Ethylcyclohexane

Endpoint summary

Description of key information

The NOAEL of ethylcyclohexane administered by oral gavage to rats for 28 consecutive days was determined as 40 mg/kg bw based on effects on livers and kidneys, and in addition, mild effects on hematology and clinical chemistry.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Molecular weight: 112.22
Purity: 99.9%
Appearance: clear and colorless liquid with a naphthene-like odor

Species:

rat

Strain:

other: Crl: CD (SD) SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Japan Charles River Co., Ltd.
- Age at study initiation:Four-week
-Weight: 162 g (151 to 168 g) in males and 142 g (133 to 151 g) in females
- Fasting period before study: no
- Housing:individually in stainless-steel wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days for males and 6 days for females

DETAILS OF FOOD AND WATER QUALITY:
diet: solid Feed Labo MR Stock, Nippon Nosan Kogyo Co. Ltd., Lot No. 061255, 061272
Drinking water: sterilized tap water irradiated with ultraviolet rays, after filtering with a cartridge filter with a pore size of 1 μm

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7°C to 22.6°C
- Humidity (%): 55% to 62%

Route of administration:

oral: gavage

Details on route of administration:

5 mL of the dosing solution per 1 kg of body weight was administered orally once a day (in the morning) for 28 days, using a syringe fitted with a Teflon stomach tube. The test substance was provided as the 0.8 w/v% solution (40 mg/kg group), 4.0 w/v% solution (200 mg/kg group) or 20 w/v% solution (1000 mg/kg group).

Vehicle:

olive oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The initially prepared dosing solution was analyzed to ensure that it was prepared at the defined concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once a day

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4 main groups: 5 males and 5 females (0,40, 200 and 1000 mg/kg)
2 satellite groups: 5 males and 5 females (0 and 1000 mg/kg), recovery period of 14 days

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were set in accordance with the results of dose range-finding studies. 14-day oral administration was performed at 0 (vehicle control), 30, 100, 300, or 1000 mg/kg per day to the respective groups each consisting of 4 male and 4 female rats, and observation of clinical signs, measurements of body weight and food consumption, urinalysis, hematology, clinical chemistry, necropsy, and weighing of organs were carried out. The results revealed increases or a tendency toward increases in liver weight and γ-GTP in males and females in the 1000 mg/kg group, which suggested effects on the liver. A tendency to increase liver weight was also noted in males in the 300 mg/kg group. In addition to these changes, salivation immediately after dosing was shown in males and females in the1000 mg/kg group. Based on the above results, 1000 mg/kg per day was defined in this study as the highest dose that could definitely cause repeated dose toxicities and 40 mg/kg per day as the lowest dose that could cause no toxicity effects. Between these 2 dose levels, 200 mg/kg per day was set, and thus a total of 3 dose levels were used.
To investigate reversible, persistent and/or late-onset toxicities in emerging changes, additional 2 satellite groups, each containing 5 male and 5 female rats were set for the control group and the 1000 mg/kg group.

Positive control:

no

Observations and examinations performed and frequency:

Clinical Signs:
4 times daily during the administration period and at least once daily during the recovery period.

Detailed Clinical Observations:
on the day before the starting date, and thereafter once weekly

Sensory Function Tests (eye sight reaction, hearing response, sense of touch reaction, pain reaction, pupil reflex, righting reflex):
Animals to be sacrificed at the end of the administration period and those in the satellite groups were examined at week 4 (day 27) of the administration period and at week 2 (day 13) of the recovery period, respectively.

Grip Strength and Motor Activity Measurements (forelimb and hind limb grip strengths, umber of the animal’s movement within the system compartment for 60 minutes):
Animals to be sacrificed at the end of the administration period and those in the satellite groups at week 4 (day 27) of the administration period and at week 2 (day 13) of the recovery period, respectively.

Body Weight and Food Consumption Measurements:
Body weights: on days 1 (immediately before administration on day 1), 7, 14, 21, and 28 of the administration period, days 7 and 14 of the recovery period, and the day of sacrifice
Food consumption: once weekly

Urinalysis (appearance, and pH, occult blood, protein, glucose, ketone body, bilirubin and urobilinogen qualitative tests):
animals to be sacrificed at the end of the administration period and those in the satellite groups at week 4 (day 22) of the administration period and at week 2 (day 8) of the recovery period, respectively.

Hematology:
at the end of the administration period (for animals to be sacrificed at the end of the administration period) or at the end of the recovery period (for animals to be sacrificed at the end of the recovery period).

Clinical Chemistry:
at the end of the administration period (for animals to be sacrificed at the end of the administration period) or at the end of the recovery period (for animals to be sacrificed at the end of the recovery period).

Sacrifice and pathology:

Necropsy and Weighing of Organs:
Animals were sacrificed by exsanguination after blood sampling on the day following the end date of the administration period or of the recovery period and were subject to gross necropsy, including the body surface, orifice membranes, and various internal organs. The absolute weights of the following organs were measured: brain, thymus, heart, liver, spleen, kidneys, adrenals, thyroid, pituitary gland, testes and epididymides (males), and ovaries (females). The relative weight (against body weight) was calculated based on the body weight measured on the sacrifice day. Kidneys, adrenals, testes, epididymides, and ovaries were weighed bilaterally together. Thyroid and pituitary gland were weighed after fixation.

Histopathological Examinations:
The following organs were taken, fixed in 10% neutral phosphate buffered formalin (testes and epididymides were prefixed in Bouin's fluid) and were stored.
Brain, eye balls, pituitary gland, thyroid (including parathyroid), spinal cord (neck, chest and lumbar), heart, trachea and lung (injected with fixative and immersed), liver, kidneys, thymus, spleen, adrenals, stomach, intestine (including duodenum, jejunum, ileum, cecum, colon, rectum and Peyer's patch), reproductive organs (testes or ovaries), accessory reproductive organs (epididymides, prostate gland and seminal vesicle, or uterus and vagina), urinary bladder, sciatic nerve, lymph node (cervical lymph node and mesenteric lymph node), and bone marrow (femur).

Statistics:

The mean and standard deviation were calculated for parametric data (grip strength, motor activity level, body weight, body weight gain, food consumption, hematology data, clinical chemistry data, and organ weight) for each group. The Bartlett's variance test was used in the case of 3 or more study groups. If variances were homogeneous, one-way analysis of variance was performed. If the variances were not homogenous or for non-parametric data (differential leukocyte count, and urinalysis data), the Kruskal-Wallis’s rank test was used. If there was a significant difference as a result, the significant difference between the control group and the treatment groups was analyzed by the Dunnett's test or the Dunnett-type test. The F-test was conducted for parametric data in the case of 2 study groups. The Student’s t-test was conducted for equal distribution data, and Aspin-Welch’s t-test was used for unequal distribution data. The Mann-Whitney’s U-test was conducted for non-parametric data. Fisher’s exact probability test was used for categorical data (incidence of abnormalities in observation of clinical signs, detailed clinical observations, sensory function tests, necropsy, or histopathological examinations). The significance level was set to 5% in all analyses.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All males and females in the1000 mg/kg group showed salivation immediately after dosing during the administration period, which was of mild intensity. Moist fur around the mouth with salivation became dry approximately 15 minutes after dosing. No salivation was noted during the recovery period. No deaths were observed throughout the observation period.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant changes were observed in the body weight at each time point and in the body weight gain during the administration period and the recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant changes were observed in the food consumption during the administration period and the recovery period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology findings included significant prolongation in prothrombin time in males and significant decreases in hemoglobin
concentration and hematocrit level in females in 1000 mg/kg group. Note that a general tendency to slightly decrease prothrombin time was noted in females in each treatment group compared to the control group, and a significant difference was seen for the 1000 mg/kg group. Also, significant increases in reticulocyte count were shown in males in the 40 mg/kg group. However, no dose-related reticulocyte count changes were observed in males, indicating no effects of the test substance. As for animals to be sacrificed at the end of the recovery period, prolongation in prothrombin time still existed in males with a significant difference remaining. However, the range of variability compared to the control group was smaller than that of animals to be sacrificed at the end of the administration period. No changes were observed in females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry findings included significant increases in total bilirubin level in females in the 200 and 1000 mg/kg groups, and a similar tendency, but with no significant difference was found in males in the 200 and 1000 mg/kg groups. In addition, significant increases in γ-GTP activity and calcium and sodium concentrations in males and significant decreases in alkaline phosphatase activity, blood sugar level and blood urea nitrogen concentration in females were observed in the 1000 mg/kg group. Note that although significant increases in LDH activity were found in males in the 200 mg/kg group, no dose-related LDH changes were observed in males. For animals to be sacrificed at the end of the recovery period, no significant changes were observed in each test item.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

significant increases in relative weight of kidneys were noted in males in the 200 mg/kg group and significant increases in both absolute and relative weights of kidneys in males in the 1000 mg/kg group. Significant increases only in the absolute weight of kidneys were noted in females in the 200 and 1000 mg/kg groups. Also, significant increases in both absolute and relative weights of the liver were noted in males and females in the 1000 mg/kg group. As for animals to be sacrificed at the end of the recovery period, no significant changes were observed in absolute and relative weights of each organ.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

White areas in the liver and small size of testis (right side) were seen in 1 male each in the 1000 mg/kg group. Also, reddish areas in thymus were seen in 1 male in the control group and the 1000 mg/kg group, respectively.
As for animals to be sacrificed at the end of the recovery period, no changes were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examinations revealed increases in hyaline droplets of proximal tubular epithelium in male kidneys in the 200 and 1000 mg/kg groups, increases in eosinophilic bodies in the proximal tubular epithelium, positive for α2μ-globulin immunostaining in male kidneys in the 1000 mg/kg group, and centrilobular hypertrophy of hepatocytes in male and female livers in the 1000 mg/kg group.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The NOAEL of ethylcyclohexane administered by oral gavage to rats for 28 consecutive days is considered to be 40 mg/kg bw based on effects on livers and kidneys, and in addition, mild effects on hematology and clinical chemistry.

Executive summary:

Following 28-day repeated oral administration of ethylcyclohexane to rats at 40, 200, and 1000 mg/kg dose levels, toxicity effects on livers and kidneys were observed. Effects on livers included increases in both absolute and relative weights of the livers in males and females in the 1000 mg/kg group. Histopathological examinations revealed centrilobular hypertrophy of hepatocytes in livers in the 1000 mg/kg group, which was considered effects related to the test substance. Hematology and clinical chemistry showed increases or a tendency to increases in total bilirubin level in males and females in the 200 and 1000 mg/kg groups, and prolongation in prothrombin time and increases in γ-GTP activity in males in the 1000 mg/kg group; these changes were also considered to be toxicity effects on livers related to the test substance.

To the contrary, decreases in prothrombin time were noted in females in the 1000 mg/kg group. Decreases in prothrombin time are of little toxicological significance, and also there was no tendency to augment the changes in a dose-related manner; thus the event was unlikely to be a toxicity effect related to the test substance.

Effects on kidneys included increases in relative weight of kidneys in males in the 200 mg/kg group and increases in both absolute and relative weights of kidneys in males in the 1000 mg/kg group. Histopathological examinations revealed increases in hyaline droplets of proximal tubular epithelium in male kidneys in the 200 and 1000 mg/kg groups, as well as increases in eosinophilic bodies in the proximal tubular epithelium in male kidneys in the 1000 mg/kg group.

Proximal tubular epithelium allows proteins filtered at glomerulus, to be reabsorbed by endocytosis and broken down into amino acids by lysosomal proteolytic enzymes for subsequent use. Hyaline droplets are accumulation forms of a high molecular weight protein, lysosomal albumin, and are likely to be developed by increases in protein leakage from glomerulus or inhibition of catabolism by lysosomal enzymes. Eosinophilic bodies are huge lysosomes produced by resorption of a low molecular weight protein, α2μ-globulin which is synthesized in male rat liver, and are considered as a change specific to castrated or estrogen-suppressed rats.It was confirmed that eosinophilic bodies observed in this study were also deposits of α2μ-globulin based on immunostaining.

Increases in sodium and calcium concentrations in males in the 1000 mg/kg group shown in clinical chemistry findings were possibly effects on kidneys related to the test substance. Note that although increases only in absolute weight of kidneys were noted in females in the 200 and 1000 mg/kg groups, considering that no relevant histopathologic changes were detected in female livers and that their final body weights were slightly higher than those of the control group, these changes were considered unrelated to toxicities.

Decreases in hemoglobin concentration and hematocrit level in females in the 1000 mg/kg group (13.6 g/dL and 41.1%, respectively) were both changes within the baseline levels of background data (hemoglobin concentration: 13.5 to 15.9 g/dL; hematocrit level: 41.2% to 47.9%). With no changes noted in peripheral blood reticulocyte count or hematopoietic tissue, a similar tendency of changes was seen as mild findings in males in the 1000 mg/kg group; therefore, these changes were considered suggestive of mild effects on blood related to the test substance.

Regarding clinical chemistry changes in females in the 1000 mg/kg group, decreases in alkaline phosphatase activity (337 IU/L) were slightly lower than the baseline levels of background data (356 to 1037 IU/L); however decreases in blood sugar (114 mg/dL) and blood urea nitrogen (9.2 mg/dL) were changes within the baseline levels of background data (96 to 148 mg/dL and 8.9 to 18.6 mg/dL, respectively). These were of mild intensity as effects on clinical chemistry. Note that salivation in males and females in the 1000 mg/kg that occurred immediately after dosing and resolved approximately 15 minutes after dosing, were considered findings of avoidance response against the dosing solution; thus, the event was unlikely to be a toxicity effect related to the test substance absorption.

These changes seen during the administration period or at the end of the administration period all resolved, except high prothrombin time remaining in males that also tended to resolve, during the recovery period or at the end of the recovery period. Thus, these toxicity effects were considered reversible changes. Furthermore, no late-onset toxicity effects were observed. The above results indicate that toxicities following 28-day repeated oral administration of ethylcyclohexane to rats were mainly effects on livers and kidneys, and in addition, mild effects on hematology and clinical chemistry were observed. It was concluded that the NOEL was 40 mg/kg per day in males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

hepatobiliary

Organ:

kidney

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study 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

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the outcome of the subacute oral study with rats the following can be stated on the necessity for ECH to be categorized into class 2 for STOT RE:

• The NOEL was determined to be 40 mg/kg bw per day
• A clear NOAEL is difficult to establish because at the next higher dose of 200 mg/kg bw per day only small changes were seen (increase of total bilirubin in females only, changes in organ weights and relative kidney weights only in males and hyaline droplets in kidneys also only in males) which were inconsistent by sex without a clear target organ and absence of evidence for dysfunction of organs.
• All effects observed were reversible (besides persisting prothrombin levels in males).
• Generalized toxic effects (like food and water consumption) with regard to the well-being of the animals (besides for salivation directly after dosing) were not observed even at the highest dose level of 1000 mg/kg bw per day.  

This indicates that these findings are neither significant nor severe to the extent that classification Cat. 2 for STOT RE is warranted and justifiable.