Registration Dossier

Administrative data

Description of key information

Repeat-dose oral toxicity studies in rats were performed with treatment durations of 28  and 90 days. A NOAEL was established at 50 mg/kg/day in the subchronic study. Liver and thyroid glands were  identified as the main target organs, although the findings obviously reflect adaptive liver changes of the rat to exogenous substances, secondarily affecting the thyroid gland.  The increased kidney weights were considered as treatment-related although a histopathological correlate was observed only in the dose-range finding study at excessive dosages.

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:
11.11.1994 - 13.07.1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions. Performed under GLP. Deviation from the current OECD 408 (adopted 21 September, 1998): no functional observations were performed, not all organ as specified in the guideline were weighed and/or microscopically examined.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CRL:CD BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Production, Ciba-Geigy Ltd., 4332 Stein, Switzerland
- Age: approx. 4 weeks old at delivery
- Weight at study initiation: 162 - 196 g (males) and 135 - 165 g (females)
- Housing: groups of 5 animals in Macrolon cages
- Diet: pulverised, standard diet
- Water: tap water, ad libitum
- Acclimation period: 7 days prior to random group assignment. A further period of acclimatisation of 7 days was allowed between allocation of animals to groups and the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C (overall range: 16-24 °C due to slight deviations)
- Humidity: 55 ± 10 % (overall range: 40-70 % due to slight deviations)
- Photoperiod: 12 hours dark/12 hours light

IN-LIFE DATES: From: 14-December 1994 to 19-April 1995
Route of administration:
oral: gavage
Vehicle:
other: 0.5 % sodium carboxymethylcellulose and 0.1 % Tween 80 in distilled water.
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in the vehicle by gradually adding the vehicle into the test substance by stirring. Once made up to volume the formulations were mixed using a high shear homogeniser. Suspensions of the test substance at the appropriate concentrations were freshly prepared every day immediately prior to the dosing of the animals and administered as soon after preparation.

VEHICLE
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the commencement of the study the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable and that the stability of the formulation was satisfactory under the conditions of the study. Samples of the formulations prepared for use in study Weeks 1, 4, 8 and 12 were also analysed to check the accuracy of preparation. In addition at the start and end of the treatment period test substance characterisation was carried out.
The analysed concentrations of the test item in the formulations were within +2.0%/-11% of nominal and were considered to be within acceptable limits for use on the study. Spectral analysis of the test item at the start and end of the study showed that the compound was stable over the treatment period.
Duration of treatment / exposure:
13 weeks (main group animals)
13 weeks + 4 week recovery period (recovery group animals)
Frequency of treatment:
Once daily, approximately the same time each day, 7 days per week.
Remarks:
Doses / Concentrations:
0, 10, 50, 250 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females (in control, treatment, and recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results obtained in a 28-d oral gavage repeated dose toxicity study in rats.
- Post-exposure recovery period in satellite groups: 4 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Individual animals were observed at least once daily for any signs of behavioural changes, reaction to treatment or ill health.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed clinical observation was performed daily for the first 4 weeks of the study. After 4 weeks of treatment detailed clinical observations were performed weekly due to the absence of treatment-related clinical signs noted during the first 4 weeks of the study. Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at the time of allocation of animals to groups, on the day of commencement of treatment and once a week thereafter, including the day of death or sacrifice.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each cage of rats was recorded on a weekly basis. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each cage in each group and the number of rats surviving in each cage.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily monitoring by visual appraisal of the water bottles was maintained throughout the study. Water consumption was measured accurately, by weight, for 7 consecutive days during Week 12 for all cages in all groups.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
Before treatment commenced the eyes of all animals were examined by means of a Keeler indirect ophthalmoscope. During Week 13 the eyes of all surviving animals in the control and high dosage level groups were examined. Prior to examination, the pupils of each animal were dilated using a Tropicamide ophthalmic solution.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 13 and after recovery, samples of blood were withdrawn, under light ether anaesthesia, from the orbital sinus of all rats from each group. In addition, blood samples were collected into tubes containing heparin anticoagulant for analysis of the thyroid hormones T3, T4 and TSH from all rats in Weeks 4, 13 and 17. The plasma for thyroid hormone assay was stored frozen prior to analysis.
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes, food was removed overnight from animals sampled for the routine laboratory investigations. Animals were not deprived of food prior to sampling for the thyroid function tests, which were taken in Weeks 4, 13 and 17.
- How many animals: all animals
The following parameters were analyzed:
Packed cell volume, haemoglobin, red blood cell count, total white cell count, platelet count, differential white blood cell count, cell morphology, thrombotest.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: (see above, as described under "Haematology")
- Animals fasted: Yes (see above, as described under "Haematology")
- How many animals: all animals
- Parameters examined: total protein, albumin, globulin, urea nitrogen, creatinine, sodium, potassium, calcium, inorganic phosphor, chloride, cholesterol, alkaline phosphatase, total bilirubin, glucose, alanine aminotransferase, aspartate aminotransferase, triglycerides, tri-iodothyronine, thyroxine, thyroid stimulating hormone.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Hepatic enzyme investigations
For 5 rats/sex/group at the terminal and recovery sacrifices, whole livers were removed and a 4 g sample was used for the preparation of hepatic 3000 x g supernatant and microsomes for subsequent biochemical measurements of microsomal protein, total cytochrome P450 and UDP glucuronyltransferase.
Sacrifice and pathology:
GROSS PATHOLOGY:
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pimitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastrointestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver was sectioned at iiltervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded.
The following organs from all animals killed at the scheduled sacrifices were dissected free of fat and weighed (NB: the liver and thyroids were weighed for all animals on the study): adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroid, uterus

HISTOPATHOLOGY:
Microscopical examinations: adrenals, alimentary tract, aorta, brain (medullary, cerebellar and cortical sections), nasopharynx, heart, kidneys, liver, lungs, lymph nodes, ovaries, pancreas, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles, spleen, testes (with epididymides), thymus, thyroid (with parathyroid), trachea, urinary bladder, uterus (corpus and cervix).

The microscopic examinations consisted of the following:
The specified list of tissues including all macroscopically abnormal tissues from all animals from the control group and all animals from the high dosage level group, whether dying during the smdy or killed at 13 weeks. Any macroscopically abnormal tissue found in any animal. Lungs, liver and kidneys from all animals in the intermediate and low dosage level groups. The investigations were extended to cover the following tissues, based on the organ weight data prior to completion of the microscopic examination in order to cover possible treatment-related effects:
Thyroid (both sexes), ovaries and testes from the intermediate and low dosage groups for Main and Recovery group animals. Liver and kidneys from all recovery group animals.
Statistics:
The following sequence of statistical tests was used for food consumption, water consumption, bodyweight, clinical pathology and organ weight data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75 %), the proportion of animals with values different from the mode was analysed, Fisher (1950) and Mantel (1963). Otherwise:
A test was applied to test for heterogeneity of variance between treatments, Bartlett (1937). Where significant (at the 1 % level) heterogeneity was found, 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. If significant heterogeneity of variance was present, and could not be removed by a transformation, an analysis of ranks was used, Kruskal-Wallis (1952/3).
Analyses of variance were followed by Students t test and Williams' test (Williams 1971/2) for a dose-related response, although only the one thought most appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of these tests, Shirley, 1977. Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, analysis of variance was performed using terminal bodyweight as covariate when the within-group relationship between organ weight and bodyweight was significant at the 10% level in an attempt to allow for differences in bodyweight may have influenced the organ weights.
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no deaths attributable to treatment. There were two deaths which occurred during the recovery period for humane reasons due to eye damage following blood sampling. There were no clinical signs indicative of a reaction to treatment.

BODY WEIGHT AND WEIGHT GAIN
Overall, there was not considered to be a conclusive adverse effect of treatment with the test article on bodyweight gain. Bodyweight gain for males receiving 250 mg/kg/day was marginally lower than that of controls from Week 2 of treatment. Prior to Week 2 bodyweight gain for this group had been
essentially similar to that of the male controls. However, during the first two weeks of treatment bodyweight gain was superior to that of controls for females receiving 250 mg/kg/day, although subsequent bodyweight gain was similar to that of controls during the remainder of the treatment period. Bodyweight gain amongst both sexes receiving 10 or 50 mg/kg/day were considered to have been unaffected by treatment. During the 4-week recovery period bodyweight gain for females previously treated with 250 mg/kg/day was superior to that of the female controls, while weight gain among males at this dose level was comparable to that of the male controls.

FOOD CONSUMPTION
Overall, there was not considered to be a conclusive adverse effect on food consumption. During the 13 week treatment period group mean cumulative food consumption was slightly higher than that of control for females receiving 250 mg/kg/day, which accounted for the increased bodyweight gain noted for this group. Group mean food consumption among the remaining male and female treated groups was essentially comparable with concurrent controls during the treatment period. During the 4 week recovery period, food intake for females previously treated with 250 mg/kg/day remained superior to that of control, reflecting the superior bodyweight gain noted for the group over this period. Food intake for males previously treated with 250 mg/kg/day was essentially similar to that of controls.

FOOD EFFICIENCY
The efficiency of food utilisation, as assessed during the 13 week treatment period by calculation of the food conversion ratios, was essentially similar for the control and treated groups. However, during the recovery period the efficiency of food utilisation was slightly superior to that of control for females previously treated with 250 mg/kg/day, which reflected the increased bodyweight gain noted for this group. The efficiency of food utilisation for males previously treated with 250 mg/kg/day was essentially similar to controls during the recovery period.

WATER CONSUMPTION
Measurement of water consumption during Week 12 revealed slightly increased water consumption for females receiving 250 mg/kg/day compared with that of controls. Group mean water consumption among all other male and female treated groups were considered to be similar to that of their respective controls.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmoscopic examination performed in Week 13 did not reveal any treatment-related ocular changes.

HAEMATOLOGY
The investigations performed in Week 13 revealed slightly, but significantly, decreased red blood cell parameters of packed cell volume (PCV), haemoglobin (Hb) and red blood cell coimt (RBC) for females receiving 250 mg/kg/day when compared with controls. Haemoglobin values, together with mean corpuscular haemoglobin concentration (MCHC), were also slightly, but significantly, decreased, together with a marginal reduction in PCV values among males receiving 250 mg/kg/day in comparison with controls. The investigations performed at the end of the recovery period in Week 17 continued to show slight reductions in PCV, Hb and RBC counts among both sexes previously treated with 250 mg/kg/day when compared with controls, although not all parameters attained statistical significance. The other minor differences from controls noted at these investigations, which on occasion attained statistical significance, were only slight and were not consistent between the sexes, thus these differences were not considered to be of toxicological importance.

CLINICAL CHEMISTRY
Analysis of T3 and T4 values revealed a dosage-related decrease in T3 values amongst male treated groups during Week 4 when compared with controls. However this change was not evident in Week 13, the T3 values for male treated groups being essentially similar to those of the controls. Thus in the absence of any treatment-related organ weight changes or pathological findings in the thyroid among male treated groups this change in male T3 values noted during Week 4 was considered to be transitory and not to be of toxicological importance. Among females receiving 250 mg/kg/day, however, T3 values were significantly reduced during Weeks 4 and 13 of treatment, together with a marginal reduction in T4 values for females at this dose level during Week 13, when compared with controls. Conversely, after a 4-week recovery period the T3 and T4 values for females previously treated with 250 mg/kg/day were slightly increased when compared with controls, indicating a reversal of the suppressed T3 and T4 values apparent during Week 13 of treatment. The Week 13 investigations revealed slightly, but significantly, increased cholesterol values for both sexes, together with increased triglyceride values for females, among animals receiving 250 mg/kg/day when compared with those of their respective controls. After the 4-week recovery period, similar slightly increased cholesterol values and marginally, but not significantly, increased triglyceride values were still apparent among females previously treated with 250 mg/kg/day when compared with controls. The other minor changes from controls noted at these investigations, which on occasion attained statistical significance, were only slight, were not consistent between the sexes and did not show a dosage related trend, thus these differences were not considered to be a result of treatment or of toxicological importance.

ORGAN WEIGHTS
Analysis of organ weight data from animals killed after completion of 13 weeks of treatment revealed increased liver weights (adjusted for final bodyweight) for both sexes receiving 250 mg/kg/day and for the females receiving 50 mg/kg/day when compared with controls. In addition thyroid weights
(adjusted for final bodyweight) were increased among females receiving 250 mg/kg/day when compared with controls. Kidney weights (adjusted for final bodyweight) were increased for both sexes receiving 250 mg/kg/day when compared with their respective controls. A slight, but not dosage-related, increase in testes weights was apparent among males receiving 50 or 250 mg/kg/day, together with an increase in ovary weights among females receiving 250 mg/kg/day, when compared with controls. However in the absence of any treatment-related pathological findings and the lack of dosage dependency these latter findings were not considered to be of toxicological importance. Analysis of organ weights obtained from animals killed after 4 weeks of recovery also revealed increased liver weights (adjusted for final bodyweight) and kidney weights for both sexes and thyroid weights (adjusted for final bodyweight) in females, among animals previously treated with 250 mg/kg/day when compared with controls. However, the degree of difference between control and the high dose recovery animals was slightly less than that noted for animals killed after completion of 13 weeks of treatment indicating a slight degree of recovery following cessation of treatment. Slightly increased pimitary, heart and adrenal weights were noted among recovery female animals previously treated with 250 mg/kg/day, when compared with control. However, these latter increases were attributable to the increased bodyweights noted amongst this dose group when compared with control and in the absence of any previous treatment-related organ weight or pathological changes in these organs, were not considered to be related to treatment.

GROSS PATHOLOGY
The macroscopic examination performed on rats killed after completion of 13 weeks of treatment revealed liver enlargement among 3/10 males and 5/10 females receiving 250 mg/kg/day compared with none in controls. The incidence and distribution of all other macroscopic findings from animals killed after 13-weeks of treatment or after completion of an additional 4-week recovery period were considered characteristic of animals of this species, strain and age, and none were considered to be attributable to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Examination of animals killed after completion of 13 weeks of treatment revealed an increased incidence of centrilobular hepatocyte enlargement among both sexes receiving 250 mg/kg/day and in males receiving 50 mg/kg/day; together with thyroid follicular hypertrophy noted among females receiving 250 mg/kg/day. No such changes in the liver and thyroid were noted among animals killed at the end of the 4-week recovery period, indicating that the effects of the test item on these tissues were reversible once treatment was stopped.

OTHER FINDINGS
Hepatic enzyme analysis (Hepatic Enzymology Report)
On the basis of the effects of the test substance on hepatic enzyme activities measured in this study, it can be concluded that the test substance is acting as a modest cytochrome P450 and Phase II drug metabolising enzyme inducer when administered at a level of 250 mg/kg bw/d under present conditions. Female rats appeared to be more sensitive to the effects of the test substance than were their male counterparts. The largest increases in enzyme parameters were observed in 7-pentoxyresorufin O-depentylase activity (in females) and in palmitoyl CoA oxidase activity. The relatively large induction of 7-pentoxyresorufin O-depentylase activity (CYP2B) in female rats, with smaller increases of other P450-reIated parameters, together with induction of p-nitrophenol UDPGT, indicate that the test substance is acting as a phenobarbitone-type inducer, at least in this sex. In addition to its phenobarbitone-like effects, there was evidence from induction of palmitoyl CoA oxidase activity that the test substance could act as a moderately weak peroxisome proliferator.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: haematology; organ weights (kidney)
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reversible effects in liver at 50 mg/kg bw/d: increased weight and centrilobular hypertrophy
Critical effects observed:
not specified

The liver and the thyroid were considered as target organs. Increased liver weights in the high dose groups and in females receiving 50 mg/kg bw/day were observed together with increased thyroid weights in females receiving 250 mg/kg bw/day. These findings were substantiated by microscopical findings as increased incidences of centrilobular hepatocyte enlargement and thyroidal follicular hypertrophy. The blood chemistry showed slightly increased cholesterol and triglyceride levels, reduced T3 and T4 values and an increase of cytochrome P450, EROD, PROD and UDPGA enzyme activities. These changes are considered to be the result of an adaptive response to the chemical and not to be indicative of a toxic effect. The liver effects observed characterise the test item as an inducer of cytochrome P450 and phase II drug metabolising enzymes and as a moderately to weak peroxisome proliferator. A possible effect on the kidney was evident at 250 mg/kg, which together with the haematological changes noted at this dose level, is considered to be an indication of renal toxicity among high dose level animals. After the recovery period effects on the haematological and some biochemical parameters were still evident, together with liver, kidney and thyroid organ weight changes, albeit not as marked as seen at the end of the treatment phase. The changes observed by microscopical investigation were reversed after the 4-week treatment-free recovery period. Based on these results the NOEL was established at 10 mg/kg bw/day and the NOAEL at 50 mg/kg bw/day.

Conclusions:
Oral administration of the test article to rats at dosage levels of 0, 10, 50 or 250 mg/kg/day for 13 weeks, followed by a 4 week recovery period in selected animals previously treated with 0 or 250 mg/kg/day revealed treatment-related changes among both sexes receiving 50 or 250 mg/kg/day. The treatment-related changes were confined mainly to animals receiving 250 mg/kg/day and to a lesser extent animals receiving 50 mg/kg/day. These changes were manifested as disturbances in the haematological and biochemical parameters, together with liver, kidney and thyroid organ weights, liver enzyme induction and pathological changes, evident as centrilobular hepatocyte enlargement and follicular epithelial hypertrophy. After a 4-week recovery period disturbances of the haematological and some biochemical parameters were still evident, together with liver, kidney and thyroid organ weight changes, albeit not as marked as seen during the treatment phase. Furthermore, there were no micropathological changes consistent with those recorded at the end of the treatment period, indicating some recovery following withdrawal of treatment. The principal target organs were the liver, together with a related treatment effect on the thyroid. However, these changes were considered to be the result of an adaptive response in the liver caused by the test material acting as an inducer of hepatic drug metabolising enzymes and not to be indicative of a toxic response. Conversely, a possible effect on the kidney was evident at 250 mg/kg/day, which together with the haematology changes noted at this dose level, was considered to be an indication of possible minor toxicity among high dose level animals. Thus on the basis of these data 50 mg/kg/day was considered to be the no observed adverse effect level (NOAEL) in the rat in this study. However, although centrilobular hepatocyte enlargement was noted in one low dose male, in the absence of any treatment-related biochemical, liver weight or macroscopic changes in this animal and an absence of hepatic drug metabolising enzyme induction occurring at 10 mg/kg/day, this finding was considered to be fortuitous and not to be of toxicological importance. Thus 10 mg/kg/day was considered to be the no observable effect level (NOEL) in this study.
Executive summary:

In a GLP-compliant subchronic in vivo study performed according to OECD guideline 408, the test material was administered by gavage to three groups, each of ten male and ten female rats, for 90 consecutive days, at dose levels of 10, 50 and 250 mg/kg/day. One group of rats received the vehicle alone and acted as the control. A further 10 animals/sex (Recovery groups) received the test substance at dosage levels of 0 (Control) and 250 mg/kg/day for 13 weeks and were then maintained for a further 4 weeks untreated and killed. Clinical signs, bodyweight and food consumption were monitored throughout the study for all animals. Water consumption was measured, together with an ophthalmoscopic examination and laboratory investigations which were performed at intervals during the study on Main and Recovery group animals. Selected organ weights were recorded and a wide range of tissues were preserved and subsequently examined microscopically.

There were no deaths attributable to treatment. There were two deaths which occurred during the recovery period for humane reasons due to eye damage following blood sampling. No clinical signs of a reaction to treatment were noted. The overall bodyweight gain and the overall group mean cumulative food consumption of treated groups were not considered to have been adversely affected by treatment. The efficiency of food utilisation was essentially comparable for control and treated groups during the 13 -week treatment period. However, it was slightly superior to control among females previously treated with 250 mg/kg/day during the recovery period, reflecting the increased weight gain exhibited by this group. Water consumption for females receiving 250 mg/kg/day was superior to that of control when measured during Week 12 of treatment. The ophthalmoscopic examination performed in Week 13 did not reveal any treatment-related ocular changes. The Week 13 laboratory investigations revealed slightly reduced values for red blood cell parameters among animals receiving 250 mg/kg/day, consisting of reduced packed cell volume, haemoglobin and red blood cell counts among females and haemoglobin (plus MCHC values) together with a marginal reduction in PCV values noted among males at this dosage. At the end of the recovery period similar reductions in the red blood cell parameters were still apparent among both sexes previously treated with 250 mg/kg/day. Slightly increased cholesterol values for both sexes and triglyceride values for females among animals receiving 250 mg/kg/day were recorded. At the end of the recovery period female cholesterol values were still slightly increased, together with a marginal increase in female triglyceride values for animals previously treated with 250 mg/kg/day, thus indicating recovery from the changes seen during Week 13. Analysis of the thyroid hormones revealed significantly reduced T3 values among females receiving 250 mg/kg/day during Weeks 4 and 13, together with reduced T4 values in Week 13. However, at the end of the recovery period, the female T3 and T4 values were slightly increased, indicating recovery once treatment was stopped.

Increased liver weights were reported for both sexes receiving 250 mg/kg/day and for females receiving 50 mg/kg/day, together with increased thyroid weights among females receiving 250 mg/kg/day after 13 weeks of treatment, which corresponded to pathological findings in these tissues. In addition group mean kidney weights for both sexes receiving 250 mg/kg/day were noted to be increased, but with no corresponding pathological change. Similar increases in liver, thyroid and kidney weights were noted among animals previously treated with 250 mg/kg/day, killed after completion of the 4-week recovery period.

The test article was acting as a modest cytochrome P450 and Phase II drug metabolising enzyme inducer, when administered at a level of 250 mg/kg/day. Female rats appeared to be more sensitive to the test compound than their male counterparts, with the increases in 7-pentoxyresorufin 0-depentylase and induction of p-nitrophenol UDPGT, indicating that the test article was acting as a phenobarbitone-type inducer in females. In addition evidence from induction of palmitoyl Co A oxidase activity indicates that the test article could also act as a moderately weak peroxisome proliferator.

During macroscopic examinations the principal treatment-related finding was of liver enlargement noted in animals receiving 250 mg/kg/day, killed after completion of 13 weeks of treatment. No such change was noted among animals killed at the end of the 4-week recovery period. Microscopic examination of animals killed after completion of 13 weeks of treatment revealed an increased incidence of centrilobular hepatocyte enlargement among both sexes receiving 250 mg/kg/day and in males receiving 50 mg/kg/day; together with thyroid follicular hypertrophy noted among females receiving 250 mg/kg/day. No such changes in the liver and thyroid were noted among animals killed at the end of the 4-week recovery period, indicating that the effects of the test item on these tissues were reversible once treatment was stopped.

Thus, on the basis of these data 50 mg/kg/day was considered to be the no observed adverse effect level (NOAEL) in the rat in this study. However, although centrilobular hepatocyte enlargement was noted in one low dose male, in the absence of any treatment-related biochemical, liver weight or macroscopic changes in this animal and an absence of hepatic drug metabolising enzyme induction occurring at 10 mg/kg/day, this finding was considered to be fortuitous and not to be of toxicological importance. Thus, 10 mg/kg/day was considered to be the no observable effect level (NOEL) in this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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

Additional information

In a GLP-compliant subchronic repeated dose study the test substance was administered daily to rats by oral gavage at dosage levels of 0, 10, 50 or 250 mg/kg/day for 13 weeks, followed by a recovery period of 4 weeks amongst selected male and female rats previously treated with 0 or 250 mg/kg/day. Treatment-related changes were evident among male and female treated groups receiving 50 or 250 mg/kg/day. Among animals receiving 250 mg/kg/day the liver was the principal target organ. Biochemical changes consisted of increased cholesterol values in both sexes, increased triglyceride values noted in females, at this dose level. At termination, increased liver weights, liver enlargement and centrilobular hepatocyte enlargement, were noted in male and female animals at this dose level. In addition, induction of hepatic drug metabolising enzymes was noted among both sexes, with females showing greater sensitivity and a phenobarbital like response. Also there was evidence that the test article was acting as a moderately weak peroxisome proliferator in both sexes receiving 250 mg/kg/day. The thyroid among females receiving 250 mg/kg/day was the other main target organ, with decreased T3 and T4 values evident biochemically, along with increased thyroid organ weights and follicular epithelial hypertrophy. These changes in the thyroid were considered a secondary effect resulting from the enhanced clearance of T3 and T4 caused by the hepatic enzyme induction.

A possible effect of treatment on the kidney was also noted, manifested as increased kidney weights in males and females receiving 250 mg/kg/day. The laboratory investigations also revealed haematological changes consisting of slight reductions in red blood cell parameters, noted among males and females receiving 250 mg/kg/day. A similar increase in kidney weights was apparent among animals receiving 1000 mg/kg/day in the previous 28 day study conducted with the test article. However in the absence of supportive pathological changes in the kidney, this finding is considered to be of uncertain toxicological importance.

At 50 mg/kg/day treatment-related changes consisting of increased liver weights in females and an increased incidence of centrilobular hepatocyte enlargement in the livers of male animals only, were observed. The changes at this dose level also confirm that the liver is the principal target organ for treatment with the test item, although a clear secondary effect on the thyroid gland was not evident at this dosage. There were no clear treatment-related changes apparent among animals receiving 10 mg/kg/day, apart from centrilobular hepatocyte enlargement which was noted in one male animal. However, in the absence of any treatment-related biochemical changes, liver weight change or macroscopic findings in this animal, together with no treatment-related hepatic enzyme changes noted in this group, the centrilobular hepatocyte enlargements in this single animal was considered to be fortuitous and as such not to be of toxicological importance.

After a 4-week recovery period the red blood cell parameters were still slightly reduced for both sexes, together with slightly increased cholesterol values in females, for animals previously treated with 250 mg/kg/day. Organ weight analysis also revealed increased liver and kidney weights for both sexes and thyroid weights in females which were still increased among recovery animals previously treated with 250 mg/kg/day, although the degree of the difference between the control and treated groups was less at the end of the recovery period than the difference noted for animals killed after 13 weeks of treatment. Conversely, T3 and T4 values for females previously treated with 250 mg/kg/day were slightly increased at the end of the recovery period, indicating a reversal of the suppression noted for these values during the treatment period. In addition, there were no micropathological changes evident which were consistent with those recorded previously at the end of the treatment period. Thus there is some degree of reversal of the effects on the liver and thyroid once treatment has stopped.

Thus on the basis of these data 50 mg/kg/day was considered to be the no observed adverse effect level (NOAEL) in the rat, while the lower dose level of 10 mg/kg/day was considered to be the no observable effect level (NOEL) in this study.

In a previously conducted 28-day repeated dose study with rats, the test article was administered to groups of five rats per sex by gavage at dose levels of 10, 50 and 1000 mg/kg body weight (Ciba-Geigy, 1992). Two satellite groups were treated with the high dose and the vehicle and kept for another four weeks after the treatment period (recovery groups). During this study neither deaths nor treatment-related clinical signs were noted. At treatment end red blood cell parameters were changed in males (50 and 1000 mg/kg) and in females (1000 mg/kg). The liver was identified as target organ by macroscopical and microscopical findings, liver weight changes and findings in clinical chemistry in males of group 3 (50 mg/kg) and in group 4 animals (1000 mg/kg). It can be inferred from the observations made during the above study, that a "no observable effect level" for the test article when offered to rats continuously in their food over a period of 4 weeks is 10 mg/kg body weight in the male sex (based on changes in clinical chemistry, organ weights and histopathological findings at 50 mg/kg bodyweight) and 50 mg/kg bodyweight in the female sex..


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP-compliant guideline study

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the present data, classification for repeated dose toxicity is not warranted under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for repeated dose toxicity is not warranted under Regulation (EC) No.1272/2008.