higher concentrations. No mortality occurred in the study in any of the test groups, and no significant effects on body weight gain or studied haematologic parameters were reported. Exposure to the two higher concentrations resulted in lethargy, distention of the superficial blood vessels in the ear, signs of conjunctival irritation, lacrimation and salivation, whereas exposure to the moderate concentration caused only slight conjunctival irritation and exposure to the lowest concentration did not cause any adverse effects. The gross and microscopic histopathology examination found unspecified signs of vascular injuries and inflammatory response to these injuries.

The study was assigned a Klimisch reliability rating 3, and is not considered reliable for the purposes of risk assessment or classification.

Dermal route

The repeated dose toxicity via the dermal route was studied in a single young white rabbit in a non-standard, pre-OECD guideline experiment (Treon, 1943), which is considered as supporting information due to the methodological deficiencies. The test substance was applied twice daily at a single fixed dose, separated by 30min before removal, for a period of 6 days. The total dose per day was ca. 3133 mg/kg bw. The animal died 15min after dosing was completed on the sixth day. Effects on heart, liver, kidney and vascular degeneration in the lung were observed, along with narcosis, tremors and convulsions.

The study was assigned a Klimisch reliability rating 3, and is not considered adequate or reliable for the purposes of risk assessment or classification.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 November 2016 to 09 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Appendix to Director General Notification, No. 12-Nousan-8147, JMAFF

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han Wistar rats Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 6-7 week
- Weight at study initiation: 171-233 g (males), 114-161 g (females)
- Fasting period before study: No
- Housing: 2 or 3 per cage per sex in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings.
- Diet: SDS Rat and Mouse (modified) No. 1 Diet SQC Expanded ad libitum
- Water: Mains water ad libitum
- Acclimation period: Approximately 2 weeks

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for bedding, food, water and environmental enrichments were provided by suppliers to the Test Facility. The suppliers analysed the materials for various substances including, where appropriate, nutritive components, significant contaminants, dissolved materials, heavy metals, pesticide residues, pH, nitrates, nitrites, and also conducted a microbiological screen. Materials were used within the manufacturers' designated shelf-life. It was considered that there were no known contaminants in any of these that could have interfered with the objectives of the study. Copies of all certificates of analysis are retained at the Test Facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 53-64
- Air changes (per hr): Minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09 November 2016 To: dd month year

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/w) Methylcellulose (400 cPs) and 0.5% (w/w) TWEEN 80 in Milli-Q Water.

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The dosing formulations were prepared at least weekly, stored at 4°C, protected from light, and dispensed daily. The required amount of test substance was added to the vehicle and magnetically stirred until a homogeneous, white suspension was achieved. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing.

- VEHICLE
- The control substance/vehicle was prepared at least weekly, stored at 4°C, and dispensed daily. It was prepared by heating 50% of the required Milli-Q water to between 70-90°C, and slowly adding the methylcellulose while stirring. The required weight of Tween 80 was added to the solution, while stirring. The heat was switched off and the formulation was magnetically stirred until homogeneous. The formulation was then made up to the final volume and stirred until homogeneous. It was removed from the refrigerator and stirred for at least 30 minutes before use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis on day 1 (week 1 preparations) and week 4. Achieved concentration was analysed for all groups and homogeneity was analysed on samples collected from the top, middle and bottom of the Group 2 to 4 preparations. Analysis was by HPLC with LC-MS/MS detection.
Analytical results: The majority of study samples examined had mean and individual concentrations within or equal to the acceptance criteria of ± 10% (actual values +8.6% to -10.0%) of their theoretical concentrations. The only exception was for Week 4, Group 4 where 2 out of 6 of the individual values of 10.8% and -10.4% were attained, however the mean was within the acceptance criteria of ± 10%. The analytical samples were reanalysed, and the result confirmed that the individual values were outside acceptance criteria (-7.2% to -14.2%), as well as the mean during reanalysis. Consequently, the backup samples for the Group 4 dose formulation were analysed in triplicate, and the results (-32.2% mean concentration and individual concentrations of 25.0% to -43.8%) were found to be outside the acceptance criteria. However, it was noted that the first Group 4 backup sample container was incorrectly stored without a lid, resulting in questionable analytical results. The backup samples were therefore considered unreliable due to this deviation to the protocolled storage conditions. As the original Group 4 samples were only marginally outside the acceptance criteria and only 2/6 from the original run were outside the ±10%, this event is considered not to have had an impact on the outcome of the study.
Homogeneity results: The RSD of concentrations for all samples in each group was within the acceptance criteria of less than or equal to 5%, except for back-up formulation Week 4, group 4 (8.8%). As the backup formulation samples were considered to be suspect, this was considered not to have had an outcome on the study

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The test substance was tested previously in a 14-day tolerability study in rats with adverse clinical findings observed in males and females at the 750 mg/kg/day group. Therefore, 500 mg/kg/day was selected as the high dose level with the other lower dose levels included to assess the dose-response relationship.
- Rationale for animal assignment: Animals were assigned to groups by a stratified randomisation scheme designed to achieve similar group mean body weights. Males and females were randomised separately.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (general health, mortality and moribundity)
- Time schedule: Twice per day
- Post dose: All animals were observed regularly throughout dosing days for reaction to treatment. The onset, intensity and duration of any signs was recorded, as appropriate, with particular attention being paid during dosing and for the first hour after dosing.
-Detailed clinical observations: All animals were removed from their cage and subjected to a detailed clinical examination on a weekly basis beginning Week -1.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice during Week -1, and then once daily throughout
the dosing period. A weight was also recorded on the day of scheduled necropsy.

FOOD CONSUMPTION:
- Time schedule for examinations: Twice during Week -1, and then once daily throughout the dosing period.

WATER CONSUMPTION:
- Time schedule for examinations: Monitored by visual inspection on a regular basis throughout the
study.

OPHTHALMOSCOPIC EXAMINATION: No

DETAILED FUNCTIONAL OBSERVATIONS: Yes
- Time schedule for examinations: Week 4
- Dose groups that were examined: All
- Battery of functions tested:
Home cage observations (Prostration, Stereotypy / bizarre behaviour, Tremors (head, limbs, whole body), Convulsions, Ease of removal from the cage)
Body temperature
Handling observations (Condition of the eyes, checked for Pupillary function, Miosis / Mydriasis, Enophthalmos/ Exophthalmos, Lacrimation, Evaluation of diameter of the pupil); Condition of the coat; Body tone; Pinna response; Presence of salivation; Overall ease of handling; Respiration rate and pattern
Extensor thrust
Observation in the standard arena - 2 min observation period (Rearing, Grooming, Urination and defecation, Arousal (level of alertness), Posture, Tremor (head, limbs, whole body), Convulsions, Vocalisation, Piloerection, Palpebral, closure, Gait abnormalities, Stereotypy (excessive repetition of behaviours) and/or unusual behaviours)
Functional tests: Reaction to sound and touch, fore- and hind-limb grip strength, pain perception, landing foot splay, motor activity

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: All
- Parameters examined.: Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Red blood cell count, Red Blood Cell Distribution Width, Reticulocyte count (absolute), Platelet count, White blood cell count (total), Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute), Activated partial thromboplastin time, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: All
- Parameters examined.: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Total bilirubin, Urea /BUN, Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride, Bile Acids

URINALYSIS: Yes
- Time schedule for collection of urine: During Week 4 (6 hour collection period)
- Metabolism cages used for collection of urine: Yes
- Animals fasted during collection: Yes
- Parameters examined.: Colour, Appearance/Clarity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Blood

Sacrifice and pathology:

Animals surviving until scheduled euthanasia were euthanised by exposure to rising levels of carbon dioxide, had a terminal body weight recorded, followed by exsanguination. The animals were euthanised in a rotating order across dose groups such that similar numbers of animals from each group, including controls, were necropsied throughout the day.

GROSS PATHOLOGY: Yes (evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues).

ORGAN WEIGHTS: Yes (brain, epididymes, adrenal glands, prostate glands, seminal vesicles, thyroids, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus

HISTOPATHOLOGY: Yes
Groups: Controls and high dose
Tissues examined: Aorta, Bone marrow, Bone - femur, Bone - sternum, Brain, Epididymis x 2, Eye x 2, Adrenal x 2, Mammary gland, Parathyroid x 2, Pituitary, Prostate, Salivary x2, Seminal vesicle with coagulating gland, Thyroid x2, Gut-associated lymphoid tissue (Peyer’s Patches), Heart, Kidney x 2, Caecum, Colon, Rectum, Lesions/masses, Liver, Lung, Lymph node - mandibular, Lymph node - mesenteric, Muscle - skeletal, Optic nerve, Sciatic nerve, Oesophagus, Ovary x 2, Pancreas, Skin, Duodenum, Ileum, Jejunum, Spinal cord, Spleen, Stomach, Testis x 2, Thymus, Trachea, Urinary bladder, Uterus with cervix, Vagina

ADDITIONAL LIVER SAMPLING: Yes
- For all animals, a representative section from the left lateral lobe, the right median lobe and the caudate lobe were taken and fixed in 10% neutral buffered formalin for 36-48 hours and then processed to paraffin wax block. A small piece of duodenum was incorporated into each block.
Multiple samples of liver (8 x approximately 150 mg) were taken from two 5 mm sections of the left lateral lobe. The samples were snap frozen in liquid nitrogen in individual RNA-ase free tubes. All samples were taken as quickly as possible and stored in a freezer set to maintain -80ºC pending possible future analysis.

The remainder of the liver was cut into chunks (approximately 2 g) and snap frozen in liquid nitrogen. Samples were stored in a freezer set to maintain -80ºC pending possible future analysis.

BONE MARROW SMEAR EVALUATION: Two bone marrow smears were collected from the femur at necropsy. Bone marrow smears were allowed to air dry and were fixed in Methanol. Both smears were stained using May-Grunwald-Giemsa as soon as practical after necropsy.

Statistics:

All analyses were two-tailed for significance levels of 5% and 1%.
All means for ANOVA are presented with standard deviations.
Means for ANCOVA are presented with standard error.
If the variances are clearly heterogeneous, appropriate transformations (e.g. log, square root, double arcsine) were used in an attempt to stabilise the variances.
Body weights, cumulative body weight changes, selected functional observation battery and motor activity parameters, absolute organ weights, selected urinalysis, haematology, coagulation and clinical chemistry were analysed initially by a one-way analysis of variance (ANOVA).
Organ weights were also analysed by analysis of covariance (ANCOVA) on final body weight. This statistical analysis provided an Adjusted Organ Weight value, which is displayed in the results table along with flags for statistical significance.
Summary values of organ to body weight ratios were presented but these were not analysed statistically.
For all of the parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett’s test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant, and statistical flags are presented in the tables of results in the final report.
Functional observational battery parameters that yielded discontinuous or descriptive data were analysed using Fisher’s Exact Test.
Micropathology incidence data were analysed using Fisher’s exact test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Decreased activity, partial closing of the eyes and irregular respiration were noted in all males and females receiving 500 mg/kg/day from almost immediately post dose on Day 1. This observation was noted until approximately 5 hours post dose on Day 1, and daily until approximately Day 10 of the study for males and Day 24 for females. However, the duration of the observation decreased over time from 5 hours post-dose to 1 hours post-dose suggesting the animals were better at a tolerating the test compound.
Ploughing was noted in all males and females receiving 500 mg/kg/day on most days from Day 2 until the end of the study; it tended to be noted only immediately post dose. Salivation was noted intermittently in all males and females receiving 500 mg/kg/day from Day 2 and 4, respectively, until the end of the study; it tended to be noted between immediately post dose and 15 min post dose. Erect fur was seen in all males and females receiving 500 mg/kg/day on Day 1, then intermittently until Day 12 of the study. Two males and 2 females suffered non-sustained convulsions shortly after dosing, 1 male on each of Days 2 and 24 and 1 female on each of Days 1 and 26. All animals recovered and did not require euthanasia.
There were additional findings at the 500 mg/kg dose level that were observed inconsistently between sexes. For males these findings were noted: a wet lower jaw was noted in 1 male on Day 1 between 3 and 4 hours post dose, 1 male was noted as subdued 1 hour post dose on Days 11 and 12, and abnormal breathing was noted in 1 male from immediately post dose on Day 19 until 15 min post dose on Day 21. This latter finding was also recorded prior to dosing on Days 20 and 21. On Day 1, these findings were noted for females: chewing action in 3 females 1 hour post dose and head twitches in 2 females immediately post dose, with 1 animal still displaying this sign 1 hour post dose. On Day 19, excessive grooming was noted in 1 female immediately post dose and an abnormal gait in 2 females 15 min post dose. Ploughing was noted intermittently in all males and females receiving 150 mg/kg/day from Days 12 and 11 respectively until study completion. Salivation was also noted intermittently in 1 male on Day 26 and 2 females from Day 20 until completion of the study.
There were no clinical observations noted in animals receiving 50 mg/kg/day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gain higher in females at 500 mg/kg/day. No effect in males.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Overall food consumption higher in females at 500 mg/kg/day. No effect in males.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmological findings were reported.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was an increase in white blood cells at the 150 mg/kg/day group, but as it was not present at the 500 mg/kg/day dose level this is considered not related to treatment.
There were a few statistically significant findings that were not attributed to treatment. Mean corpuscular volume was increased in the 150 and 500 mg/kg group in a dose-related manner and monocytes were increased in the 500 mg/kg/day group only. Prothrombin time was increased in the 50mg/kg/day group, based on the 3 animals analyzed for that group, but the higher dose levels were similar to the control value.
Any differences in the hematology parameters were within the range of the overall control group and historical control data or due to biological variation or lacked a dose-response and were therefore considered not to be test substance related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alanine aminotransferase was up to approximately 40% and alkaline phosphatase was up to approximately 50% higher in males receiving 150 or 500 mg/kg/day compared with controls, attaining statistical significance at 500 mg/kg/day. Alanine aminotransferase was up to 140% higher in females receiving 50, 150 or 500 mg/kg/day compared with controls.
There was an increase in potassium concentration in the 150 and 500 mg/kg/day males, attaining statistical significance at 500mg/kg/day. There was a decrease in chloride concentration in the 150 and 500 mg/kg/day males, attaining statistical significance at 500mg/kg/day. Females had statistically significantly increased triglycerides and calcium at all dose levels, although not showing a clear dose response. Females had statistically significantly decreased chloride at 50mg/kg/day, but there was no dose response, so this finding can be considered not related to treatment.
There were no other changes in the clinical chemistry parameters related to administration of the test substance. Any differences in the clinical chemistry parameters were within the range of the overall control group and historical control data, or due to biological variation or lacked true dose relationship and were therefore considered not to be test substance related.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urine volume was 68% and 164% higher in males and females, respectively, receiving 500 mg/kg/day compared with controls, however as there were no other correlating changes, this finding is considered to be incidental. There were no other changes in the urinalysis parameters noted.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Handling of males and females receiving 500 mg/kg/day tended to be easier compared with controls, this finding may correlate with the decreased activity noted in these animals in the clinical observations.
Hunched posture, piloerection/erect fur and fur staining were noted in some males and females receiving 500 mg/kg/day during the functional observation battery. These findings were similar to the clinical observations recorded for animals at this dose level. There were no other changes in any quantitative or qualitative functional observation parameters that were considered to be related to administration of the test substance.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher liver weights were recorded in males and females ≥ 50 mg/kg/day; this change was present in a dose dependent manner, and was statistically significant at 150 mg/kg/day and above.
There were other isolated organ weight values that were different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of the test substance.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no differences in qualitative or quantitative functional observations or motor activity assessments noted during the neurotoxicity assessment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The livers were examined across all dose groups. Centrilobular hepatocyte hypertrophy was present in males and females at 500 mg/kg/day, correlating with the increased liver weights and increased alanine aminotransferase in males and females and increased alkaline phosphatase in males. One of the male’s hyperthrophy was rated as mild, whereas all other males and females were rated minimal.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this age of rat, and were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity: There were no changes in motor activity that were considered to be related to administration of the test substance.
There were occasional changes in motor activity, some of which were statistically significant; however, these changes were isolated, showed no clear trend and lacked a true dose relationship, therefore were considered to be incidental.

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Intergroup comparison of overall bodyweight gain (g) – Days 1-29

Dose level (mg/kg/day)
Males				Females
0	50	150	500	0	50	150	500
132.00	125.00	125.60	133.40	53.40	60.80	60.80	66.0*

* Statistically significant difference from control group mean, p<0.05

 

Table 2: Intergroup comparison of selected clinical chemistry parameters

Parameter	Dose level (mg/kg/day)
	Males				Females
	0	50	150	500	0	50	150	500
ALT	55.6	46.2	62.8	79.6**	29.0	50.4*	50.4*	68.4**
ALP	127.4	144.8	154.2	191.2*	92.6	84.0	80.6	90.4
K	4.42	4.44	4.62	4.80**	3.90	4.14	4.20	3.94
Cl	100.2	100.2	99.4	98.4**	102.0	100.0*	101.2	100.8
Trigs	1.718	1.700	2.256	2.052	0.562	1.516**	1.272*	1.734**
Ca	2.696	2.684	2.712	2.734	2.628	2.740**	2.708*	2.726**

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

Units: ALT and ALP measured in U/L; K, Cl, Trigs and Ca measured in mmol/L

 

Table 3: Intergroup comparison of liver weight

	Dose level (mg/kg/day)
	Males				Females
	0	50	150	500	0	50	150	500
Absolute value (g)	13.16	12.83	14.15	15.71	6.95	7.7	8.32*	9.54**
Absolute (covari- ance analysis)	12.86	13.13	14.50*	15.36**	7.37	7.63	8.19**	9.31**
% of body weight	4.01	4.06	4.50	4.75	3.81	3.96	4.24	4.81

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

Table 4: Intergroup comparison of treatment-related microscopic findings in liver

	Dose level (mg/kg/day)
	Males				Females
	0	50	150	500	0	50	150	500
No. animals per group	5	5	5	5	5	5	5	5
Liver (No. examined)	(5)	(5)	(5)	(5)	(5)	(5)	(5)	(5)
Hyper- trophy; centri- lobular, hepato- cellular	0	0	0	4*	0	0	0	3
Minimal	0	0	0	3	0	0	0	3
Mild	0	0	0	1	0	0	0	0

* Statistically significant difference from control group mean, p<0.05

Conclusions:

In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) following 28 days of dosing with the test substance is 150 mg/kg/day. The NOAEL is based on increased body weight in females and decreased activity, irregular respiration and convulsions in males and females at 500 mg/kg/day. In addition centrilobular hepatocyte hypertrophy of the liver with increased liver weights and liver enzymes levels was noted in males and females at 500 mg/kg/day.

Executive summary:

Groups of 5 male and 5 female Han Wistar Crl:WI (Han) rats received the test substance at dose levels of 50, 150, and 500 mg/kg/day. A concurrent vehicle control group received 0.5% (w/w) methylcellulose and 0.5% (w/w) TWEEN 80 in Milli-Q Water.

The animals were observed for general health/mortality and moribundity, as well as clinical signs and functional observations. Body weights, food consumption and motor activities were measured and recorded at pre-determined intervals. Blood samples were collected from all animalsat necropsy to evaluate haematology, coagulation and clinical chemistry parameters. Urine was collected from animals during Week 4 and urinalysis parameters were evaluated.

All animals were terminated after completion of 28 days of treatment and underwent a detailed necropsy examination with selected organs weighed. Tissues from the control and high dose groups were subjected to a comprehensive histological examination with the liver being examined from all four dose groups.

There were no premature decedents. Decreased activity, partial closing of the eyes and irregular respiration were noted in all males and females receiving 500 mg/kg/day from approximately Day 1 until Day 10 of the study; however, the duration of the observation decreased over time suggesting a tolerance had developed. Erect fur was also noted in all animals receiving 500 mg/kg/day. These findings correlated with increased ease of handling in all animals and the hunched posture. They also correlated with the piloerection/erect fur and fur staining noted in some males and females receiving 500 mg/kg/day during the functional observation battery. In addition, ploughing and salivation were noted in all animals receiving 150 or 500 mg/kg/day. Overall body weight gain and food consumption was higher in females, but not males, receiving 500 mg/kg/day compared with controls. There were no differences in qualitative or quantitative functional observations or motor activity assessments noted during the neurotoxicity assessment.

Haematology and coagulation parameters and gross pathology findings in males and females were unaffected by administration of the test substance. Alanine aminotransferase (40%) and alkaline phosphatase (50%) were higher in males receiving 150 or 500 mg/kg/day compared with controls. Alanine aminotransferase was up to 140% higher in females receiving 50, 150 or 500 mg/kg/day compared with controls. Clinical chemistry changes were associated with dose-dependent increased liver weights recorded in all treated males and females and centrilobular hepatocyte hypertrophy present in males and females at 500 mg/kg/day. Liver weights were statistically significantly higher for males and females at 150 mg/kg/day and above. Clinical chemistry and liver weight changes in animals at 150 mg/kg/day and below, are not associated with the pathology changes, so considered to be adaptive and non-adverse.

Under the conditions of this study the No Observed Adverse Effect Level (NOAEL) following 28 days of dosing with the test substance is 150 mg/kg/day.  The NOAEL is based on increased body weight in females and decreased activity, irregular respiration and convulsions in males and females at 500 mg/kg/day. In addition centrilobular hepatocyte hypertrophy of the liver with increased liver weights and liver enzymes activity was noted in males and females at 500 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

An adequate, relevant and reliable study on the repeated dose toxicity via the oral route is available that provides robust information on the no-adverse effect levels in the rat, which can be used for the derivation of DNELs.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

No repeat dose studies via the inhalation route which are considered to be adequate and reliable for the purposes of risk assessment or classification were available.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

No repeat dose studies via the dermal route which are considered to be adequate and reliable for the purposes of risk assessment or classification were available.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Under the conditions of the study the NOAEL following 28 days of dosing with 4 -methylcyclohexanone was 150 mg/kg/day. Increased body weight in females and decreased activity, irregular respiration and convulsions in males and females were observed at the LOAEL of 500 mg/kg/day. In addition, centrilobular hepatocyte hypertrophy of the liver with increased liver weights and liver enzymes activity was noted in males and females at 500 mg/kg/day.

On the basis of the guiding values set out for significant toxic effects in CLP, Annex I: 3.9.2.9.7 and 3.9.2.9.5, and interpolation of the NOAEL and LOAEL, 4-methylcyclohexanone is not considered to be classified for STOT-RE.