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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:
1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
SHORT-TERM TOXICITY OF ISOAMYL SALICYLATE IN RATS
Author:
J. J.-P. DRAKE, I. F. GAUNT, K. R. BUTTERWORTH, JEAN HUDSON,
JOAN HARDY and S. D. GANGOLLI
Year:
1974
Bibliographic source:
Fd Cosmer. Tooxfcol. Vol. 13. pp. 185-193. Pergamon Press 1975. Printed in Great Britain

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
The test chemical is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of at least 90 days. During the period of administration, the animals are observed closely for signs of toxicity. Animals which die or are humanely killed during the test are necropsied and at the conclusion of the test, remaining animals are also humanely killed and necropsied after the full dosing period.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Isopentyl salicylate
EC Number:
201-730-4
EC Name:
Isopentyl salicylate
Cas Number:
87-20-7
Molecular formula:
C12H16O3
IUPAC Name:
3-methylbutyl salicylate
Test material form:
liquid
Specific details on test material used for the study:
Isoamyl salicylate was supplied by Bush Boake Allen Ltd.. London, and complied with the following specification: Specific gravity (at 2O”C), 1.050-1.052; refractive index (20°C) 1.507-1.509; free a$d, max 0.1%; ester content, min. 99%; dryness to toluene, no turbidity. It was stated that the composition of the alcohols in the sample used was: Isobutyl, 3.5%; n-butyl, 0.3%; isoamyl, 95.1%; optically active amyl, 1.1%.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rats of the Wistar strain obtained from a specified-pathogen-free colony were given ground Spillers’ Laboratory Small Animal Diet and tap water ad lib.
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animal room was maintained at 20 f 1°C with a relative humidity of 50-70%.

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
As the highest dietary concentration of the ester to be used in the feeding study was 0,5%, it was considered that administration in the diet was appropriate. Fresh diet was prepared twice weekly
Details on oral exposure:
Diets containing 1 and 2% isoamyl salicylate were prepared. Samples of these were placed immediately in a sealed container while other samples were placed in animal-feeding pots and exposed to the atmosphere in an animal room for 48 hr.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were extracted with methanol and the isoamyl salicylate content of the extract was estimated using a Pye 104 dual flame gas chromatogram fitted with a 5 ft glass column packed with 10% Carbowax 20M on Celite.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
50 ppm
Dose / conc.:
500 ppm
Dose / conc.:
5 000 ppm
No. of animals per sex per dose:
15 animals/sex/dose

Examinations

Observations and examinations performed and frequency:
The animals were observed frequently for abnormalities of condition or behaviour and were weighed initially, at days 1, 2, 6, 9 and 13 and then at weekly intervals up to day 91.were weighed initially, at days 1, 2, 6, 9 and 13 and then at weekly intervals up to day 91. The consumption of food and water was measured over a 24-hr period preceding the day of weighing.
Sacrifice and pathology:
At the end of the appropriate period of feeding, the animals were killed by exsanguination from the aorta under barbiturate anaesthesia, following a 24-hr period without food. An autopsy was conducted, during which any macroscopic abnormalities were noted, and the brain, heart, liver, stomach, small intestine, caecum, spleen, kidneys, adrenal glands, gonads, pituitary and thyroid were weighed. Samples of these organs and of lung, lymph nodes, salivary gland, trachea, oesophagus, aorta, thymus, urinary bladder, colon, rectum, pancreas, uterus and skeletal muscle were preserved in 10% buffered formalin. Paraffin-wax sections of these tissues were stained with haematoxylin and eosin for microscopic examination. The histopathological examination was carried out on the tissues from all rats given 5000 ppm isoamyl salicylate, from half the control animals and from half of those given diet containing 50 ppm of the ester. The examination was confined to the liver, kidneys, spleen and heart in the rats given the intermediate level (500 ppm). Blood taken at autopsy was examined for haemoglobin concentration, packed cell volume and counts of erythrocytes and leucocytes. Slides were prepared from all blood samples to demonstrate reticulocytes and the different types of 1eucocyteB but counts of these were confined to the samples from the control rats and those given 5000 ppm isoamyl salicylate. Serum was analysed for the content of urea, glucose, total protein and albumin and for the activities of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and lactic dehydrogenase.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Throughout the experiment, the rate of body-weight gain in both sexes of rats given 5000 ppm isoamyl salicylate showed a statistically significant reduction compared with the controls. This was evident after 1 day of treatment and by the end of the study the weight of the males and females at this dietary level was lower than the controls by 15 and 9%, respectively. In addition the food intake was reduced throughout the study at the highest level of feeding (5000 ppm) so that the mean intake over the experimental period was less than the control values to a statistically significant degree. The overall reductions were 20 and 10% in males and females respectively. The most marked reductions in food intake were in the first few days of treatment, when the differences from controls were statistically significant despite being based on only three observations for each group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean intakes of the flavouring over the entire test period, calculated from body-weight and food-intake data, were 41, 46.0 and 415 mgJkg/day for males and 4.8, 46.9 and 475 mg/kg/day for females fed dietary levels of 50, 500 and 5000 ppm, respectively.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
On the first day of treatment, the water intake was reduced in rats of all treatment groups compared with that of the controls, the differences being statistically significant at the highest dietary level in both sexes. For the remainder of the study the water intake of the male rats given any level of the ester was similar to that of the controls, whereas the females given the highest dietary level showed an increase in water intake. This increase was statistically significant on day 2 of treatment and on occasions during the rest of the test, and the overall mean water intake by this group was significantly increased (P < 0.01; ranking method of White, 1952) compared with that of the controls.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were isolated statistically significant differences between treated and control animals in the results of the haematological examinations (Table 2). The only reduced values were in the erythrocyte counts of the females given 500 and 5000 ppm isoamyl salicylate for 2 wk. There were no comparable findings in males at the same time or in either sex at subsequent examination.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No abnormal constituents were found in the urine, but the rats given the highest dietary level tended to produce urine of a lower specific gravity following prolonged dehydration. The difference from controls was statistically significant (P < 0.05; White, 1952) in both sexes at wk 6 but only in females at wk 13. During wk 2 the females given this dietary level produced an increased volume of urine in the 6-hr collection.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were scattered statistically significant changes in organ weights and relative organ weights. The relative brain weight was increased at the highest dietary level in males at wk 6 and 13, while in the same animals the actual liver weight was lower than that of the controls. However, because of lower body weights, this latter difference was not evident when the weights were expressed relative to body weight. Conversely, in the females given 5000 ppm the relative liver weight was increased at all examinations. An increase in relative liver weight was also found at wk 2 in males given the highest dietary level. The relative spleen weights were increased compared with the controls in male rats given 5000 ppm isoamyl salicylate for 6 wk and in females on the same diet for 13 wk, whilst the spleen weight and relative spleen weight were increased in males given 500 ppm for 6 wk. Statistically significant changes in kidney weight were confined to rats treated for 13 wk. when there were increases in relative weight in both sexes given the highest level offlavouring and in the kidney weight and relative kidney weight of males on the intermediate level (500 ppm). There was an isolated increase in the small-intestine weight in males given 500 ppm for 6 wk but the remainder of the changes in organ weight were confined to rats given the highest level. These consisted of increases in the relative small-intestine weight in females at wk 13 and in the relative caecum weight in males at wk 6, a. decrease in adrenal-gland weight in males at wk 6, an increase in relative testis weight at wk 6 and
13, an increase in pituitary and relative pituitary weight in males at wk 2 and a decrease in pituitary weight in males at wk 6.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The histopathological examination revealed lung changes consisting of thickening of the alveolar walls together with lymphocyte cuffing of the blood vessels and bronchi. Also there were changes in the kidney consisting of lymphocyte infiltration and a protein exudate into the renal tubules. However, the distribution of these findings was similar in treated and control rats.
Details on results:
The rate of body-weight gain was similar in the two groups throughout the 98 days of the study. There were no statistically significant differences at any time.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
ca. 4.7 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 46 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
46 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The reduced rate of body-weight gain seen in rats given 5000 ppm isoamyl salicylate was associated with a reduced food intake. In addition, it was noticed that the greatest reduction in food intake was in the early stages of the experiment, suggesting that the reduced growth rate was due to a failure to eat a distasteful diet. This suggestion was confirmed by the paired-feeding study in which the differences in weight gain were eliminated by restricting the food intake of the controls to that of the treated rats. Such a reduced weight gain, attributable to an unpalatable diet, cannot be taken to indicate a toxic effect. The majority of the differences between the treated and control rats in the haematological examination did not indicate any adverse effect. The isolated decreases in the erythrocyte count at wk 2 in females given 500 and 5000 ppm isoamyl salicylate were small, being approximately 10% of the control value, and there were no comparable decreases in the haemoglobin concentration or packed cell volume. In addition the absence of any change in the number of reticulocytes indicated that the rate of erythropoiesis was not altered. It is concluded therefore that these decreases in erythrocyte count did not reflect any adverse effect of isoamyl salicylate. The increases in relative spleen weight could have resulted from an increased erythrophagocytosis in the spleen. However, the degree of splenic haemosiderosis detected in the histopathological examination did not differ in the control and treated animals. Moreover, under conditions of increased splenic weight due to an increased erythrophagocytosis there is usually a rapid compensatory reticulocytosis (Gaunt, 1973). but this did not occur in these animals. The finding that the female rats given 5000 ppm isoamyl salicylate excreted increased volumes of urine with a lower specific gravity than that produced by the controls indicates a functional injury to the kidney. This change was not seen in the male rats, but in both sexes there was an increased relative kidney weight without any histopathological change.
In addition, increased relative kidney weights were also evident in males given 500 ppm. These observations were taken, in the absence of any contrary evidence, to indicate some toxic effect of the ester on the kidney, particularly since one of the possible metabolites, salicylic acid, is known to be nephrotoxic (Joint FAO/WHO Expert Committee on Food Additives, 1962).
Since functional changes of the kidney were detected in females alone, it is possible that the increased water intake by the females given 5000 ppm, without similar changes in the males, may have been a result of the renal changes. Hatton & Bennett (1970) consider that the urge to drink in rats is controlled. by plasma osmolality and, if this is so, the excretion of a more dilute urine could result in an increased water intake. Alternatively, it has been suggested (Maller & Wank, 1971) that the total volume of water plus solid diet remains constant in the rat. On this basis, the increased water intake could have resulted from the decreased food intake but this fails to account for the restriction of the increased water intake to females, since the amount of food consumed was more markedly reduced in the males.
Apart from the increased kidney weight there were increases in the relative liver weight, again with no indication of any histopathological changes. The liver is known to be involved in the metabolism of some salicylate esters (Davison et al. 1961) as well as in the metabolism of the acid, and it is possible that the increased relative liver weight may have reflected an increased metabolic demand. Such increases in relative liver weight have been shown to occur with other materials and are thought to be of little toxicological significance (Golberg, 1967). The finding of an increase in relative brain weight in the animals with lower body weight was not surprising, since Peters & Boyd (1966) showed that in partially starved animals there was no loss of brain weight. Under such conditions of normal organ weight but reduced body weight, the calculation of relative organ weights will be expected to give increased ratios. In a number of studies in these laboratories this sitnation has been found to apply in the case of the testis and it may well account for other increases in relative organ weights at the highest level of treatment. The remainder of the changes in organ weight were scattered and cannot be said to represent any effect of the test material.
On the basis of the results of this study it is concluded that the no-untoward-effect level for isoamyl @icylate is 50 ppm of the diet. The average intake of the compound at this level was 4.7 mg/kg/day and allowing a lOO-fold safety factor this would suggest a safe daily intake of O-047 mg/kg or 2.8 mg/day for a 60-kg adult and correspondingly less in children. From data supplied by seven of the leading flavouring manufacturers, it was calculated that the maximum likely intake of isoamyl salicylate in man was 1.7 mg/day, so the no-untoward-effect level in this study is at least 150 times the likely intake in man. As the lowest effect level in the present study was ten times the no-untoward-effect level and the effects seen were very slight, it seems probable that the margin of safety may be even greater.
Executive summary:

Isoamyl salicylate was given to groups of 15 male and 15 female rats at dietary concentrations of 0 (control), 50, 500 or 5000 ppm for 13 wk. There was a decrease in weight gain at the highest dietary level accompanied by a reduced food intake, but a paired-feeding study showed that this was due to the diet’s unpalatability. The females given the highest dietary level drank more water than the controls and produced slightly greater volumes of more dilute urine. The relative kidney weight was increased in rats on the 500 and 5000 ppm levels without any histopathological changes. It is concluded that isoamyl salicylate was exerting a mildly nephrotoxic effect. The relative liver weight was increased at the highest level of feeding but there were no other effects attributable to treatment. The no-untoward-effect level from this study was 50 ppm of the diet, providing a mean intake of approximately 4.7 mg/kg/day.