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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August to November 1984
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Deviations:
not specified
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
The animals' health was assessed on the day of delivery, after which they underwent a 5-d period of acclimation to the conditions in the animal room. Females were nulliparous and nonpregnant. Only healthy animals without any clinical signs were included in the study. Neither prior to delivery nor during the acclimation or trial periods were the animals vaccinated or treated with anti-infectives.
The rats were about 6 to 8 weeks old at the start of the study. Average body weights were 140 g for males and 123 g for females.
The animals were housed individually under conventional conditions in Makrolon(R) Type-II cages (SPIEGEL and GÖNNERT, Zschr. Versuchstierkunde, 1, 38, 1961) on low-dust wood granules (BOGNER Inc., Solingen, Germany). The wood granules were regularly checked for contaminants by random sampling. Analytical results did not give any indication of an influence on the outcome of the trial. Clean cages with fresh bedding were provided on a weekly basis. During the acclimation period, animals were held in groups of the same sex in Makrolon (R) Typ-III cages.
The climate in the animal room was standardized: Room temperature = 22±2°C; relative humidity = about 50 %; light/dark cycle = 12 h (artificial light from 6 a.m. to 6 p.m.); change of air = approximately 10 times per h.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
During the study period, animals received their weekly ration of pelleted feed with the change of cage (fixed-formula Altromin(R) 1324 standard diet, produced to specifications by Altromin Inc., Lage, Germany); tap water was provided ad libitum. The tap water was of drinking quality. The available data on the analysis of water did not show any signs of an influence on the study outcome.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Concentrations in the application medium were not analyzed during the experiment.
Duration of treatment / exposure:
The animal experiments were performed over a period of three months (August to November 1984).
Frequency of treatment:
Groups of 15 male and 15 female rats each received single daily doses of isopentyl p-methoxycinnamate, formulated in polyethylene glycol 400 [Lutrol (R)], by gavage, seven days per week. Control animals were given the equivalent volume of Lutrol (R).
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0
Basis:
other: mg/kg body weight
Remarks:
Doses / Concentrations:
20
Basis:
other: mg/kg body weight
Remarks:
Doses / Concentrations:
200
Basis:
other: mg/kg body weight
Remarks:
Doses / Concentrations:
2000
Basis:
other: mg/kg body weight
No. of animals per sex per dose:
Groups of 15 male and 15 female rats per dose level were used.
Control animals:
yes, concurrent vehicle
Details on study design:
The test article was applied at a constant application volume of 5 mL/kg body weight. The amount applied was based on animal weights determined shortly before application.
Positive control:
No positive control was used.

Examinations

Observations and examinations performed and frequency:
The animals were examinated twice daily (once on weekends and holidays) and any changes or clinical signs were recorded. A detailed weekly report on the condition of individual animals concentrated on the following evaluations: body surfaces, stance, general behavior, respiration, and excretory products. The experimental animals were weighted before the first application and weekly thereafter. Body weights were also recorded directly before the scheduled necropsy, to calculate relative organ weights.

Weekly feed intakes and water consumption per dose, sex, and group were determined from the difference between the rations offered and that not consumed. Based on these primary feed-intake data, the following calculations were made: mean feed intake per animal and day, and cumulative feed intake per animal. Analogous to feed intakes, drinking-water consumption was determined by taking the weight difference between the water offered and that not being consumed. The nutritional composition and degree of contamination of the standard diet were routinely checked (by random sampling) and analyzed.

Clinical laboratory investigations were performed on ten animals of each dose group after four experimental weeks and at the end of the study. Blood samples intended for investigations of blood sugar were taken from one of the caudal veins of nonanaesthetized animals; for the other determinations, samples were taken from the retroorbital venus plexus of animals anaesthetized with diethyl ether.

Urinalyses: Urine was collected in periods of about 16 hours, during which time tap water was available ad libitum, but no feed was offered.
Semiquantitative = glucose, blood, protein, and pH values; ketone bodies, bilirubin, and urobilinogen
Quantitative = Protein, volume, specific gravity

Haematology: A Coulter Counter was used to determine erythrocyte, leukocyte, and thrombocyte counts, as well as MCV values and haemoglobin concentrations.

Clinical chemistry:
Enzymes = alkaline phosphatase (AP), glutamic-oxaloacetic transaminase (GOT), and glutamic-pyruvate transaminase (GPT)
Substrate = Uric acid in serum or plasma, blood sugar, cholesterol, total bilirubin, urea, Na+, K+, Ca++, phosphate
Sacrifice and pathology:
Gross pathology:
Unscheduled necropsies = rats dying during the study were necropsied as soon as possible and subjected to a thorough gross-anatomical evaluation.
Necropsies at end of the study = After the 13-week treatment, all surviving animals were anaesthetized with diethyl ether and killed by exsanguination. They were then necropsied and evaluated gross-phatologically. The following organ weights were determined for the sacrified rats: adrenal glands (in pairs), heart, kidneys (in pairs), liver, lung, spleen, testes (in pairs).

Histopathology: The following organs and tissues were histopathologically investigated in the 0 and 2000-mg/kg dose group (males and females), as were the livers and spleens of male and female animals in the 20 and 200-mg/kg dose group : adrenal glands, aorta, brain, epididymides, eyes, femur en bloc with skeletal muscle and sciatic nerve, heart, intestines (duodenum, jejunum, ileum, caecum, colon, rectum), kidneys, larynx, liver, lung, lymph nodes (mesenteric and mandibular), ovaries, pancreas, pituitary, prostate gland, salivary glands, seminal vesicle, skin (mammary region), spinal cord (cervical, thoracic, and lumbar sections), spleen, sternum, stomach, testes, thymus (if present), (thyroid glands, oesophagus, and trachea en bloc), urinary bladder, uterus, and all other organs with noticeable changes.
Other examinations:
no data
Statistics:
The following calculations were made: arithmetic group means, standard variations, upper and lower confidence limits at a confidence level of 1 - a = 95% and 1 - a = 99%. The values of treatment groups were compared with those of the control group using the two-sided significance test (U-test) at a significance level of a = 5% and a = 1%.
Since the U-test compares all groups treated with isopentyl p-methoxycinnamate with the control group, without adjusting the level of significance, increased false positives can be expected, which need not be based on biologically relevant differences between the groups.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
see section below

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 200 mg/kg bw (total dose)
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Clinical signs: "Weight loss" was given as a finding. No direct treatment-related effect was recognizable, since this finding occured in only a few animals at isolated times, and one control male was also affected.

Feed and water intakes: Male and female rats of all dose groups consumed about the same amount of pelletted feed as the control animals. Drinking-water consumption was comparable to that of the control group for animals receiving doses up to and including 200 mg/kg/day. At the dose level of 2,000 mg/kg/day, drinking-water consumption increased about 13 % in male animals and about 26% in females.

Body weights: Doses of up to and including 200 mg/kg/day isopentyl p-methoxycinnamate did not have any influence on body weight development. At the dose level of 2,000 mg/kg/day, body weights of male rats were lower than those of control animals.

Mortality: The following animals died during the study: one control female, one female of the 20 mg/kg/day dose group, and one male each of the 20 and 200 mg/kg/day dose groups. These deaths were not considered to be related to the treatment.

Ophthalmology: Ophthalmological investigations, performed at the beginning and end of the treatment period, did not reveal any changes related to treatment with isopentyl p-methoxycinnamate in either the refractory media, the ocular fundus, or the pupillary reflex.

Haematology: After one month, male rats in the 2,000 mg/kg/day dose group exhibited slightly lower leukocyte counts, and both males and females had slightly higher mean corpuscular haemoglobin concentrations than the control animals. After three months, male rats in the 2000 mg/kg/day dose group showed a higher haemoglobin content than control animals, as well as a higher mean haemoglobin content and mean corpuscular haemoglobin concentration. Female animals revealed higher leukocyte and erythrocyte counts, a higher haemoglobin content, elevated haematocrit values and mean corpuscular haemoglobin concentration, and a lower thrombocyte count. The differential blood count for male rats in the 2,000 mg/kg/day dose group showed a lower proportion of eosinophils than control animals after one and three months. In females of this group, the proportion of segmented neutrophils was higher, and that of lymphocytes was lower than the control group. Since these differences were only small, however, they were not considered to be of toxicological relevance.

Clinical chemistry: A dose of 20 mg/kg/day isopentyl p-methoxycinnamate did not cause any toxicologically relevant differences from the control animals in the parameters determined. After one month of treatment, the 200 mg/kg/day dose resulted in slightly lower cholesterol and higher phosphate concentrations in male animals, and lower uric acid concentrations in females, as compared to control animals. After three months, males exhibited slightly lower creatinine concentrations and females showed a very slight increase in the Na+-ion concentration. The 2,000 mg/kg/day dose caused the following differences from controls after one month: higher alkaline-phosphatase activity in male and female animals, higher GOT activity in females, and higher creatinine, potassium, and phosphate concentrations in males, lower uric acid and cholesterol concentrations in males and females, and a reduced calcium concentration in females. After three months, male and female animals in the 2,000 mg/kg/day dose group exhibited the following changes compared to controls: higher alkaline phosphatase and GOT activities and Na+-concentration, lower cholesterol and Ca2+-concentrations. In addition, males exhibited an elevated bilirubin concentration and lower creatinine concentration. In females, GPT activity and phosphate concentration were higher than in control animals. Since the differences in electrolyte concentrations between treated and control animals were very slight after one and three months, and no dose-effect-relationship was recognizable for the drop in creatinine concentration in male animals after three months, these findings were not considered to be of toxicological relevance.

Urinalyses: After one and three months of study, findings of urine investigations of ten rats per sex and dose did not show any differences between control animals and treated rats in dose groups up to and including 2,000 mg/kg/day.

Gross pathology: Terminal necropsies after three months = None of the necropsies on rats sacrified after the three-month study indicated treatment-related organ damage in any of the dose groups.

Organ weights = Organ weights of animals treated with doses up to and including 200 mg/kg/day did not differ relevantly or dose-dependently from those of control animals. Male and female animals in the 2,000-mg/kg dose group exhibited higher absolute and relative liver weights and relative kidney weights than the corresponding control animals, and lower absolute and relative spleen weights. Only male animals showed slightly higher relative heart weights than the controls.

Histopathology: Nearly all animals in the 2,000 mg/kg/day group revealed enlarged hepatocytes with only lightly stained cytoplasm, mainly in the peripheral zones of lobules. Some of these cells exhibited enlarged nuclei. Male and female animals in the 2,000 mg/kg/day group exhibited an increase in pigment containing iron in the siderocytes of the spleen; seven of 15 females revealed an increase in pigment containing iron in Kupffer cells of the liver. Very slight changes (e.g. small retention cysts in the pars glandularis of the stomach, small vacuoles in cells of the zona fasciculata of the adrenals, cystic atrophy of the epiphysis) were seen in some animals of all group and considered to be normal findings.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions described, the animals tolerated doses up to and including 200 mg/kg isopentyl p-methoxycinnamate. Accordingly, the no observed adverse effect level (NOAEL) for isopentyl p-methoxycinnamate was determined to be 200 mg/kg.
Executive summary:

The effects of repeated oral doses of isopentyl p-methoxycinnamate administered to rats over a period of 13 weeks were assessed in this study.

In this study, groups of 15 male and 15 female rats each received isopentyl p-methoxycinnamate daily by gavage in dosages of 20, 200, and 2,000 mg/kg body weight.

The animals thus treated did not differ in appearance, behaviour, or mortality from the control animals.

Feed and water intakes were not affected in dose groups up to and including 200 mg/kg/day. At the dose level of 2,000 mg/kg/day, male and female animals showed a markedly higher drinking-water consumption than control animals.

Doses up to and including 200 mg/kg/day did not have any effect on body weight development of treated rats. At the dose level of 2,000 mg/kg/day, male rats showed a retardation in body weight development.

Haematological investigations did not provide any indication of a haemotoxic effect of isopentyl p-methoxycinnamate in dose groups up to and including 200 mg/kg/day. At the dose level of 2,000 mg/kg/day, an increase in pigment deposits containing iron in the spleen and liver indicated an increased breakdown of erythrocytes.

Neither clinico-chemical analyses nor gross-anatomical or hispathological organ findings indicated any liver damage in dose groups up to 200 mg/kg/day. At the dose level of 2,000 mg/kg/day, increased enzyme activities and bilirubin concentrations, and a decreased concentration of cholesterol in plasma indicated an effect on liver function. Histopathologically, the condition corresponded to that of a metabolically activated liver. This was also implied by the higher absolute and relative liver weights.

Neither the urinalysis or clinico-chemical findings nor the gross-anatomical or hispathological organ findings indicated any kidney damage at doses up to 2,000 mg/kg/day.

Necropsies and gross-anatomical investigations of the remaining organs did not provide any indication of specific organ lesions, resulting from treatment with isopentyl p-methoxycinnamate in doses up to and including 2,000 mg/kg/day.

Ophthalmological investigations, conducted before and at the end of the study on ten male and ten female animals in both the control group and 2000 mg/kg/day dose group, did not reveal any treatment-related changes due to isopentyl p-methoxycinnamate.

Thus, under the experimental conditions described, the animals tolerated doses up to and including 200 mg/kg/day isopentyl p-methoxycinnamate. Accordingly, the no observed adverse effect level (NOAEL) for isopentyl p-methoxycinnamate was determined to be 200 mg/kg/day.