A mixture of: 4-(2,2,3-trimethylcyclopent-3-en-1-yl)-1-methyl-2-oxabicyclo[2.2.2]octane; 1-(2,2,3-trimethylcyclopent-3-en-1-yl)-5-methyl-6-oxabicyclo[3.2.1]octane; spiro[cyclohex-3-en-1-yl-[(4,5,6,6a-tetrahydro-3,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]furan]; spiro[cyclohex-3-en-1-yl-[4,5,6,6a-tetrahydro-4,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]]furan]

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:

30 March 1994 to 14 April 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study is reliable and adequate for fulfilling the endpoint.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: 4-(2,2,3-trimethylcyclopent-3-en-1-yl)-1-methyl-2-oxabicyclo[2.2.2]octane; 1-(2,2,3-trimethylcyclopent-3-en-1-yl)-5-methyl-6-oxabicyclo[3.2.1]octane; spiro[cyclohex-3-en-1-yl-[(4,5,6,6a-tetrahydro-3,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]furan]; spiro[cyclohex-3-en-1-yl-[4,5,6,6a-tetrahydro-4,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]]furan]

EC Number:

422-040-1

EC Name:

A mixture of: 4-(2,2,3-trimethylcyclopent-3-en-1-yl)-1-methyl-2-oxabicyclo[2.2.2]octane; 1-(2,2,3-trimethylcyclopent-3-en-1-yl)-5-methyl-6-oxabicyclo[3.2.1]octane; spiro[cyclohex-3-en-1-yl-[(4,5,6,6a-tetrahydro-3,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]furan]; spiro[cyclohex-3-en-1-yl-[4,5,6,6a-tetrahydro-4,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]]furan]

Cas Number:

426218-78-2

Molecular formula:

C16H26O

IUPAC Name:

1-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)-2-oxabicyclo[2.2.2]octane; 3,6',6',6'a-tetramethyl-2',3'a,4',5',6',6'a-hexahydrospiro[cyclohexane-1,3'-cyclopenta[b]fura]-3-ene; 4,6',6',6'a-tetramethyl-2',3'a,4',5',6',6'a-hexahydrospiro[cyclohexane-1,3'-cyclopenta[b]fura]-3-ene; 5-methyl-1-(2,2,3-trimethylcyclopent-3-en-1-yl)-6-oxabicyclo[3.2.1]octane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England, on 16 March 1994.
- Age at study initiation: 28 ± 1 days old
- Weight at study initiation: 70 - 89 g on arrival
- Housing: All rats were initially caged, as far as possible, in groups of five according to sex in metal cages with wire mesh floors. The cages (each containing five rats) constituting each group were distributed in batteries in such a manner that possible environmental influences arising from their spatial distribution were equilibrated, as far as possible, for all treatments.
- Diet (e.g. ad libitum): A standard pelleted laboratory rodent diet (Special Diet Services Rat and Mouse Maintenance Diet) was provided ad libitum, except as noted under CLINICAL PATHOLOGY.
- Water (e.g. ad libitum): Drinking water was provided ad libitum, except as noted under CLINICAL PATHOLOGY.
- Acclimation period: A seven-day acclimatisation period was allowed between delivery of the animals and start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 to 22.0°C
- Humidity (%): Relative humidity was generally controlled in the range 35 to 58% RH. A few incidences of low humidity (20% RH) were recorded early in the acclimatisation period, however, the animals were not harmed and the integrity of the study was not affected.
- Air changes (per hr): Air exchange was maintained at approximately 19 air changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was controlled to provide 12 hours artificial light (0700 - 1900 hours) in each 24-hour period.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed out and mixed with the vehicle (corn oil) using a high shear homogeniser. For each concentration, a series of formulations was prepared by further direct dilution of the test substance.
Formulations were prepared freshly each day.
The chemical stability and, for suspensions, homogeneity of test substance formulations were assessed prior to the start of treatment and concentration analyses of formulations prepared for administration on Day 1 (and Day 15 for Groups 1 and 2) were performed by the Department of Analytical Chemistry at Huntingdon Research Centre Ltd.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A representative sample (approximately 1 ml) of test formulation was accurately weighed and dissolved in a suitable volume of acetone. The extract was appropriately diluted using acetone to provide a solution containing cassifix at an expected concentration of 60 ug/ml. The concentration of cassifix in the final solution was quantified by gas liquid chromatography using flame ionisation detection.

Instrument: Gas liquid chromatograph Perkin Elmer 8500
Instrument: Autosampler Perkin Elmer 8300
Instrument: Detector Flame ionisation
Instrument: Data handling Perkin Elmer Access*Chrom
Analytical column: Fused silica
Configuration 25 m x 0.53 mm ID
Analytical column: Stationary phase CP-Sil 8CB 95 % -dimethyl-5 % -Analytical column: Film thickness 2 um
Temperatures: Injector 150 ° C
Temperatures: Oven 100 ° C ramped at 8 °C/min to 240 °C
Temperatures: Detector 300 °C
Gases: Carrier Helium, 10 ml/min
Gases: Flame Air, 25 psi
Hydrogen, 20 psi
Injection volume 2 uL
Sensitivity 10 – 25 mV
Retention time The sum of the peaks between 12 - 14.7 min

Calibration
A primary standard solution was prepared for each analytical occasion by dissolving an accurately weighed quantity (50 mg) of cassiflX in acetone. Solutions to assess the linearity of detector response, containing cassifix in the concentration range 20 - 100 ug/ml, were prepared by appropriate dilution of the primary standard using acetone.
A solution for instrument calibration, containing cassifix at a concentration of 60 ug/ml, was prepared by appropriate dilution of the primary standard solution using acetone.
The calibration solution was injected onto the GLC, at regular intervals throughout the sample analysis sequence.
The response of the sum of the peaks observed at the characteristic retention time for cassifIx (between 12 and 14.7 minutes) in calibration, sample and procedural recovery chromatograms was measured and the concentration of cassifIx was determined.

Limit of detection
The limit of detection, defIned as the concentration of cassifIx in control matrix producing a peak response equivalent to 3 x baseline noise, was estimated as 0.15 mg/ml.

VALIDATION OF THE METHOD OF ANALYSIS
The analytical procedure was validated by fortifying six samples (1 ml) of control vehicle (corn oil) with cassifIx, to concentrations of 3 mg/ml and 200 mg/ml, which were analysed in accordance with the analytical procedure. The test substance, cassifIx, was added either as a solution in acetone (inclusion levels < 20 mg/ml) or as neat test material (inclusion levels ≥ 20 mg/ml).
Procedural recoveries were determined for each inclusion level and analysed concurrently with test formulations.

DETERMINATION OF CONCENTRATIONS OF CASSIFIX IN DOSE FORMULATIONS ANALYSED DURING THE STUDY
Representative samples (approximately 20 ml) of freshly prepared dose formulations were thoroughly mixed by vigorous shaking and duplicate sub-samples (1 ml) were analysed in accordance with the analytical procedure.

DETERMINATION OF THE STABILITY OF CASSIFIX IN CORN OIL FORMULATIONS
Freshly prepared specimen formulations (approximately 120 ml) , containing cassifix at nominal concentrations of 3 mg/ml and 200 mg/ml , were each thoroughly mixed by inversion and magnetically stirred . After magnetic stirring for 5 minutes (0 hour), samples (approximately 1 ml) were removed for analysis at points approximately one-quarter, one-half and three-quarters the depth (representing the top, middle and bottom) of the formulation.
The magnetic stirring was discontinued and the remainder of each formulation was stored at ambient temperature in the dark. At a time point representing 24 hours after preparation, each formulation was re-suspended and sampled for analysis as above. The stirring was continued and further samples were removed after 1 hour and 2 hours.
At each occasion, the three samples of each formulation were analysed in accordance with the analytical procedure.
The analytical results confirmed that the doses analysed during the toxicity study were accurately formulated. The results also confirm that specimen formulations were stable during ambient temperature storage for a 2 hour period, which represented the time from preparation to completion of dosing.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

five male and five female rats per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
The high dosage was selected on the basis of available toxicity data in particular an acute oral toxicity study (LD50 >2.0 g/kg bodyweight, HRC Report No.: IFF 16 1/930884/AC) and a preliminary oral toxicity study (lFF/168) performed at this laboratory. Other levels were selected on the basis of the key dosages relative to EEC labelling requirements.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed four times on Day 1 and three times per day subsequently for signs of ill health, behavioural changes or toxicosis . Any observed changes were recorded. All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. On Saturdays, Sundays and public holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Any animal showing signs of severe debility or toxicosis was sacrificed for reasons of animal welfare.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed four times on Day 1 and three times per day subsequently for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded. All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. On Saturdays, Sundays and public holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Any animal showing signs of severe debility or toxicosis was sacrificed for reasons of animal welfare.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed prior to dosing on Day 1 (Week 0) and subsequently at weekly intervals throughout the study .

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Daily monitoring by visual appraisal was maintained throughout the dosing period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Prior to termination (Week 4).
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: All
- Parameters examined: Packed cell volume (PCV), Haemoglobin (Rb), Red blood cell count (RBC), Absolute indices: Mean corpuscular haemoglobin concentration (MCHC), Calculated: Hb (g/dl) x 1 00 + PCV ( % ), Mean corpuscular volume (MCV), Calculated : PCV ( % ) x 10 + RBC (x 1 06/mm3), Platelet count (pIts), Total white cell count (WBC)
The following estimations were measured using the appropriate methodology: Thrombotest (TT) - Method of Owren, P . A . (Lancet, 1959, ii, 754) Differential white blood cell count (Di ff) - namely: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes
The percentage distribution of each cell type is determined by standard microscopy of a blood smear stained with modified Wright's stain counting 1 00 cells. Percentage values are then converted to absolute values by computer inevitably involving a "rounding off" in a proportion of the results. Hence, the measured total WBC may differ slightly from the calculated total for the differential count. Additionally, blood film slides were examined for morphological abnormalities . No abnormal cells were seen that were considered to be treatment-related.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Prior to termination (Week 4).
- Animals fasted: Yes
- How many animals: All
- Parameters examined: Glucose - using BCL Test Kit (hexokinase mediated), Total Protein, Albumin (Alb), Globulin (Glob), Calculated : Total Protein (g/dl) minus Albumin (g/dl), Albumin/Globulin ratio (A/G), Calculated from Total protein and Albumin concentrations, Urea nitrogen (Urea Nitr), Creatinine, Alkaline phosphatase (AP), Reaction temperature 30°C Glutamic-pyruvic transaminase (GPT), also known as 'alanine aminotransferase (ALT)' - using BCL Test Kit, reaction temperature 30°C Glutamic-oxaloacetic transaminase (GOT), also known as 'aspartate aminotransferase (AST), - using BCL Test Kit, reaction temperature 30°C, Total bilirubin (Bilirubin), Sodium (Na), Potassium (K), Calcium (Ca), Chloride (Cl), Inorganic Phosphorus (P), Cholesterol (Chol)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: None

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After 28 days of treatment (Day 29), all animals surviving treatment were killed by carbon dioxide asphyxiation and a complete autopsy undertaken. The order of sacrifice was determined using pre-set cage sequence. Specified organs were weighed and relevant tissue samples were fixed for
microscopic examination.
Organ weight
The following organs from each animal were dissected free of fat and weighed: adrenals, brain, kidneys, liver, ovaries, spleen, testes (with epididymides)

Macroscopic pathology:
The macroscopic appearance of the tissues of all rats was recorded and samples of the following tissues were preserved in 1 0 % buffered formalin:
adrenals*, aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), femur (with joint),
head, heart*, ileum, jejunum, kidneys*, larynx, liver*, lungs, lymph nodes (cervical and mesenteric), mammary glands, oesophagus, ovaries, pancreas,
pharynx, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spleen*, sternum (for bone and marrow sections), stomach, testes including epididymis* (Bouin's as fixative), thymus (where present), thyroid (with parathyroids), tongue, trachea, urinary bladder, uterus (with cervix), vagina, any macroscopically abnormal tissue*
* Tissues required for histopathological examination for rats from Groups 1 and 4.

HISTOPATHOLOGY: Yes
Microscopic pathology
Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin wax (m.p. 56°C); sections were cut at 4 um (with the exception of testes which were cut at 2 um) and stained with haematoxylin and eosin.
Microscopic examination of prepared slides (from tissues indicated under Macroscopic pathology) was carried out for all rats of Group 1 (Control group) and Group 4 (High dosage group) killed on Day 29.
Examinations were extended following documented approval from the Sponsor, to include the kidneys (males) and the liver (males and females) of the intermediate and low dosage groups following the observation of a treatment-related change in these tissues in the animals of the high dosage group.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following sequence of statistical tests was used for bodyweight gains, organ weight and clinical pathology data: If the data consists predominantly of one particular value (relative frequency of the mode exceeds 75 % ), the proportion of values different from the mode was analysed by Fisher's exact test (Fisher 1950) followed by Mantel's test for a trend in proportions (Mantel 1963).
Otherwise: Bartlett's test (Bartlett 1937) was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1 % level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out followed by Williams' test (Williams 1971/2) for a dose-related response.
If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks (Kruskal-Wallis 1952/3) was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test, (Shirley 1977)).
Covariate analysis of organ weight data (with final bodyweight as covariate) was also performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10 % level of significance (Angervall and Carlstrom 1963).
Significant differences between control animals and those treated with the test substance were expressed at the 5 % (* P <0. 05) or 1 % (** P <0.0 1 ) level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

At 1000 mg/kg/day, paddling of the forepaws was seen in two female rats on isolated occasions. This finding was only observed immediately following administration of the test substance and was transient. It was considered to be attributable to discomfort following dosing and was not of toxicological importance. Increased salivation after dosing and associated wet fur was seen on the majority of occasions throughout the study in male and female rats dosed at 1000 mg/kg/day and increased salivation was also seen sporadically during the study for rats receiving 150 mg/kg/day (and in one male rat receiving 15 mg/kg/day). Post-dose brown perioral staining was noted in one male rat receiving 150 mg/kg/day on Day 10. An increase in salivation following dosing (and related signs namely wet fur and brown perioral staining) are commonly observed in rat orally dosed studies and are considered to be a result of the unpalatability of the test substance and are, therefore, not of toxicological importance.
The greasy fur seen in all the rats (including controls) during this study was linked to administration of the vehicle, corn oil, and likewise not of toxicological importance.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight gains were lower than control for male rats treated at 1000 mg/kg/day and male rats treated at 150 mg/kg/day. This was mainly due to lower gains during Week 3 to 4, the week during which clinical investigations were performed. There was considerable variation in overall bodyweight gain (up to 7% higher for females at 15 mg/kg/day or 11% lower for males at 150 or 1000 mg/kg/day). There was no dosage relationship and individual gains were satisfactory, these small differences for control were not considered to be treatment-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable for control and treated rats.

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

no effects observed

Description (incidence and severity):

Water consumption was assessed by visual appraisal throughout the study and was found to be satisfactory.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no statistically significant differences from control in any of the haematology parameters measured that were considered to be treatment-related. The monocyte count was statistically significantly lower than control for male rats treated at 1000 mg/kg/day. As the magnitude of the difference was small and the total white blood cell count remained unaffected, a treatment-related effect was not suspected.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Lower glucose levels for male and female rats receiving 1000 mg/kg/day achieved statistical significance in comparison with controls.
Cholesterol levels were statistically significantly higher than control for male rats treated at 150 or 1000 mg/kg/day. For rats at 150 mg/kg/day, the mean value was elevated by two particularly high values (105 for 110, 104 for 140). At the intermediate dosage there was considerable variation and in the absence of a dosage relationship and a histopathological effect on the liver at this dosage level, a treatment-related effect on cholesterol was considered unlikely.
Lower than control calcium ion concentration was noted for all male groups receiving treatment. As there was no dosage relationship and the difference from the control was small and the values were within the expected background range for rats of this age and strain (Ca mq/l male: 5 percentile 5.0, median 5.4, 95 percentile 5.7) this was not considered to be related to treatment.
There were no other statistically significant differences from control for groups of treated rats.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly higher than control liver weights (bodyweight adjusted) were recorded for male and female rats treated at 1000 mg/kg/day (36 and 47%, respectively). Liver weight (bodyweight adjusted) was higher than control for female rats receiving 150 or 15 mg/kg/day (12%). Individual values were within expected background range for rats of this age and strain (liver rat female: 5 percentile 8.7, median 10.5, 95 percentile 13 .6) and in the absence of any histopathological change a treatment-related effect on the liver at the low and intermediate dosage was not suspected.
The kidney weights were statistically significantly higher than control for male and female rats receiving 1000 mg/kg/day and for males at 150 mg/kg/day (10%). For males at 1000 mg/kg/day a minor histopathological change was seen that supports a slight increase in kidney weight. For rats of the intermediate dosage group this tissue was normal and in the absence of a dosage-related effect on kidney weight a kidney effect at this dosage was considered unlikely. Likewise, the kidneys of female rats from the high dosage group were histopathologically normal and this small difference from control in kidney weight was considered to have arisen by chance.
The ovary weight for rats receiving 1000 mg/kg/day was statistically significantly higher than control (absolute ns the increase was 21% and body weight adjusted it was 34%.
This was considered to be partly caused by a particularly high individual value (156 mg for animal no. 39 (female) and not considered related to treatment.
The bodyweight adjusted adrenal weights for all male rats receiving treatment were statistically lower than control _22%). All the values were within the expected background range for rats of this age and strain (adrenal both mg male: 5 percentile 40, median 49, 95 percentile 60) this apparent finding was considered to be caused by a particularly high group mean control value.
There were no other differences from control in the organ weights measured.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed at termination revealed no changes attributable to treatment with the substance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minimal hepatocyte enlargement was seen in centrilobular zones in all males and was generalised in all females receiving 1000 mg/kg/day. This change was associated with the higher weights recorded for this treatment group.
A marginal increase in incidence and degree of eosinophilic inclusions in proximal tubular epithel ium was seen in males receiving 1000 mg/kg/day when compared to controls.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Other tables than those presented below and further information can be found in the attached study report.

 

The analytical results confirm that the doses analysed during the study were accurately formulated.

The results also confirm that specimen formulations were stable during ambient temperature storage for a 2 hour period, which represented the time from preparation to completion of dosing.

 

Microscopic pathology: Liver

Treatment-related findings	Control		15 mg/kg/day		150 mg/kg/day		1000 mg/kg/day
	M	F	M	F	M	F	M	F
Centrilobular hepatocyte enlargement (minimal)	0	0	0	0	0	0	5**	0
Generalised hepatocyte (minimal)	0	0	0	0	0	0	0	5**
Total number of animals examined	5	5	5	5	5	5	5	5

** P <0. 0 1 with Fisher's exact test

 

 

Microscopic pathology: Kidney

Treatment-related findings in males	Control	15 mg/kg/day	150 mg/kg/day	1000 mg/kg/day
Eosinophilic inclusions in proximal cortical tubular epithelium - Trace	2	3	2	2
Eosinophilic inclusions in proximal cortical tubular epithelium - Minimal	0	0	0	2
Total no. of animals examined	5	5	5	5

 

The substance, a fragrance additive, was administered to rats by oral gavage for 28 days at dosages of 15, 150 or 1000 mg/kg/day. There was a treatment-related effect in the liver of male and female rats from the high dosage group. Centrilobular (males) or generalised (females) hepatocyte enlargement, with associated increased liver weight, were recorded. Associated minor biochemical ch anges (lower glucose, higher cholesterol level) were also recorded. This liver fi nding was considered to be adaptive in nature and probably related to metabolism of the test substance. The microscopic finding in the kidneys of high dosage group male rats, increased incidence and degree of eosinophilic inclusions in proximal tubular epithelium, is characteristic of light hydrocarbon nephropathy syndrome. This syndrome is specific to male rats and is a common finding with compounds of this nature. This finding, whilst treatment-related, is not considered predictive for a similar effect in man. The treatment-related finding in the liver of the high dosage group rats was considered to be adaptive and the treatment-related finding in the male kidneys not predictive for man, thus not representing a potential to cause serious damage to health. The rats were in general good health and no other treatment-related effects were seen. Consequently the high dosage of 1000 mg/kg/day was defined as the no-observed-adverse effect level (NOAEL) in the rat for Cassiffix. No treatment-related findings were seen for rats of the intermediate or low dosage groups and therefore 150 mg/kg/day was defined as the no-observed-effect level (NOEL) in the rat for Cassiffix.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD TG 407), the NOAEL was determined to be ≥1000 mg/kg bw/day for males and females, as no adverse effects were observed at a dose of 1000 mg/kg/day. This value will be used for the risk assessment.

Executive summary:

In a 28 -days repeated dose toxicity study (OECD TG 407, GLP) with a 2 -week recovery period, the substance was administered via oral gavage to Sprague Dawley rats (five males and five females) at dosages of 15, 150 and 1000 mg/kg/day. The substance was prepared as a suspension in corn oil at concentrations of 0.3, 3.0 or 20% w/v. Control animals (five males and five females) received the vehicle (corn oil) only, at the same dose volume (5mL/kg/day). All parameters from OECD TG 407 were recorded. Blood samples for clinical investigations were taken on day 27 and all animals were killed and examined macroscopically on day 29. Histopathological examination of the tissues was then initiated. The following results were found: Clinical signs: no adverse effects considered to be associated with treatment were observed for mortality, clinical signs, bodyweight, food consumption or efficiency of food utilisation. Haematology: no effects observed. Biochemical parameters: lower than control glucose levels were recorded specifically for male and to a lesser extent for female rats receiving 1000 mg/kg/day. Higher cholesterol levels for male rats receiving 1000 mg/kg/day were seen. Organ weights: higher than control liver weights (bodyweight adjusted) were recorded for male and female rats treated at 1000 mg/kg/day. Kidney weights were higher than control for male rats treated at 1000 mg/kg/day. Macroscopically no effects were seen. Microscopic pathology: Minimal centrilobular hepatocyte enlargement (males) and minimal generalised hepatocyte enlargement (females) were seen in the liver of rats receiving 1000 mg/kg/day. A marginally increased incidence and degree of eosinophilic inclusions were seen in the proximal tubular epithelium of the kidneys of male rats receiving 1000 mg/kg/day. The changes in the liver are considered to be of an adaptive nature due to administration of the test substance which is readily absorbed and are therefore not considered for the derivation of the NOAEL. The minor kidney effects are related to alpha-hydrocarbon nephropathy and are not taken into account for deriving the NOAEL for man because this is a male rat specific phenomenon. Based on the findings, the NOAEL was determined to be ≥1000 mg/kg bw/day for males and females.