Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 February 1993 to 01 April 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health and Welfare (M.H.W.) Guidelines 1986 for a twenty-eight day repeat dose oral toxicity study as required by the Japanese Chemical Control Law 1978 of the Ministry of International Trade and Industry (M.I.T.I.) amended 1987

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

EC Number:

412-540-8

EC Name:

Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Cas Number:

22471-55-2

Molecular formula:

C12 H22 O2

IUPAC Name:

ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Constituent 2

Reference substance name:

Ethyl cis-2,2,6-trimethylcyclohexane-1-carboxylate

Molecular formula:

C12H22O2

IUPAC Name:

Ethyl cis-2,2,6-trimethylcyclohexane-1-carboxylate

impurity 1

Reference substance name:

Unidentified impurities

Molecular formula:

Not specified.

IUPAC Name:

Unidentified impurities

Test material form:

liquid: viscous

Details on test material:

Test material identification : ET-344 SP
Chemical name : Ethyl 2,2,6-trimethyl cyclohexanecarboxylate
Description: Clear, colourless liquid
Date received: 2 November 1992
Container: Metal bottle x3
Storage conditions: Room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
A sufficient number of male and female Sprague-Dawley CD strain rats were obtained from Charles River (U.K.) Limited, Manston, Kent. On receipt the animals were examined for signs of ill-health or injury.The animals were acclimatised for eight days during which time their health status was assessed. A total of sixty animals (thirty males and thirty females) were accepted in to the study. At the start of the treatment the males weighed 132 to 176g, and the females weighed 124 to 148g, and were approximately five to six weeks old. The animals were housed in groups of five by sex in polypropylene grid floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food, except during urine collection when food was withdrawn overnight.A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diet , Services Limited, Witham, Essex, U.K.) was used. Certificates of analysis of the batches of diet are provided in the report. The animals were allowed free access to mains water from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. The animals were housed in a single air-conditioned room within the Safepharm Barrier Maintained Rodent Facility. The rate of air exchange was at least 15 air changes per hour and the low intensity fluorescent ilighting was controlled to give 12 hours continuous light and 12 hours darkness. Environmental conditions were continuously monitored by a computerised system and print-outs of the mean temperatures and humidities were included in the study records. During the study the room temperature was maintained within the range of 18 - 24°C and relative humidity of 43 - 93%. On two isolated occasions the room temperature or humidity fell outside protocol limits but this was considered not to have affected the purpose or integrity of the study.The animals were randomly allocated to dose groups using random letter tables and the group mean bodyweights were then determined to ensuresimilarity between the dose groups. The cage distribution within the holding rack was also randomised.The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories. Colour coded cage labels were used to assist recognition of dose groups.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test material was administered daily, for twenty-eight consecutive days, by gavage using a metal cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 2 ml/kg/day of Arachis oil B.P. Animals from satellite groups were maintained for a further 14 days treatment-free period following termination of treatment.The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Vehicle:

arachis oil

Details on oral exposure:

For the purpose of this study the test material was prepared at the appropriate concentrations, as a solution in Arachis oil B.P. The stability and homogeneity of the test material formulations were determined by the Analytical Laboratory. Results have showed that the formulations were stable for at least ten days.Formulations were therefore prepared weekly and stored at 4°C in the dark. Samples were taken of each test material formulation and were analysed for concentration of ET-344 SP at the Analytical Laboratory. The results indicate that the prepared formulations were within +/- 9% of the nominal concentration.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Male: 5 animals at 0 mg/kg bw/day
Male: 5 animals at 15 mg/kg bw/day
Male: 5 animals at 150 mg/kg bw/day
Male: 5 animals at 1000 mg/kg bw/day
Female: 5 animals at 0 mg/kg bw/day
Female: 5 animals at 15 mg/kg bw/day
Female: 5 animals at 150 mg/kg bw/day
Female: 5 animals at 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

Six groups, each of ten rats (five males and five females) were dosed as shown below in the table. The test material was administered daily, for twenty-eight consecutive days, by gavage using a metal cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner w ith 2 mL/ kg/ day of Arachis oil B.P. Animals from satellite groups were maintained for a further 14 days treatment-free period following termination of treatment. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

a) Clinical Signs
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends. All observations were recorded. During the treatment-free period, satellite group animals were observed twice daily, morning and afternoon (once daily at weekends).
b) Bodweight
Individual bodyweights were recorded on the day before the start of treatment (day 0) and on days seven, fourteen, twenty-one and twenty-eight and in the case of satellite group animals on days thirty-five and forty-two. Bodyweights were also recorded at necropsy.
c) Food ConsumptionFood consumption was recorded for each cage group at weekly intervals throughout the study.
d) Water ConsumptionPossible inter group differences were detected during the first week of treatment and therefore, water intake was measured and recorded daily for each cage group from day 8 onwards.
e) Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from groups 1 to 4 at the end of the treatment period (day 28). Parameters showing possible treatment-related changes in these animals were examined in satellite group animals at the end of the treatment-free period (day 42). Blood samples were obtained from the lateral tail vein. A few repeat samples were also obtained prior to necropsy on day 29 by exsanguination. Animals were not fasted prior to sampling.
Urinalysis was performed on all animals from groups 1 to 4 during week 4 and on animals from groups 5 and 6 during the final week of the study.Urine samples were collected over a period of approximately 16 hours, by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection, but without access to food.
i ) Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:Haematocrit (Hct)Haemoglobin (Hb)Erythrocyte count (RBC)Total leucocyte count (WBC)Differential leucocyte countPlatelet count (PLT)Erythrocyte indices - mean corpuscular haemoglobin (MCH)- mean corpuscular volume (MCV)- mean corpuscular haemoglobin concentration (MCHC)Clotting time (CT) was assessed by 'Hepato Quick' time using samples collected into sodium citrate solution (0.11 mol/l).ii)Blood ChemistryThe following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:Blood ureaTotal proteinAlbuminAlbumin/globul in ratio (by calculation)SodiumPotassiumChlorideCalciumInorganic phosphorusAlkaline phosphatase (AP)Alanine aminotransferase (ALAT)Aspartate aminotransferase (ASAT)GlucoseGamma glutamyl transpeptidase (y-GT)TriglyceridesTotal cholesterolTotal bilirubinCreatinineiii) UrinalysisThe following parameters were measured on freshly collected urine.Volume Specific gravitypHProtein GlucoseKetonesBilirubinUrobilinogenReducing substancesBlood

Sacrifice and pathology:

f) Pathology
On completion of the dosing period or, in the case of satellite group animals, at the end of the treatment-free period; all animals were killed by intravenous overdose of sodium pentobarbitone (Sagatal 60 mg/ml ; May and Baker Limited, Dagenham, Essex, UK) followed by exsanguination.All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
i) Orqan WeiqhtsThe following organs from animals that were killed at the end of the study, dissected free from fat, were weighed before fixation :Adrenals Brain Gonads Heart Kidneys Liver Pituitary Spleenii) HistopatholoqySamples of the following tissues were removed from all animals and preserved in 10% buffered formalin.
Adrenals
Aorta (thoracic)
Bone & Bone Marrow (femur)
Brain
Caecum
Colon
Duodenum
Eyes
Gross lesions
Heart
Ileum
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands
Sciatic nerve
Seminal vesicles
Skin (hind limb)
Spleen
Stomach
Jejunum
Testes
Kidneys
Thymus
Liver
Thyroid/parathyroid
Lungs
Trachea
Lymph nodes (cervical and mesenteric)
Urinary bladder
Muscle (skeletal)
Uterus
Oesophagus
All tissues were despatched to Experimental Pathology Services, Willow Court, Netherwood Road, Rotherwas, Hereford, U.K. for processing. The following preserved tissues from all test and control group animals (groups 1 t o 6) were prepared as paraffin blocks, sectioned at nominal thickness of 5 um and stained with haematoxylin and eosin. Microscopic examination was initially performed on high dose and control group tissues (groups 1 and 4)Adrenals Spleen Heart Kidneys Liver
Macroscopically observed lesions were also processed.Since there were indications of treatment-related hepatic and renal changes, examination was subsequently extended to include sections of liver and kidneys from all animals in the remaining dose groups.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.Absolute and relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating'F-max' test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal Wallis non-parametric analysis ofvariance and Mann Whitney U-Test.Probability values were calculated as:p < 0.001 ***p < 0.01 **p < 0.05 *p >= 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High dose animals showed clinically observable signs from day 2 onwards. Increased salivation was apparent immediately before or one hour after dosing together with associated signs of wetness of the fur.
Red/brown staining of the external body surface was also seen but this was confined to the females and was more prevalent during the first two weeks of treatment. Individual animals also showed incidents of fur loss during the study. One satellite high dose male developed a small wound around the shoulder from day 14 which was apparent throughout the study period. This was a minor physical injury and was not related t o administration of the t e s t material.
No clinically observable signs were detected in the remaining dose groups during the treatment period or in satellite high dose animals during the fourteen day recovery period.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

All animals showed normal gains in bodyweight throughout the study period. Bodyweight gain in test animals was comparable with that seen in controls.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

High dose animals showed increased water consumption over the treatment period, in comparison with controls, which recovered completely in the satellite high dose group during the treatment-free period.
No adverse effects on water intake were detected in the remaining dose groups throughout the study

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in the haematological parameters measured. Statistically significant reductions in haemoglobin, haematocrit and erythrocyte counts were detected for high dose females in comparison with controls. All values were within the respective normal ranges (female haemoglobin: 11.2 - 15.5 g/ dL , haematocrit : 31.7 - 45.0%, erythrocytes: 5.6 - 7.9 X 10^12/ L) and the reductions were probably a consequence of several unusually high values in the control group, and, as such, were considered not to be toxicologically significant. A similar reduction in erythrocyte counts was seen in satellite high dose females, but again there were several unusually high values in the control group and this was considered to be of no toxicological importance.The remaining statistically significant difference detected between test and control groups was confined to increased eosinophil counts in intermediate dose females. No dose-relationship was apparent and, as such, the increase was considered not to be related to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

High dose females showed a statistically significant increase in plasma total protein and plasma albumin together with a reduction in albumin/globulin (A/G) ratio. Intermediate dose females also showed increased total protein and reduced A/G ratio in comparison with controls. All values were with in the respective normal ranges (total protein: 5.04 - 7.94 g/dL, albumin: 3.25 - 4.63 g/dL, A/G ratio : 1.11 - 2.32), however, in view of the hepatic changes seen microscopically it is possible that these increases/reductions may be associated with a liver effect. Further blood chemical changessupporting hepatic abnormalities were seen in high dose females with statistically significant increases in alanine aminotransferase, bilirubin and gamma glutamyl transpeptidase, the latter parameter also elevated i n high dose males.A statistically significant reduction in plasma glucose was seen in all female treatment groups together with an increase in inorganic phosphate for the low and high dose animals of this sex. No convincing dose relationship was apparent and the differences in both parameters were considered to be of no toxicloical significance. Aspartate aminotransferase (ASAT) \ showed a statistically significant reduction in high dose males but all values were entirely witthin the normal range for rats of this strain and age (ASAT: 55 -172 IU/ L) and a reduction in this enzyme cannot be regarded as toxicologically important. Plasma bilirubin was reduced in satellite high dose animals of either sex, but again a reduction is unlikely to be toxicologically significant .The remaining statistically significant differences detected between test and control groups were confined to low or intermediate dose animals and, as such, showed no dose-related response

Urinalysis findings:

no effects observed

Description (incidence and severity):

High dose females showed increased reducing substances in the urine. This was probably due to excretion of the test material or its metabolite(s) and, in the absence of a specific increase in urine glucose, was considered not to be indicative of an adverse effect to treatment.
No abnormalities were detected in the remaining dose groups or in satellite high dose animals after fourteen days without treatment.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases in absolute and relative liver weight were seen in high dose animals of both sexes in comparison with controls with many values outside the normally expected range for rats of this strain and age (male absolute l iver: 8.6965 - 19.3336 g, male relative liver: 2.9118 - 4.7183%, female absolute liver: 6.4946 - 12.7188 g, female relative liver: 3.0780 - 4.8918%). Intermediate dose males also showed elevated relative liver weights with one abnormally high individual value. Relative liver weights were still elevated in satellite high dose animals after fourteen days without treatment but statistical significance was only achieved in the males. Absolute and relative kidney weights were significantly increased in high dose males with relative weights also elevated in intertermdiate dose animals of this sex.High dose females showed a statistically significant increase in absolute kidney weight only and all values were entirely within the normal range (absolute kidney; 1.3898 - 2.1328 g ) . This isolated increase in females, was considered to be of no toxicological significance.Absolute adrenal weights showed a statistically significant reduction for high dose males when compared with controls. All values were within the normal range (0.0218 - 0.0719 g) and with no effect on relative adrenal weights the reduction was considered to be fortuitous. Similar reductions were apparent for satellite high dose male absolute adrenal and pituitary weights, but again these were considered not to be associated with treatment. Statistically significant increases in satellite high dose male relative heart weight and satellite high dose female relative ovary weight were also detected but no adverse effect was seen on these organ weights for main group animals at the end of the dosing period and, as such, the increases were considered not to be toxicologically significant.The remaining statistically significant differences detected between test and control groups were confined to low and/ or intermediate animals and, as such, showed no dose-related response.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

NecropsyHigh dose animals showed dark and/or enlarged livers. Patchy pallor of the kidneys was also noted in animals from this group but, with one exception, was confined to males. Four out of the five intermediate dose males also showed patchy pallor of the kidneys.No macroscopic abnormalities were detected in the low dose group.
The remaining incidental finding recorded for one high dose male, identified as a small seminal vesicle, was consistent with a normally expected, low incidence finding in laboratory maintained rats and was, therefore, of no toxicological significance.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related hepatic and renal changes were observed
LIVER: Hepatocellular enlargement and increased hepatocytoplasmic density were observed in relation to treatment for male rats dosed at 1000 mg/kg/day. Female rats, and male rats from the remaining dose levels were unaffected. The condition was observed to have regressed from satellite 1000 mg/kg/day rats following an additional fourteen days without treatment.
KIDNEYS: Eosinophilictubular degeneration was observed in relation to treatment for male rats dosed at 1000 mg/kg/day, 150 mg/kg/day, and at 15 mg/kg/day. Female rats were unaffected. Such renal changes are typical of hydrocarbon induced nephropathy observed in the renal tubules of male rats. The condition had regressed in favour of regenerative basophilic tubules in the kidneys of satellite 1000 mg/kg/day male rats following an additional fourteen days without treatment.All remaining morphological changes were those commonly observed in laboratory maintained rats o f the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Histopathological findings: neoplastic:

not specified

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects noted on kidney and liver

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material, ET-344 SP, to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in toxicologically significant effects at 1000 and 150 mg/kg/day.The kidney changes detected in male rats from all treatment groups were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health and for the purposes of hazard evaluation the "No Observed Effect Level" (NOEL) is considered to be 15 mg/ kg/ day.

Executive summary:

The study was designed in accordance with OECD guideline #407.

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley CD strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil B.P.). Two satellite groups, each of five males and five females were treated with the high dose (1000 mg/kg/day) or the vehicle alone throughout the twenty-eight day study period and then maintained without treatment for a further fourteen days.

Clinical signs, bodyweight, food and water consumptions were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all main group animals during the final week of dosing. Parameters showing possible abnormalities were examined in satellite group animals at the end of the treatment-free period.

All animals were subjected to a gross necropsy examination with histopathological evaluation of selected tissues.

The results are summarised as follows:

Mortality Data

There were no deaths during the study.

Clinical Observations

High dose animals showed clinical signs from day 2 onwards. Observations included increased salivation immediately after dosing together with associated signs of wet and stained fur. No clinically observable signs were detected in satellite high dose animals during the recovery period or in the low or intermediate dose groups during the twenty-eight day treatment period.

Bodyweight

Bodyweight gain in test animals was comparable with that seen in controls.

Food Consumption

No adverse effects on food consumption were detected.

Water Consumption

Group mean water consumption was measured from day 8 o f the study onwards and an increase in water intake was apparent in the high dose group when compared with that of controls. This recovered completely during the treatment free period and no adverse effects on water consumption were seen in the remaining dose groups.

Haemotology

No treatment-related effects were detected.

Blood Chemistry

High dose females showed statistically significant increases in total protein and albumin and a reduction in albumin/globul in ratio. Statistically significant increases in a1anine aminotransferase and bilirubin were detected in high dose females and gamma glutamyl transpeptidase was increased in both males and females treated at this dose level.

Urinalysis

High dose females showed increased reducing substances in the urine.

No abnormalities were detected in the remaining dose groups or in satellite high dose animals after fourteen days without treatment.

Necropsy

High dose animals showed dark and/ or enlarged livers and patchy pallor of the kidneys the latter change confined to the males. Intermediate dose males showed similar kidney changes to those of the high dose group.

No treatment-related macroscopic abnormalities were detected in low dose animals.

Organ weights

Statistically significant increases in absolute and relative liver weight were seen in high dose animals of either sex with many values outside the normal ranges for rats of this strain and age. Intermediate dose males also showed elevated relative liver weights and relative liver weights were significantly increased i n satellite high dose males after fourteen days without treatment. Absolute and relative kidney weights showed statistically significant increases in high dose males with intermediate dose males also showing raised relative weights in comparison with controls.

Histopathology

LIVER: Hepatocellular enlargement and increased hepatocytoplasmic density were observed in relation to treatment for male rats dosed at 1000 mg/ kg/ day. Female rats, and male rats from the remaining dose levels were unaffected. The condition was observed to have regressed from satellite 1000 mg/ kg/ day rats following an additional fourteen days without treatment.

KIDNEYS: Eosinophilic tubular degeneration was observed in relation to treatment for male rats dosed at 1000 mg/ kg/ day, 150 mg/ kg/ day and at 15 mg/ kg/ day. Female rats were unaffected. Such renal changes are typical of hydrocarbon induced nephropathy observed in the renal tubules of male rats.

The condition had regressed in favour of regenerative basophilic tubules in the kidneys of satellite 1000 mg/ kg/ day male rats following an additional / fourteen days without treatment.

Conclusion

Oral administration of the test material, ET-344 SP, to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/ kg/ day resulted in toxicologically significant effect s at 1000 and 150 mg/ kg/ day.

The kidney changes detected in male rats from all treatment groups were consistent with well documented changes that are peculiar to the male ratin response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health and for the purposes of hazard evaluation the "No Observed Effect Level" (NOEL) is considered to be 15 mg/ kg/ day.