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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:
not documented
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Secondary source from public literature
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Short-Term Toxicity Of Isoamyl Salicylate In Rats.
Author:
Drake J.J-P., Gaunt, I.F., Butterworth, K.R., Hoosen, J., Hardy, J., Gangolli, S.D.
Year:
1974
Bibliographic source:
Fd. Cosmet. Toxicol. Vol. 13, pp. 185-193
Report date:
1974

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance was administered to male and female rats at 4 doses of increasing concentration, administered via the diet, for a period of 13 weeks.
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Reference substance name:
Isoamyl salicylate
IUPAC Name:
Isoamyl salicylate
Details on test material:
- Name of test material (as cited in study report): Isoamyl salicylate
- Composition of test material, percentage of components: Isobutyl - 3.5%, n-butyl - 0.3%, isoamyl - 95.1%, optically active amyl - 1.1%
- Other: Specific gravity at 20°C: 1.050 - 1.052
Refractive index 20°C: 1.507 - 1.509
Free acid: max of 0.1%
Ester content: min of 99%
Dryness to toluene
No turbidity

The structural and functional similarity between isopenyl salicylate and hexyl salicylate means that read-across between the two materials is justified.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Specified pathogen free colony
- Age at study initiation: Not documented
- Weight at study initiation: Males: 51 - 86g
Females: 58 - 84g
- Fasting period before study: Not documented
- Housing: Five rats were housed together in a cage.
- Diet (e.g. ad libitum): Spillers' Laboratory Small Animal Diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: No information

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 1°C
- Humidity (%): 50 - 70%
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): No information

IN-LIFE DATES: From: To: Not documented

Administration / exposure

Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
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 feeding room for 48 hours. 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 5ft glass column packed with 10% Carbowax 20M Celite.

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diet was prepared twice daily.
- Mixing appropriate amounts with (Type of food): Not documented
- Storage temperature of food: Not documented

Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not documented
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily in feed.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 50, 500, 5000ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 4.7, 46, 415 mg/kg bw/day - males and 0, 4.8, 46.9, 475 mg/kg bw/day - females
Basis:
actual ingested
No. of animals per sex per dose:
15 animals
Control animals:
yes, plain diet
Details on study design:
Groups of 15 males and females were given diets containing 1 (control), 50, 500 or 5000 ppm isoamyl salicylate for 13 weeks. Additional groups of 5 rats of each sex of similar body weight were given diet containing 0, 500 or 5000 ppm for 2 or 6 weeks.
The nominal ppm dose levels equated to 0, 4.7, 46, 415 mg/kg bw/day - males and 0, 4.8, 46.9, 475 mg/kg bw/day - females in the 13 week study.
A paired group was included in the study design to investigate palatability one group receiving control diet and the other a treated diet at the highest inclusion level.

In a paired feeding study, two groups of ten rats, caged individually, were given either a diet containing 5000 ppm isoamyl salicylate or the control diet for 98 days. The groups consisted of rats from the same litters and each treated rat was paired with a litter-mate control. Each day each control rat was given a quantity of food equal to that consumed during the previous day by its treated litter-mate. The rats were weighed at intervals.
Positive control:
No information provided

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed frequently for abnormalities of condition or behaviour.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Days 1, 2, 6, 9 and 13 and at weekly intervals up to day 91.

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

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not documented
- Anaesthetic used for blood collection: Yes (barbiturate anaesthesia used for exsanguination)
- Animals fasted: Yes (24 hours prior to sacrifice)
- How many animals: All the rats in the 5000ppm group, half the control animals, and half the animals given 50ppm of the ester. The liver, kidneys, spleen and heart were examined in the 500ppm group.
- Parameters examined: 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.
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 leucocytes 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.

CLINICAL CHEMISTRY: No data

URINALYSIS: Yes
- Time schedule for collection of urine: During the last 2 days of treatment
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: Appearance, microscopic constituents and content of glucose, ketones, bile salts and blood. A renal concentration test was also conducted at this time, consisting of measurements of the specific gravity and volume of urine produced during a 6 hour period of water deprivation and of that produced during the 2 hour period following a water load of 25ml/kg. At week 6 and 13, the same measurements were made on the urine produced from 16-20 hours after the water load. A count of the number of cells in the urine was carried out on the 24 hour sample.

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: No additional information
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
No additional information
Statistics:
No information provided

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
The rats given 5000ppm isoamyl salicylate were visibly smaller than the controls and approximately half of them showed signs indicative of respiratory infection from week 3, consisting of pilo-erection, a tendency to adopt a hunched position and noisy, croaking respiration. In most animals these signs regressed but in one female they continued and the rat was killed during week 6, exhibiting signs of lethargy, hypothermia, and rapid shallow breathing.

BODY WEIGHT AND WEIGHT GAIN
The rate of body weight gain in both sexes of rats given 5000ppm isoamyl salicylate showed a statistically significant reduction compared with the controls which was evident after 1 day of treatment. On completion of the study, the weight of the males and females at this dietary level was lower than the controls by 15 and 9%, respectively.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food intake was reduced throughout the study at the highest level of feeding (5000ppm) 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 3 observations for each group.

FOOD EFFICIENCY
No information provided.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
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 compared with that of the controls.

OPHTHALMOSCOPIC EXAMINATION
No information provided

HAEMATOLOGY
There were isolated statistically significant differences between treated.and control animals in the results of the haematological examinations. The only reduced values were in the erythrocyte counts of the females given 500 and 5000 ppm isoamyl salicylate for 2 weeks. There were no comparable findings in males at the same time or in either sex at subsequent examination. There were no differences between the treated and control groups in the serum analyses.

CLINICAL CHEMISTRY
No data

URINALYSIS
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 in both sexes at week 6 but only in females at week 13. During week 2 the females given this dietary level produced an increased volume of urine in the 6-hr collection.

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
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 week 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 week 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 week and in females on the same diet for 13 week, whilst the spleen weight and relative spleen weight were increased in males given 500 ppm for 6 week. Statistically significant changes in kidney weight were confined to rats treated for 13 weeks when there were increases in relative weight in both sexes given the highest level of flavouring 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 week 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 week 13 and in the relative caecum weight in males at week 6, a decrease in adrenal-gland weight in males at week 6, an increase in relative testis weight at week 6 and 13, an increase in pituitary and relative pituitary weight in males at week 2 and a decrease in pituitary weight in males at week 6.

GROSS PATHOLOGY
No information provided

HISTOPATHOLOGY: NON-NEOPLASTIC
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.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not applicable

HISTORICAL CONTROL DATA (if applicable)
No information provided

OTHER FINDINGS
In the paired feeding study, 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

Dose descriptor:
NOAEL
Remarks:
500 ppm
Effect level:
46.9 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No additional information

Applicant's summary and conclusion

Conclusions:
The results of this study with isoamyl salicylate indicate that the no effect level (NOEL or no untoward effect dose) is 50 ppm inclusion in diet (equivalent to an achieved intake of 5 mg/kg bw/day).

The endpoint conclusions for this study were re-evaluated by the registrants resulting in a clarification of the study NOAEL and consequently a revision of the starting dose decision for hazard assessment. The NOAEL was concluded to be 500pm, based on local and systemic effects, rather than the original 50 ppm initially concluded by the reviewer. The NOAEL/NOEL terminology is not included by the report author – NOEL & NOAEL were not terms used at the time of the study conduct - but the report author concluded a no un-towards effect level of 50 ppm. Following review of the criteria for determining the no-untoward effect level and applying that to current terminology, the registrants consider that 50 ppm is in fact a NOEL. At 500 ppm, the only effect observed was on kidney weight in male rats. This was possibly due to more variability in the kidney weights or possibly attributable to the contribution of individual outlying animals. The increased kidney weight was not statistically linked to any histopathological change. The 5000 ppm dose is considered to be the LOAEL for this study.

The revised No Observed Adverse Effect Level (NOAEL), based on observation of adverse effects in the high dose group, was concluded to be 500 ppm, and equates to a systemic dose of 50 mg/kg bw/day after adjusting for the molecular weight ratio of hexyl and isoamyl salicylates. This NOAEL conclusion is also in accordance with other salicylate data presented by Belsito.

Due to the structural and functional similarities of hexyl and iospentyl salicylate, it is considered appropriate to use the NOAEL from this study as the starting point for derivation of DNEL values for hexyl salicylate

Based on these results, hexyl salicylate should not be classified according to Regulation EC No. 1272/2008. According to Directive 67/548/EEC, the test substance does not require classification.
Executive summary:

In the study conducted by Drake et al (1974), the test substance, isoamyl salicylate, was examined for its ability to induce toxicity following repeated exposure for a period of 13 weeks. The test substance was administered to male and female Wistar rats via oral administration in the diet at 4 dose levels, specifically, 0, 50, 500 and 5000ppm. Observations included mortality, food and water consumption, body weight and body weight gain, haematology, urinalysis, histopathological examinations and an examination of organ weights.

One mortality was observed in the top dose level of 5000ppm. 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.

Under the conditions of this study, the no observed adverse effect level (NOAEL) was 500 ppm in diet or circa 50 mg/kg bw/day. Based on these results, the test substance should not be classified according to Regulation EC No. 1272/2008. According to Directive 67/548/EEC, the test substance does not require classification.

Due to the similar structural, physical and chemical properties of Isoamyl Salicylate, it was considered appropriate for the purposes of read-across to Hexyl Salicylate