Registration Dossier

Administrative data

Description of key information

No repeated dose toxicity data are available for hexyl salicylate; however read-across data are available for the category substances methyl salicylate (17-week rat study, 2-year rat study, 8-week dog study, 2-year dog study) and isoamyl salicylate (90-day rat study). The data are relatively old but together provide a consistent weight of evidence.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
not documented
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This is an old study (1963), predating GLP. The study was well conducted and reported but it was limited in endpoints evaluated. It was used as preliminary study for a chronic study in rat (2 years).
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
no guideline followed
Principles of method if other than guideline:
17-week dietary toxicity study in the rat, intended as a range-finding study
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Osborne-Mendel
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Not documented
- Age at study initiation: Weanling
- Weight at study initiation: Approximately 50 g
- Fasting period before study: Not documented
- Housing: Not documented
- Diet (e.g. ad libitum): Not documented
- Water (e.g. ad libitum): Not documented
- Acclimation period: Not documented

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not documented
- Humidity (%): Not documented
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): Not documented

IN-LIFE DATES: From: To: Not documented
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
17 weeks
Frequency of treatment:
Continuous (daily), dietary exposure
Dose / conc.:
0 ppm
Remarks:
0%; control
Dose / conc.:
1 000 ppm
Remarks:
0.1%; ~50 mg/kg bw/d
Dose / conc.:
10 000 ppm
Remarks:
1%; ~500 mg/kg bw/d
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, concurrent no treatment
Details on study design:
No information provided
Positive control:
Not required for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Each rat's weight was recorded weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
No

FOOD EFFICIENCY:
No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No information provided
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
No additional information provided
Statistics:
No information provided
Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced weight gain was reported at the highest dose level
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No information provided

BODY WEIGHT AND WEIGHT GAIN
There was a reduction in body weight gain in males and females at the high dose. The differences were considered to be statistically significant

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No information provided

FOOD EFFICIENCY
No information provided

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No information provided

OPHTHALMOSCOPIC EXAMINATION
No information provided

HAEMATOLOGY
No information provided

CLINICAL CHEMISTRY
No information provided

URINALYSIS
No information provided

NEUROBEHAVIOUR
No information provided

ORGAN WEIGHTS
No information provided

GROSS PATHOLOGY
No gross abnormalities were observed

HISTOPATHOLOGY: NON-NEOPLASTIC
All microscopic findings were negative

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not applicable

HISTORICAL CONTROL DATA (if applicable)
No information provided

OTHER FINDINGS
Bones were not examined microscopically
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No gross or microscopic findings were observed at this dose level.
Key result
Critical effects observed:
not specified

No additional information provided

Conclusions:
Under this test condition, a reduction of bodyweight gain was observed at high dose, with no gross or microscopic findings. No effects were reported in the other dose-groups. An NOAEL of 0.1% (50 mg/kg bw/d) was identified.
Executive summary:

A sub-chronic oral toxicity study with methyl salicylate was performed to investigate its potential to induce toxicity following repeated exposure. The study was conducted using groups of 10 male and 10 female Osborne-Mendel rats. The animals were fed methyl salicylate at three dietary concentrations (0%, 0.1% or 1.0%), equivalent to intakes of 0, 50 and 500 mg/kg bw/d in the diet for 17 weeks. Bodyweight gain and selected organ weight and pathology were assessed. The high dose (1.0%) was associated with reduced bodyweight gain but had no effect on organ weight or histopathology. No effects were reported in the other dose groups. The results of this study support a NOAEL value of 0.1% in the diet, equivalent to 50 mg/kg bw/d.

Endpoint:
chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
no data provided
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This is an old study (1963), predating GLP however the protocol and results were reported in adequate detail and included haematological studies, gross pathology, and limited histopathological examinations of key organs and tissues.
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
no guideline followed
Principles of method if other than guideline:
2-year oral toxicity study in the dog
GLP compliance:
no
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
No information provided
Route of administration:
oral: capsule
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
No information provided
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information provided
Duration of treatment / exposure:
2 years
Frequency of treatment:
Daily (6 days a week) for 2 years
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
350 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
2 per sex per dose
Control animals:
yes
yes, concurrent no treatment
Positive control:
Not required for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
No

FOOD EFFICIENCY:
No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Collections were made at 2 weeks, 1, 3, and 6 months and 1 and 2 years
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No additional information
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
No additional information
Statistics:
No information provided
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two dogs died of unrelated disease: one high-dose female died of infectious canine hepatitis after 33 days on the experiment. Her replacement died of canine distemper after 19 weeks on the experiment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The administration of 350 and 150 mg/kg/day retarded the growth of the dogs. The dogs on the highest level lost an average of 1.90 kg while the control dogs gained an average of 1.85 kg. The dogs on 150 mg/kg/day gained an average of only 0.5 kg
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological analyses at 1, 3, 6, 12 and 24 months were normal.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Enlarged livers were seen at necropsy of the dogs on the 150 and 350 mg/kg/day levels. The heavier livers plus reduced bodyweight gain produced higher ratios of liver weight to body weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At necropsy, the dogs treated at 150 and 350 mg/kg body weight/day had enlarged livers.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopically, the livers of dogs at 150 and 350 mg/kg bw/d had larger hepatic cells than those seen in the control dogs. Fatty metamorphosis was not greater in the livers of the treated dogs than the very small amounts seen in the livers of the control dogs.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No clinical signs were observed. Two dogs died of unrelated disease: one high-dose female died of infectious canine hepatitis after 33 days on the experiment. Her replacement died of canine distemper after 19 weeks on the experiment.

BODY WEIGHT AND WEIGHT GAIN
The administration of 350 and 150 mg/kg/day retarded the growth of the dogs. The dogs on the highest level lost an average of 1.90 kg while the control dogs gained an average of 1.85 kg. The dogs on 150 mg/kg/day gained an average of only 0.5 kg

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No data

FOOD EFFICIENCY
No data

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

OPHTHALMOSCOPIC EXAMINATION
No data

HAEMATOLOGY
Haematological analyses at 1, 3, 6, 12 and 24 months were normal.

CLINICAL CHEMISTRY
No data

URINALYSIS
No data

NEUROBEHAVIOUR
No data

ORGAN WEIGHTS
Enlarged livers were seen at necropsy of the dogs on the 150 and 350 mg/kg/day levels. The heavier livers plus reduced bodyweight gain produced higher ratios of liver weight to body weight.

GROSS PATHOLOGY
At necropsy, the dogs treated at 150 and 350 mg/kg body weight/day had enlarged livers.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopically, these livers had larger hepatic cells than those seen in the control dogs. Fatty metamorphosis was not greater in the livers of the treated dogs than the very small amounts seen in the livers of the control dogs.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No data

HISTORICAL CONTROL DATA (if applicable)
No data

OTHER FINDINGS
No data
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: There were no mortalities observed at this dose level. No effects on body weight or organ weights were observed and no clinical signs noted at this concentration level.
Key result
Critical effects observed:
no

No additional information

Conclusions:
Under these test conditions, animals of the 150 and 350 mg/kg groups had retarded growth and enlarged livers were observed in these animals. No effects were reported at 50 mg/kg bw/day. Based on this study, the NOAEL /oral/dogs is 50 mg/kg body weight/day.
Executive summary:

In this study, groups of two male and two female beagles were administered methyl salicylate in capsules at dose levels of 0, 50, 150, or 350 mg/kg bw/d on 6 days/week for 2 years. One high-dose animal died of hepatitis apparently unrelated to methyl salicylate. Haematological analyses at 1, 3, 6, 12 and 24 months and complete necropsy examination were normal. Dogs treated at 150 and 350 mg/kg bw/d had enlarged livers, seen microscopically as enlarged hepatic cells. No other pathology was reported in any of the animals. Reduced body weight was reported in the 350 and 150 mg/kg bw/d groups. Based on this chronic oral study, the NOAEL value is 50 mg/kg bw/d.

Endpoint:
chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
no data available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
This is an old study (1963), predating GLP however the protocol and results were reported in adequate detail and included hematological studies, gross pathology, and limited histopathological examinations of key organs and tissues.
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
no guideline available
Principles of method if other than guideline:
2-year chronic toxicity in the rat
GLP compliance:
no
Remarks:
predates GLP
Limit test:
no
Specific details on test material used for the study:
No further details
Species:
rat
Strain:
Osborne-Mendel
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Not documented
- Age at study initiation: Weanling
- Weight at study initiation: Approximately 50 g
- Fasting period before study: Not documented
- Housing: Not documented
- Diet (e.g. ad libitum): Not documented
- Water (e.g. ad libitum): Not documented
- Acclimation period: Not documented

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not documented
- Humidity (%): Not documented
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): Not documented

IN-LIFE DATES: From: To: Not documented
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Every other week
- Mixing appropriate amounts with (Type of food): Not documented
- Storage temperature of food: All diets were stored in sealed cans under refrigeration

VEHICLE
None
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
2 years
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
0.1% in diet
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
0.5% in diet
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
1% in diet
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
2% in diet
No. of animals per sex per dose:
25/sex/dose (except for 24 males, 26 females in 2% group)
Control animals:
yes
Positive control:
Not required for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The rats were weighed weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
No

FOOD EFFICIENCY:
No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 11, 17 and 22 months
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 rats/dose

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No additional information
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
No other examinations reported
Statistics:
Not documented
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of the 1.0% and 2.0% groups developed rough hair coats.
Mortality:
mortality observed, treatment-related
Description (incidence):
In the high-dose group, half of the animals died by week 8 and all of the animals died by week 49 of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Animals of the 1.0% and 2.0% groups had statistically significant growth inhibition.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Average organ weights were similar for all animals. However, relative organ to body weight ratios for the testes of male animals and for the heart and kidneys of the female animals of the 1.0% group were significantly increased.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross lesions of the pituitary gland were observed in 10 animals of the 0.5% group as compared to four animals in the control group. In the 2% group, 29 of the 50 animals had pneumonia, which appeared to be more acute than regularly observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopically, the only induced lesion was a pronounced change in the bones of the rats on the 2.0% diet. Cancellous bone in the metaphysis was increased as compared to same-age controls; this was observed to a moderate degree in five and a marked degree in four of the nine bones examined from animals of the 2.0% group. Bone lesions were slight in 2 of 11 and 1 of 11 bones examined from animals of the 1.0% and 0.5% groups, respectively. The affected bones had fewer osteoclasts, and the number was inversely proportional to the degree of change.
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Animals of the 1.0% and 2.0% groups developed rough hair coats. In the high-dose group, half of the animals died by week 8 and all of the animals died by week 49 of the study.

BODY WEIGHT AND WEIGHT GAIN
Animals of the 1.0% and 2.0% groups had statistically significant growth inhibition.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Not documented

FOOD EFFICIENCY
Not documented

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not documented

OPHTHALMOSCOPIC EXAMINATION
Not documented

HAEMATOLOGY
No haematological effects were observed

CLINICAL CHEMISTRY
Not documented

URINALYSIS
Not documented

NEUROBEHAVIOUR
Not documented

ORGAN WEIGHTS
Average organ weights were similar for all animals. However, relative organ to body weight ratios for the testes of male animals and for the heart and kidneys of the female animals of the 1.0% group were significantly increased.

GROSS PATHOLOGY
Gross lesions of the pituitary gland were observed in 10 animals of the 0.5% group as compared to four animals in the control group. In the 2% group, 29 of the 50 animals had pneumonia, which appeared to be more acute than regularly observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopically, the only induced lesion was a pronounced change in the bones of the rats on the 2.0% diet. Cancellous bone in the metaphysis was increased as compared to same-age controls; this was observed to a moderate degree in five and a marked degree in four of the nine bones examined from animals of the 2.0% group. Bone lesions were slight in 2 of 11 and 1 of 11 bones examined from animals of the 1.0% and 0.5% groups, respectively. The affected bones had fewer osteoclasts, and the number was inversely proportional to the degree of change.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
The data are limited: Malignant pituitary tumours occurred in 1 male and 2 female rats on the 0.5% diet. Mammary tumours occurred in females rats on all diets.

HISTORICAL CONTROL DATA (if applicable)
No information provided

OTHER FINDINGS
No additional information
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Key result
Critical effects observed:
no

The results for a supplemental study:

One male test animal died on day 11, the two others died on day 19. The females died on days 31, 40 and 71. Rough hair coat and growth inhibition was observed for all test animals, with some animals having labored respiration.

Grossly, four of the six treated rats had slight to moderate lung damage. Focal gastric hemorrhages were present in the glandular portion of three of the test animals. Bone growth was affected.

Long bones of the limbs showed reduced length and diameter compared to controls. Density of the hypophysis of long bones was increased.

Conclusions:
Under the test conditions, growth retardation and bone lesions were reported at 1.0 and 2.0% and gross pituitary lesions at 0.5% methyl salicylate in the diet. Based on this study, the NOAEL is 50 mg/kg bw/d in the rat.
Executive summary:

In this methyl salicylate was administered to groups of male and female Osborne-Mendel rats at concentrations of 0, 0.1%, 0.5%, 1.0% or 2.0% in the diet providing doses of approximately 0, 50, 250, 500, and 1000 mg/kg bw/d for two years. All rats in the 1000 mg/kg bw/d group died by the 49th week. Body weight of both sexes was significantly decreased in the 500 and 1000 mg/kg bw/d dose groups. An increased amount of cancellous bone was present in the metaphyses in rats treated at either 500 or 1000 mg/kg bw/d, with a more marked effect at the highest dose level. The relative testis weight of males was significantly increased as were the relative weights of the heart and kidneys of females in the 500 mg/kg bw/d group. Gross pituitary gland lesions were increased at 250 but not at 500 mg/kg bw/d compared to controls. Based on this study, the NOAEL is 50 mg/kg bw/d.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
not documented
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This is an old study (1963 predating GLP. The study was well conducted and reported but it was limited in endpoints evaluated. It was used as preliminary study for a chronic study in dogs (2 years).
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Repeated oral (capsule) toxicity study in the dog, serving as a dose range-finding study for the subsequent 2-year study
GLP compliance:
no
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
No information provided
Route of administration:
oral: capsule
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
No information available
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No information reported
Duration of treatment / exposure:
59 days
Frequency of treatment:
Once a day for 6 days each week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Dose / conc.:
1 200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
1 per sex per dose
Control animals:
yes, concurrent no treatment
Details on study design:
No information provided
Positive control:
No information provided
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Not documented

BODY WEIGHT: Yes
- Time schedule for examinations: Not documented

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No additional information
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
No further information provided
Statistics:
No information provided
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 50, 150 and 250 mg/kg/day, no clinical signs and mortality were observed. At 500 mg/kg/day, all dogs died within the first month of the study. Two of these dogs had diarrhoea and weakness during their last 3 days. At 800 and 1200 mg/kg/day, all dogs died within the first month of the study. Several dogs vomited 3-4h after dosing.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 500 mg/kg/day, all dogs died within the first month of the study. At 800 and 1200 mg/kg/day, all dogs died within the first month of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
All dogs receiving 500 mg/kg/day or more of methyl salicylate lost weight and died or were killed due to moribund condition.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
On microscopic examination, the livers of both dogs on the 1200 and one on the 800 mg/kg/day levels had moderate to marked degrees of fatty metamorphosis. Of the other minor pathological changes observed, none were attributed to the administration of the test substance.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
At 50, 150 and 250 mg/kg/day, no clinical signs and mortality were observed. At 500 mg/kg/day, all dogs died within the first month of the study. Two of these dogs had diarrhoea and weakness during their last 3 days. At 800 and 1200 mg/kg/day, all dogs died within the first month of the study. Several dogs vomited 3-4h after dosing.

BODY WEIGHT AND WEIGHT GAIN
All dogs receiving 500 mg/kg/day or more of methyl salicylate lost weight and died or were killed due to moribund condition.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No data

FOOD EFFICIENCY
No data

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

OPHTHALMOSCOPIC EXAMINATION
No data

HAEMATOLOGY
No data

CLINICAL CHEMISTRY
No data

URINALYSIS
No data

NEUROBEHAVIOUR
No data

ORGAN WEIGHTS
No data

GROSS PATHOLOGY
On microscopic examination, the livers of both dogs on the 1200 and one on the 800 mg/kg/day levels had moderate to marked degrees of fatty metamorphosis. Of the other minor pathological changes observed, none were attributed to the administration of the test substance.

HISTOPATHOLOGY: NON-NEOPLASTIC
On microscopic examination, the livers of both dogs on the 1200 and one on the 800 mg/kg/day levels had moderate to marked degrees of fatty metamorphosis. Of the other minor pathological changes observed, none were attributed to the administration of the test substance.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No data

HISTORICAL CONTROL DATA (if applicable)
No information provided

OTHER FINDINGS
No information provided
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

No additional information

Conclusions:
A NOAEL of 250 mg/kg bw/day was identified.
Executive summary:

In a sub-chronic toxicity study, groups of two dogs, one male and one female, were given doses of 50 to 1200 mg/kg bw/d methyl salicylate orally in capsule form daily, 6 days per week for up to 59 days. All dogs receiving 500 mg/kg bw/d or more of methyl salicylate lost weight and died or were killed due to moribund condition within 1-month of study initiation. One animal given 800 mg/kg bw/d and both given 1200 mg/kg bw/d vomited for 3 -4 hours following each administration. Microscopically, moderate to marked fatty changes were observed in the liver of one animal at 800 mg/kg bw/d and both at 1200 mg/kg bw/d. Animals given 500 mg/kg bw/d had diarrhoea and weakness during their last 3 days. No adverse effects were observed in animals given 50, 150 and 250 mg/kg bw/d. A NOAEL of 250 mg/kg bw/day was identified for this study.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
not documented
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
no guideline followed
Principles of method if other than guideline:
90-day rat study, broadly comparable to OECD 408
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
No further details
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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
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
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily in feed.
Dose / conc.:
0 ppm
Remarks:
control
Dose / conc.:
50 ppm
Remarks:
Actual ingested dose: 4.7 mg/kg bw/d in males and 4.8 mg/kg bw/d in females
Dose / conc.:
500 ppm
Remarks:
Actual ingested dose: 46 mg/kg bw/d in males and 46.9 mg/kg bw/d in females
Dose / conc.:
5 000 ppm
Remarks:
Actual ingested dose: 415 mg/kg bw/d in males and 475 mg/kg bw/d in females
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
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
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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 piloerection, 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.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female at 5000 ppm was sacrificed during Week 6
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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):
effects observed, treatment-related
Description (incidence and severity):
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:
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 compared with that of the controls.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
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 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.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
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 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 pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
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
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
.
Other effects:
no effects observed
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.
Key result
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:
body weight and weight gain
organ weights and organ / body weight ratios
other: see 'Remark'
Key result
Critical effects observed:
no

No additional information

Conclusions:
The 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.
Executive summary:

In this study, 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 dose levels of 0, 50, 500 and 5000 ppm. 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 5000 ppm. 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 (~50 mg/kg bw/d).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
No repeated dose toxicity data are available for hexyl salicylate; however read-across data are available for the category substances methyl salicylate (17-week rat study, 2-year rat study, 8-week dog study, 2-year dog study) and isoamyl salicylate (90-day rat study). The data are relatively old but together provide a consistent weight of evidence.
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28/07/2016 - 25/08/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
(2009)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 50064843
- Expiration date of the lot/batch: 14 April 2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 – 25 °C
- Solubility in water : 2 mg/l at 23 °C
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Young adult, male and female Wistar outbred rats (Crl:WI(Han)) were obtained from a colony maintained under specific pathogen-free (SPF) conditions at Charles River, Germany.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-9 weeks
- Weight at study initiation: mean males= 251g, mean females =171g
- Fasting period before study: overnight fasting
- Housing:Macrolon cages with wood shavings
- Diet:ad libitum except during exposure and – for animals of the main study – the overnight fasting period before sacrifice.
- Water: ad libitum except during exposure
ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From:28/07/2016 To:25/08/2016
Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.6 - <= 3.3 µm
Remarks on MMAD:
The average particle size (MMAD) of the aerosol was 3.3 μm (gsd of 1.7), 2.9 μm (gsd of 1.8) and 2.6 μm (gsd of 1.9) for the low, mid and high concentration test atmospheres, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. The test atmospheres were generated by nebulization using an air-driven atomizer which was placed at the top inlet of the exposure chamber. For generation of the low concentration test atmosphere, the atomizer was heated to reduce the aerodynamic particle size.

The amount of test material delivered to the atomizer was controlled using a motor-driven syringe pump.The atomizer was supplied with dry compressed air, controlled at a pressure of 4 bar using a reducing valve. Air flow to the atomizer was measured using a mass view meter. In the top of the exposure unit, a bypass stream of humidified compressed air was added, the flow of which was controlled and measured using rotameters which were calibrated at the settings used during the study using a volumetric flow meter. the air supply to the various rotameters was controlled by mass flow controller.From the top of the exposure chamber, the test atmosphere was directed downward and led to the noses of the animals. At the bottom of the unit the test atmosphere was exhausted.

The exposure unit for the control animals was supplied with a flow of humidified compressed air only, which was controlled using a reducing valve and measured by mass view meter
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The overall average actual concentrations of Hexyl salicylate in the test atmospheres were determined by gravimetric analysis
Duration of treatment / exposure:
Exposure was over a 28-day period, with a total number of 20 exposure days.
Frequency of treatment:
6 hours/day for 5 days/week
Dose / conc.:
0 mg/m³ air
Remarks:
Actual concentration in air
Dose / conc.:
10.9 mg/m³ air
Remarks:
Actual concentration in air
Dose / conc.:
52.3 mg/m³ air
Remarks:
Actual concentration in air
Dose / conc.:
249 mg/m³ air
Remarks:
Actual concentration in air
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes
Details on study design:
Animal selection was random
- Dose selection rationale: concentrations were based on the results of a preceding 7-day range finding study (5 exposure days in total). Results for this study are presented in Annex 10 of the study report.
- Post exposure period: Amimals were subject to necropsy on the day following the last exposure.

Positive control:
Not required for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
On days of exposure, each animal was observed daily in the morning, prior to exposure, by cage-side observations and, if necessary, handled to detect signs of toxicity. All animals were thoroughly checked again after exposure. Clinical signs listed but not limited to Annex 6 of the study report were used for the recording of clinical observations. During exposure, a group-wise observation was made about halfway through the 6-hour exposure period.

BODY WEIGHT: Yes
Body weight of each animal was recorded 2 days before the start of exposure. These pre-test weights served as a basis for animal allocation. Subsequently, the animals of the main study were weighed prior to exposure on the first day (day 0), and then every 3 or 4 days, including the day of scheduled sacrifice. The animals were also weighed on the day before overnight fasting (prior to the scheduled sacrifice).

FOOD CONSUMPTION: Yes
Food consumption was measured per cage by weighing the feeders. Food consumption was measured over three 7-day periods, starting on day 0, followed by a 6-day period. The results were expressed in g per animal per day.

HAEMATOLOGY: Yes
Hematology was conducted in samples collected from all animals at necropsy. Blood samples were taken from the abdominal aorta of the rats.
- Anaesthetic used for blood collection: Yes (pentobarbital anaesthesia) EDTA or citrate (for prothrombin time) were used as anticoagulant.
- Animals fasted: Yes (Overnight before necropsy but water was freely available)

CLINICAL CHEMISTRY: Yes
Hematology was conducted in samples collected from all animals at necropsy.
- Animals fasted: Yes (Overnight before necropsy but water was freely available)

Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were subject to gross necropsy
HISTOPATHOLOGY: Yes. A detailed assessment was made of all animals
Other examinations:
ORGANS EXAMINED AT NECROPSY: Yes
- Organ weights: adrenals , lungs with trachea and larynx, brain, spleen, heart, testes, kidneys, thymus, liver
Statistics:
-Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ with automatic data transformation. Day 0 body weight data were used as covariate in the analysis of the post-treatment data unless removed during data pre-processing.
-Pre-treatment body weight, organ weight, hematology and clinical chemistry data: ‘Generalized Anova/Ancova Test’ with automatic data transformation method
-Incidences of histopathological changes: Fisher’s exact probability test
-Food consumption: no statistics was applied on food intake (only one cage per sex)
Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical abnormalities were observed. The few signs noted were considered unrelated to the exposure to the test material. Abnormalities of the skin or fur (sparsely haired areas, encrustations, skin wounds) were observed across the groups. These are common findings, possibly caused by slight movement of the animals in the restraining tubes during exposure (resulting in slight irritation of the skin) or by placing of the subcutaneous transponder. Other clinical signs, observed incidentally in single animals, also represented background findings and were not related to exposure to the test material.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male animals of the high concentration group showed a statistically significantly lower bodyweight than controls from Day 21 until the end of the study. Overall body weight gain during the 28-day period was statistically significantly decreased in males of the high concentration group when compared to unexposed controls. There were no statistically significant differences in body weights of the female rats between the test groups and controls. At Day 14, a temporary and slight decrease in body weight was noted in all female rats, which was not considered to be treatment related. The reason for this slight decrease in all female rats remained uncertain.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was slightly decreased in all males at the higher concentrations.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no statistically significant changes in red blood cell variables, coagulation parameters and white blood cell variables in male rats. In females there were no statistically significant changes in red blood cell variables, except for an incidental decrease in reticulocytes in the low concentration group which was considered to be a chance finding because no changes were observed in the mid and high concentration group. Prothrombin time was slightly, but statistically significantly, increased in the high concentration group. The absolute and relative numbers of neutrophils were statistically significantly decreased and the percentage of lymphocytes was statistically significantly increased in females in the low concentration group. The percentage of monocytes was statistically significantly increased in females in the mid concentration group. These findings were considered chance findings because they were not confirmed at the high concentration level.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males there were no statistically significant differences in clinical chemistry variables except for a slight increase in mean albumin/globulin ratio in the high concentration group. In the absence of any changes in albumin and total protein levels, or any corroborative findings in females, a minor change in this calculated parameter was considered a chance finding. In females of the high concentration group a statistically significant increase in plasma ALP activity was observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in absolute and relative organ weights between the control group and the groups exposed to the test material
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The histopathological changes observed such as thymus microhaemorrage were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. See Table 12 and appendix 8 of the study report. These changes were therefore considered to be unrelated to the exposure to the test material.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
249 mg/m³ air
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Inhalation exposure to hexyl salicylate up to a concentration of 249 mg/m3 was tolerated well by the animals and did not result in any adverse exposure-related changes
Key result
Critical effects observed:
no

Mean body weight

Day 0

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

249.12

250.36

252.14

250.66

168.30

175.76

165.82

172.46

SD

4.89

6.81

8.54

7.21

10.38

5.58

6.94

7.23

N

5

5

5

5

5

5

5

5

Day 4

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

255.58

256.12

259.28

255.00

179.00

186.54

177.96

183.78

SD

6.44

4.83

12.36

6.04

9.62

4.91

7.54

7.68

N

5

5

5

5

5

5

5

5

Day 7

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

254.32

254.28

254.10

248.46

185.38

186.64

181.26

184.68

SD

8.12

6.50

11.93

4.12

8.82

6.06

9.40

8.00

N

5

5

5

5

5

5

5

5

Day 11

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

262.90

263.34

269.56

258.70

191.44

195.14

188.84

191.30

SD

8.60

5.60

14.84

2.94

9.01

6.18

10.46

8.21

N

5

5

5

5

5

5

5

5

Day 14

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

265.64

263.24

269.00

256.68

178.26

182.82

183.54

186.50

SD

9.30

8.00

13.79

2.96

10.57

4.86

11.88

10.14

N

5

5

5

5

5

5

5

5

Day 18

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

273.10

271.54

278.88

266.26

198.60

199.58

195.98

201.54

SD

9.09

9.39

14.02

2.75

13.61

7.26

11.44

13.12

N

5

5

5

5

5

5

5

5

Day 21

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

275.66

270.64

277.04

261.98*

199.18

204.12

194.06

202.22

SD

9.72

9.74

14.67

4.19

11.32

6.49

10.66

10.41

N

5

5

5

5

5

5

5

5

Day 25

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

285.68

279.96

291.18

274.68*

202.80

208.80

197.64

210.34

SD

10.48

10.17

14.52

2.35

11.90

7.52

9.30

10.42

N

5

5

5

5

5

5

5

5

Day 27

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

287.38

279.10

286.46

272.72*

209.28

208.52

202.60

208.22

SD

11.92

8.89

13.74

1.40

15.04

7.36

15.54

11.32

N

5

5

5

5

5

5

5

5

 * = p < 0.05

Total and differential white blood cell counts - Male (m) | Female (f)

0

mg/m3

WBC (10E9/L)

Lympho

Absolute (10E9/L)

Neutro Absolute (10E9/L)

Eosino Absolute (10E9/L)

Baso Absolute (10E9/L)

Mono Absolute (10E9/L)

Lymphocytes (%)

Neutrophils (%)

Eosinophils (%)

Basophils (%)

Monocytes (%)

Statistics

(g)

(g)

(g)

(g)

(g1)

(g)

(g)

(g)

(g)

(g)

(g1)

(g)

(g)

(g)

(g)

(g)

(g)

(g2)

(g1)

(g)

(g2)

(g)

Sex

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

 

Mean

5.16

3.60

4.04 

2.91

0.94

0.57

0.071

0.051

0.005

0.003

0.092

0.056

77.80

80.88

18.70

15.80

1.40

1.36

0.10

0.06

1.84

1.56

SD

1.00

0.80

0.95

0.65

0.10

0.15

0.014

0.027

0.004

0.004

0.010

0.015

3.95

2.72

3.57

 2.45

0.25

0.55

0.07

0.09

0.45

0.36

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

10 mg/m3

WBC (10E9/L)

Lympho

Absolute (10E9/L)

Neutro Absolute (10E9/L)

Eosino Absolute (10E9/L)

Baso Absolute (10E9/L)

Mono Absolute (10E9/L)

Lymphocytes (%)

Neutrophils (%)

Eosinophils (%)

Basophils (%)

Monocytes (%)

Mean

4.94

3.58

3.92

3.10

0.86

0.35*

0.065

0.046

0.005

0.005

0.083

0.059

79.22

86.84*

17.42

9.58**

1.30

1.32

0.10

0.12

1.68

1.66

SD

0.66

0.83

0.56

0.71

0.13

0.12

0.018

0.011

0.003

0.003

0.019

0.024

1.93

2.44

2.01

1.62

0.22

0.41

0.07

0.08

0.33

0.66

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

50 mg/m3

WBC (10E9/L)

Lympho

Absolute (10E9/L)

Neutro Absolute (10E9/L)

Eosino Absolute (10E9/L)

Baso Absolute (10E9/L)

Mono Absolute (10E9/L)

Lymphocytes (%)

Neutrophils (%)

Eosinophils (%)

Basophils (%)

Monocytes (%)

Mean

6.06

3.08

4.85

2.46

0.99

0.48

0.075

0.047

0.006

0.002

0.126

0.077

80.00

79.82

16.36

15.58

1.28

1.52

0.10

0.08

1.96

2.48*

SD

1.38

0.31

 1.09

0.23

0.31

0.08

0.032

0.017

0.001

0.002

0.078

0.018

4.31

1.76

3.50

1.77

0.52

0.41

0.00

0.08

0.87

0.48

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

250 mg/m3

WBC (10E9/L)

Lympho

Absolute (10E9/L)

Neutro Absolute (10E9/L)

Eosino Absolute (10E9/L)

Baso Absolute (10E9/L)

Mono Absolute (10E9/L)

Lymphocytes (%)

Neutrophils (%)

Eosinophils (%)

Basophils (%)

Monocytes (%)

Mean

4.76

4.38

 3.60

3.57

0.99

0.65

0.066

0.065

0.005

0.003

0.088

0.075

75.48

81.30

20.90

15.06

1.42

1.50

0.10

0.08

1.84

1.70

SD

0.34

0.61

0.36

0.63

0.14

0.13

0.027

0.020

0.003

0.002

0.034

0.024

3.67

4.07

3.19

3.73

0.65

0.44

0.07

0.04

0.69

0.41

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

[g] - Anova & Dunnett* = p < 0.05; ** = p < 0.01

[g1] - Kruskal-Wallis & Dunnett on Ranks

[g2] - Anova & Dunnett(Log)

 

Red blood cell and coagulation parameter – Male (m) | Female (f)                                                                                    

0

mg/m3

RBC

(10E12/L)

Hb

(mmol/L)

PCV

(L/L)

MCV

(fL)

MCH

(fmol)

MCHC

(mmol/L)

Reticulo

cytes

(%)

Thrombo

cytes

(10E9/L)

Prothrom

Time

(s)

Statistics

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g1)

(g)

(g)

(g1)

(g)

(g)

Sex

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

Mean

8.584

8.170

9.72

9.46

0.4552

0.4356

53.04

53.33

1.133

1.158

21.36

21.72

2.158

2.056

769.2

823.2

21.94

22.26

SD

0.172

0.151

0.16

0.17

0.0116

0.0036

1.34

0.98

0.018

0.035

0.50

0.27

0.320

0.382

34.9

120.1

1.93

0.88

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

 

5

5

10 mg/m3

RBC

10E12/L

Hb

(mmol/L)

PCV

(L/L)

MCV

(fL)

MCH

(fmol)

MCHC

(mmol/L)

Reticulo

cytes

(%)

Thrombo

cytes

(10E9/L)

Prothrom

Time

(s)

Mean

8.662

8.296

9.70

9.64

0.4600

0.4426

53.11

53.35

1.121

1.162

21.09

21.78

1.932

1.288**

776.2

863.2

22.70

22.42

SD

0.230

0.232

0.14

0.23

0.0102

0.0125

0.71

0.67

0.037

0.021

0.54

0.29

0.149

0.101

35.1

24.0

1.69

0.54

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

50 mg/m3

RBC

(10E12/L)

Hb

(mmol/L)

PCV

(L/L)

MCV

(fL)

MCH

(fmol)

MCHC

(mmol/L)

Reticulo

cytes

(%)

Thrombo

cytes

(10E9/L)

Prothrom

Time

(s)

Mean

8.308

7.856

9.52

9.28

0.4496

0.4188

54.16

53.33

1.148

 1.182

21.20

22.16

2.012

1.690

789.8

802.2

22.08

22.28

SD

0.484

0.261

0.26

0.16

0.0240

0.0087

1.85

0.77

0.046

0.024

0.55

0.21

0.478

0.223

31.6

55.2

1.43

0.95

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

250 mg/m3

RBC

(10E12/L)

Hb

(mmol/L)

PCV

(L/L)

MCV

(fL)

MCH

(fmol)

MCHC

(mmol/L)

Reticulo

cytes

(%)

Thrombo

cytes

(10E9/L)

Prothrom

Time

(s)

Mean

8.672

8.000

9.76

9.34

0.4598

0.4264

53.04

53.32 

1.126

1.168

21.23

21.91

1.936

1.696

765.8

747.4

24.14

23.70*

SD

0.213

0.322

0.21

0.15

0.0086

0.0132

1.18

1.07

0.018

0.031

0.30

0.46

0.390

0.227

30.7

75.0

2.33

0.33

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

[g] - Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[g1] - Kruskal-Wallis & Dunnett on Ranks

 

 

Clinical chemistry– Male (m) | Female (f)

0

mg/m3

ALP (U/L)

ASAT (U/L)

ALAT (U/L)

GGT (U/L)

Bilirub Total (umol/L)

Creatinine (umol/L)

Total Protein (g/L)

Albumin (g/L)

Albumin / Globulin

Glucose Plasma (mmmol/L)

Statistics

(g)

(g)

(g1)

(g)

(g)

(g)

(g1)

(g)

(g1)

(g)

(g)

(g)

(g1)

(g)

(g1)

(g)

(g)

(g2)

(g)

(g)

Sex

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

Mean

160.2

74.8

90.6

77.4

53.8

37.0

0.08

0.08

1.38

1.14

32.6

30.4

55.2

57.2

29.6

31.4

1.158

1.218

7.102

5.966

SD

37.4

19.8

3.6

11.8

8.5

7.1

0.18

0.18

0.18

0.34

2.6

4.2

0.8

1.5

0.5

0.9

0.051

0.039

0.642

0.810

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

10 mg/m3

ALP (U/L)

ASAT (U/L)

ALAT (U/L)

GGT (U/L)

Bilirub Total (umol/L)

Creatinine (umol/L)

Total Protein (g/L)

Albumin (g/L)

Albumin / Globulin

Glucose Plasma (mmmol/L)

Mean

149.2

76.4

80.8

70.0

54.6

36.0

0.12

0.10

1.44

1.28

31.8

31.0

55.4

57.2

29.8

32.2

1.166

1.290

6.992

5.750

SD

41.6 

15.7

5.5

7.0

5.6

7.3

0.27

0.17

0.36

0.62

5.4

3.2

1.9

2.6

0.8

1.1

0.051

0.048

1.253

0.854

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

50 mg/m3

ALP (U/L)

ASAT (U/L)

ALAT (U/L)

GGT (U/L)

Bilirub Total (umol/L)

Creatinine (umol/L)

Total Protein (g/L)

Albumin (g/L)

Albumin / Globulin

Glucose Plasma (mmmol/L)

Mean

129.8

94.0

83.8

74.8

46.8

35.6

0.02

0.24

1.12

1.62

30.8

31.0

55.4

56.0

29.6

31.0

1.150

1.244

7.004

5.180

SD

18.6

27.0

8.8

9.3

6.6

5.5

0.04

0.36

0.65

0.38

4.9

3.2

1.5

2.4

0.5

1.4

0.060

0.087

0.645

0.614

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

250 mg/m3

ALP (U/L)

ASAT (U/L)

ALAT (U/L)

GGT (U/L)

Bilirub Total (umol/L)

Creatinine (umol/L)

Total Protein (g/L)

Albumin (g/L)

Albumin / Globulin

Glucose Plasma (mmmol/L)

Mean

148.8

124.2*

 97.8

85.4

53.6

46.4

0.00

0.00

1.18

1.64

32.8

31.6

53.4

54.2

29.6

30.2

1.245*

1.261

7.010

4.872

SD

52.3

33.8

12.5

8.6

12.7

7.6

0.00

0.00

0.30

0.36

3.8

1.7

1.1

2.9

0.5

1.3

0.042

0.058

0.747

0.491

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

[g] - Anova & Dunnett: * = p < 0.05

[g1] - Kruskal-Wallis & Dunnett on Ranks

 

 

 

 

Conclusions:
Under the conditions of the current study, inhalation exposure to hexyl salicylate up to a concentration of 249 mg/m3 was tolerated well by the animals and did not result in any adverse exposure-related changes.
Executive summary:

The toxicity of hexyl salicylate following repeated inhalation exposure was investigated in a sub-acute (28-day) toxicity study in rats. Four groups of 5 male and 5 female rats each were exposed nose-only to concentrations of 0 (controls), 10.9, 52.3 or 249 mg/m3 hexyl salicylate for 6 hours/day, 5 days/week over a 28 -day period, with a total number of 20 exposure days. The average particle size (MMAD) of the aerosol was 3.3 μm (gsd of 1.7), 2.9 μm (gsd of 1.8) and 2.6 μm (gsd of 1.9) for the low, mid and high concentration test atmospheres, respectively. Animals were sacrificed on the day after the last exposure. The target concentration levels for this sub-acute study were selected on the basis of the results of a preceding 7 -day range finding study. Exposure was well tolerated. Exposure to 250 mg/m3 resulted in slightly decreased growth and food consumption in male rats. No exposure-related changes were observed at 10 and 50 mg/m3. No treatment-related clinical abnormalities were observed. During the last week of the exposure the average body weight of male animals of the high concentration group was slightly, but statistically significantly below controls. Since the relative difference with controls was small (about 5% at most), this finding – although likely to be treatment-related - was considered to be of little, if any, toxicological significance. The few slight, but statistically significant differences in clinical pathology parameters in females of the high concentration group (increased plasma ALP activity and prothrombin time) were considered chance findings – and therefore not treatment related – because they were observed in one sex only, the difference with controls was small, and no changes were observed in any of the associated parameters investigated. No changes were observed in absolute and relative organ weights. No macroscopic pathological changes were observed at necropsy, and treatment related histopathological abnormalities were not observed. A NOAEC of 249 mg/m3 is therefore derived for this study, in the absence of any adverse treatment-related findings at the highest exposure level.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
not documented
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Female rats were exposed for 7 hours daily via whole body inhalation chamber to 700 mg/m3, 120 ppm, for a period of 20 days.
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
other: Alderley Park
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Specified pathogen free colony
- Age at study initiation: Not documented
- Weight at study initiation: Average 200g
- Fasting period before study: Not documented
- Housing: cages
- Diet (e.g. ad libitum): ad libitum in housing cages
- Water (e.g. ad libitum): ad libitum in housing cages
- Acclimation period: Not documented

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not documented
- Humidity (%): Not documented
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): Not documented

IN-LIFE DATES: Not documented
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Not determined
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
A nearly saturated vapour obtained by passing air through a liquid contained in a bubbler with a sintered glass air-distributor disc. The volume of the liquid was usually 10-20 ml and, if the size of the sample available permitted, it was replaced daily. The bubbler was maintained in a water-bath at room temperature, about 20°C.

TEST ATMOSPHERE
- Brief description of analytical method used: The nearly saturated concentration prepared by method was estimated by weighing the sample before and after the day's run, and relating the weight loss to the volume of air passing. This concentration, expressed in milligrammes per litre, was converted to parts per million on the assumption that the sample was pure.
- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not documented
Duration of treatment / exposure:
7 hour exposure, once daily, for 20 days
Frequency of treatment:
7 hour exposure, once daily, for 20 days
Dose / conc.:
700 mg/m³ air
Remarks:
120 ppm, nominal
No. of animals per sex per dose:
4 females
Control animals:
not specified
Details on study design:
Four female rats were exposed for 7 hours daily via whole body inhalation chamber to 700 mg/m3, 120 ppm methyl salicylate, for a period of 20 days.
Dose selection rationale: The concentration was selected to produce, if possible, acute effects after short exposures and the exposure period was extended until the animals could survive 6-hour exposures, for up to four weeks.
Positive control:
No information provided
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: throughout the exposure period
- Cage side observations checked: conditions and behaviour

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: each morning

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: No
- How many animals: All
- Parameters checked: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked: No data

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No additional information.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The rats were anaesthetized with halothane and partially exsanguinated by heart puncture for haematological tests. After a gross examination of the organs, the lungs were inflated with formol-saline and immersed in the same fixative.

HISTOPATHOLOGY: Yes
The following organs were also taken for microscopical examination after fixation in formol-corrosive: lungs, liver, kidneys, spleen, and adrenals; and occasionally heart, jejunum, ileum, and thymus.
Other examinations:
No additional information
Statistics:
No information provided
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No effects, no further details given.

BODY WEIGHT AND WEIGHT GAIN
No effects, no further details given.

FOOD CONSUMPTION
No data

FOOD EFFICIENCY
No data

WATER CONSUMPTION
No data

OPHTHALMOSCOPIC EXAMINATION
No data

HAEMATOLOGY
No effects, no further details given.

CLINICAL CHEMISTRY
No data

URINALYSIS
No effects, no further details given.

NEUROBEHAVIOUR
No data

ORGAN WEIGHTS
No effects, no further details given.

GROSS PATHOLOGY
No effects, no further details given.

HISTOPATHOLOGY: NON-NEOPLASTIC
No effects, no further details given.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No data

HISTORICAL CONTROL DATA (if applicable)
No information provided

OTHER FINDINGS
No further data
Key result
Dose descriptor:
NOAEL
Remarks:
(>120 ppm)
Effect level:
700 mg/m³ air (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no toxic signs: autopsy, organs normal

No additional information

Conclusions:
A NOAEC of 700 mg/m3 can be determined for this study, in the absence of any treatment-related findings at the single exposure concentration.
Executive summary:

In this study, a group of four female rats was exposed (whole body) to an atmosphere containing methyl salicylate at a concentration of 700 mg/m2 (120 ppm) for 7 hours/day on 20 consecutive days. Observations included conditions and behaviour, body weight, haematology, urinalysis, gross pathology and histopathological examinations. No signs of toxicity were observed throughout the duration of the study and at autopsy, organs appeared normal. A NOAEC of 700 mg/m3 can therefore be determined for this study, in the absence of any treatment-related findings.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
249 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
A modern, guideline-compliant study is available for hexyl salicylate and is supported by a 20-day study with methyl salicylate.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28/07/2016 - 25/08/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
(2009)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 50064843
- Expiration date of the lot/batch: 14 April 2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 – 25 °C
- Solubility in water : 2 mg/l at 23 °C
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Young adult, male and female Wistar outbred rats (Crl:WI(Han)) were obtained from a colony maintained under specific pathogen-free (SPF) conditions at Charles River, Germany.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-9 weeks
- Weight at study initiation: mean males= 251g, mean females =171g
- Fasting period before study: overnight fasting
- Housing:Macrolon cages with wood shavings
- Diet:ad libitum except during exposure and – for animals of the main study – the overnight fasting period before sacrifice.
- Water: ad libitum except during exposure
ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From:28/07/2016 To:25/08/2016
Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.6 - <= 3.3 µm
Remarks on MMAD:
The average particle size (MMAD) of the aerosol was 3.3 μm (gsd of 1.7), 2.9 μm (gsd of 1.8) and 2.6 μm (gsd of 1.9) for the low, mid and high concentration test atmospheres, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. The test atmospheres were generated by nebulization using an air-driven atomizer which was placed at the top inlet of the exposure chamber. For generation of the low concentration test atmosphere, the atomizer was heated to reduce the aerodynamic particle size.

The amount of test material delivered to the atomizer was controlled using a motor-driven syringe pump.The atomizer was supplied with dry compressed air, controlled at a pressure of 4 bar using a reducing valve. Air flow to the atomizer was measured using a mass view meter. In the top of the exposure unit, a bypass stream of humidified compressed air was added, the flow of which was controlled and measured using rotameters which were calibrated at the settings used during the study using a volumetric flow meter. the air supply to the various rotameters was controlled by mass flow controller.From the top of the exposure chamber, the test atmosphere was directed downward and led to the noses of the animals. At the bottom of the unit the test atmosphere was exhausted.

The exposure unit for the control animals was supplied with a flow of humidified compressed air only, which was controlled using a reducing valve and measured by mass view meter
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The overall average actual concentrations of Hexyl salicylate in the test atmospheres were determined by gravimetric analysis
Duration of treatment / exposure:
Exposure was over a 28-day period, with a total number of 20 exposure days.
Frequency of treatment:
6 hours/day for 5 days/week
Dose / conc.:
0 mg/m³ air
Remarks:
Actual concentration in air
Dose / conc.:
10.9 mg/m³ air
Remarks:
Actual concentration in air
Dose / conc.:
52.3 mg/m³ air
Remarks:
Actual concentration in air
Dose / conc.:
249 mg/m³ air
Remarks:
Actual concentration in air
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes
Details on study design:
Animal selection was random
- Dose selection rationale: concentrations were based on the results of a preceding 7-day range finding study (5 exposure days in total). Results for this study are presented in Annex 10 of the study report.
- Post exposure period: Amimals were subject to necropsy on the day following the last exposure.

Positive control:
Not required for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
On days of exposure, each animal was observed daily in the morning, prior to exposure, by cage-side observations and, if necessary, handled to detect signs of toxicity. All animals were thoroughly checked again after exposure. Clinical signs listed but not limited to Annex 6 of the study report were used for the recording of clinical observations. During exposure, a group-wise observation was made about halfway through the 6-hour exposure period.

BODY WEIGHT: Yes
Body weight of each animal was recorded 2 days before the start of exposure. These pre-test weights served as a basis for animal allocation. Subsequently, the animals of the main study were weighed prior to exposure on the first day (day 0), and then every 3 or 4 days, including the day of scheduled sacrifice. The animals were also weighed on the day before overnight fasting (prior to the scheduled sacrifice).

FOOD CONSUMPTION: Yes
Food consumption was measured per cage by weighing the feeders. Food consumption was measured over three 7-day periods, starting on day 0, followed by a 6-day period. The results were expressed in g per animal per day.

HAEMATOLOGY: Yes
Hematology was conducted in samples collected from all animals at necropsy. Blood samples were taken from the abdominal aorta of the rats.
- Anaesthetic used for blood collection: Yes (pentobarbital anaesthesia) EDTA or citrate (for prothrombin time) were used as anticoagulant.
- Animals fasted: Yes (Overnight before necropsy but water was freely available)

CLINICAL CHEMISTRY: Yes
Hematology was conducted in samples collected from all animals at necropsy.
- Animals fasted: Yes (Overnight before necropsy but water was freely available)

Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were subject to gross necropsy
HISTOPATHOLOGY: Yes. A detailed assessment was made of all animals
Other examinations:
ORGANS EXAMINED AT NECROPSY: Yes
- Organ weights: adrenals , lungs with trachea and larynx, brain, spleen, heart, testes, kidneys, thymus, liver
Statistics:
-Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ with automatic data transformation. Day 0 body weight data were used as covariate in the analysis of the post-treatment data unless removed during data pre-processing.
-Pre-treatment body weight, organ weight, hematology and clinical chemistry data: ‘Generalized Anova/Ancova Test’ with automatic data transformation method
-Incidences of histopathological changes: Fisher’s exact probability test
-Food consumption: no statistics was applied on food intake (only one cage per sex)
Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical abnormalities were observed. The few signs noted were considered unrelated to the exposure to the test material. Abnormalities of the skin or fur (sparsely haired areas, encrustations, skin wounds) were observed across the groups. These are common findings, possibly caused by slight movement of the animals in the restraining tubes during exposure (resulting in slight irritation of the skin) or by placing of the subcutaneous transponder. Other clinical signs, observed incidentally in single animals, also represented background findings and were not related to exposure to the test material.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male animals of the high concentration group showed a statistically significantly lower bodyweight than controls from Day 21 until the end of the study. Overall body weight gain during the 28-day period was statistically significantly decreased in males of the high concentration group when compared to unexposed controls. There were no statistically significant differences in body weights of the female rats between the test groups and controls. At Day 14, a temporary and slight decrease in body weight was noted in all female rats, which was not considered to be treatment related. The reason for this slight decrease in all female rats remained uncertain.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was slightly decreased in all males at the higher concentrations.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no statistically significant changes in red blood cell variables, coagulation parameters and white blood cell variables in male rats. In females there were no statistically significant changes in red blood cell variables, except for an incidental decrease in reticulocytes in the low concentration group which was considered to be a chance finding because no changes were observed in the mid and high concentration group. Prothrombin time was slightly, but statistically significantly, increased in the high concentration group. The absolute and relative numbers of neutrophils were statistically significantly decreased and the percentage of lymphocytes was statistically significantly increased in females in the low concentration group. The percentage of monocytes was statistically significantly increased in females in the mid concentration group. These findings were considered chance findings because they were not confirmed at the high concentration level.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males there were no statistically significant differences in clinical chemistry variables except for a slight increase in mean albumin/globulin ratio in the high concentration group. In the absence of any changes in albumin and total protein levels, or any corroborative findings in females, a minor change in this calculated parameter was considered a chance finding. In females of the high concentration group a statistically significant increase in plasma ALP activity was observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in absolute and relative organ weights between the control group and the groups exposed to the test material
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The histopathological changes observed such as thymus microhaemorrage were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. See Table 12 and appendix 8 of the study report. These changes were therefore considered to be unrelated to the exposure to the test material.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
249 mg/m³ air
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Inhalation exposure to hexyl salicylate up to a concentration of 249 mg/m3 was tolerated well by the animals and did not result in any adverse exposure-related changes
Key result
Critical effects observed:
no

Mean body weight

Day 0

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

249.12

250.36

252.14

250.66

168.30

175.76

165.82

172.46

SD

4.89

6.81

8.54

7.21

10.38

5.58

6.94

7.23

N

5

5

5

5

5

5

5

5

Day 4

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

255.58

256.12

259.28

255.00

179.00

186.54

177.96

183.78

SD

6.44

4.83

12.36

6.04

9.62

4.91

7.54

7.68

N

5

5

5

5

5

5

5

5

Day 7

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

254.32

254.28

254.10

248.46

185.38

186.64

181.26

184.68

SD

8.12

6.50

11.93

4.12

8.82

6.06

9.40

8.00

N

5

5

5

5

5

5

5

5

Day 11

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

262.90

263.34

269.56

258.70

191.44

195.14

188.84

191.30

SD

8.60

5.60

14.84

2.94

9.01

6.18

10.46

8.21

N

5

5

5

5

5

5

5

5

Day 14

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

265.64

263.24

269.00

256.68

178.26

182.82

183.54

186.50

SD

9.30

8.00

13.79

2.96

10.57

4.86

11.88

10.14

N

5

5

5

5

5

5

5

5

Day 18

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

273.10

271.54

278.88

266.26

198.60

199.58

195.98

201.54

SD

9.09

9.39

14.02

2.75

13.61

7.26

11.44

13.12

N

5

5

5

5

5

5

5

5

Day 21

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

275.66

270.64

277.04

261.98*

199.18

204.12

194.06

202.22

SD

9.72

9.74

14.67

4.19

11.32

6.49

10.66

10.41

N

5

5

5

5

5

5

5

5

Day 25

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

285.68

279.96

291.18

274.68*

202.80

208.80

197.64

210.34

SD

10.48

10.17

14.52

2.35

11.90

7.52

9.30

10.42

N

5

5

5

5

5

5

5

5

Day 27

Male

Females

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

0 mg/m3

10 mg/m3

50 mg/m3

250 mg/m3

Mean bodyweight day( g)

287.38

279.10

286.46

272.72*

209.28

208.52

202.60

208.22

SD

11.92

8.89

13.74

1.40

15.04

7.36

15.54

11.32

N

5

5

5

5

5

5

5

5

 * = p < 0.05

Total and differential white blood cell counts - Male (m) | Female (f)

0

mg/m3

WBC (10E9/L)

Lympho

Absolute (10E9/L)

Neutro Absolute (10E9/L)

Eosino Absolute (10E9/L)

Baso Absolute (10E9/L)

Mono Absolute (10E9/L)

Lymphocytes (%)

Neutrophils (%)

Eosinophils (%)

Basophils (%)

Monocytes (%)

Statistics

(g)

(g)

(g)

(g)

(g1)

(g)

(g)

(g)

(g)

(g)

(g1)

(g)

(g)

(g)

(g)

(g)

(g)

(g2)

(g1)

(g)

(g2)

(g)

Sex

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

 

Mean

5.16

3.60

4.04 

2.91

0.94

0.57

0.071

0.051

0.005

0.003

0.092

0.056

77.80

80.88

18.70

15.80

1.40

1.36

0.10

0.06

1.84

1.56

SD

1.00

0.80

0.95

0.65

0.10

0.15

0.014

0.027

0.004

0.004

0.010

0.015

3.95

2.72

3.57

 2.45

0.25

0.55

0.07

0.09

0.45

0.36

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

10 mg/m3

WBC (10E9/L)

Lympho

Absolute (10E9/L)

Neutro Absolute (10E9/L)

Eosino Absolute (10E9/L)

Baso Absolute (10E9/L)

Mono Absolute (10E9/L)

Lymphocytes (%)

Neutrophils (%)

Eosinophils (%)

Basophils (%)

Monocytes (%)

Mean

4.94

3.58

3.92

3.10

0.86

0.35*

0.065

0.046

0.005

0.005

0.083

0.059

79.22

86.84*

17.42

9.58**

1.30

1.32

0.10

0.12

1.68

1.66

SD

0.66

0.83

0.56

0.71

0.13

0.12

0.018

0.011

0.003

0.003

0.019

0.024

1.93

2.44

2.01

1.62

0.22

0.41

0.07

0.08

0.33

0.66

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

50 mg/m3

WBC (10E9/L)

Lympho

Absolute (10E9/L)

Neutro Absolute (10E9/L)

Eosino Absolute (10E9/L)

Baso Absolute (10E9/L)

Mono Absolute (10E9/L)

Lymphocytes (%)

Neutrophils (%)

Eosinophils (%)

Basophils (%)

Monocytes (%)

Mean

6.06

3.08

4.85

2.46

0.99

0.48

0.075

0.047

0.006

0.002

0.126

0.077

80.00

79.82

16.36

15.58

1.28

1.52

0.10

0.08

1.96

2.48*

SD

1.38

0.31

 1.09

0.23

0.31

0.08

0.032

0.017

0.001

0.002

0.078

0.018

4.31

1.76

3.50

1.77

0.52

0.41

0.00

0.08

0.87

0.48

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

250 mg/m3

WBC (10E9/L)

Lympho

Absolute (10E9/L)

Neutro Absolute (10E9/L)

Eosino Absolute (10E9/L)

Baso Absolute (10E9/L)

Mono Absolute (10E9/L)

Lymphocytes (%)

Neutrophils (%)

Eosinophils (%)

Basophils (%)

Monocytes (%)

Mean

4.76

4.38

 3.60

3.57

0.99

0.65

0.066

0.065

0.005

0.003

0.088

0.075

75.48

81.30

20.90

15.06

1.42

1.50

0.10

0.08

1.84

1.70

SD

0.34

0.61

0.36

0.63

0.14

0.13

0.027

0.020

0.003

0.002

0.034

0.024

3.67

4.07

3.19

3.73

0.65

0.44

0.07

0.04

0.69

0.41

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

[g] - Anova & Dunnett* = p < 0.05; ** = p < 0.01

[g1] - Kruskal-Wallis & Dunnett on Ranks

[g2] - Anova & Dunnett(Log)

 

Red blood cell and coagulation parameter – Male (m) | Female (f)                                                                                    

0

mg/m3

RBC

(10E12/L)

Hb

(mmol/L)

PCV

(L/L)

MCV

(fL)

MCH

(fmol)

MCHC

(mmol/L)

Reticulo

cytes

(%)

Thrombo

cytes

(10E9/L)

Prothrom

Time

(s)

Statistics

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g)

(g1)

(g)

(g)

(g1)

(g)

(g)

Sex

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

Mean

8.584

8.170

9.72

9.46

0.4552

0.4356

53.04

53.33

1.133

1.158

21.36

21.72

2.158

2.056

769.2

823.2

21.94

22.26

SD

0.172

0.151

0.16

0.17

0.0116

0.0036

1.34

0.98

0.018

0.035

0.50

0.27

0.320

0.382

34.9

120.1

1.93

0.88

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

 

5

5

10 mg/m3

RBC

10E12/L

Hb

(mmol/L)

PCV

(L/L)

MCV

(fL)

MCH

(fmol)

MCHC

(mmol/L)

Reticulo

cytes

(%)

Thrombo

cytes

(10E9/L)

Prothrom

Time

(s)

Mean

8.662

8.296

9.70

9.64

0.4600

0.4426

53.11

53.35

1.121

1.162

21.09

21.78

1.932

1.288**

776.2

863.2

22.70

22.42

SD

0.230

0.232

0.14

0.23

0.0102

0.0125

0.71

0.67

0.037

0.021

0.54

0.29

0.149

0.101

35.1

24.0

1.69

0.54

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

50 mg/m3

RBC

(10E12/L)

Hb

(mmol/L)

PCV

(L/L)

MCV

(fL)

MCH

(fmol)

MCHC

(mmol/L)

Reticulo

cytes

(%)

Thrombo

cytes

(10E9/L)

Prothrom

Time

(s)

Mean

8.308

7.856

9.52

9.28

0.4496

0.4188

54.16

53.33

1.148

 1.182

21.20

22.16

2.012

1.690

789.8

802.2

22.08

22.28

SD

0.484

0.261

0.26

0.16

0.0240

0.0087

1.85

0.77

0.046

0.024

0.55

0.21

0.478

0.223

31.6

55.2

1.43

0.95

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

250 mg/m3

RBC

(10E12/L)

Hb

(mmol/L)

PCV

(L/L)

MCV

(fL)

MCH

(fmol)

MCHC

(mmol/L)

Reticulo

cytes

(%)

Thrombo

cytes

(10E9/L)

Prothrom

Time

(s)

Mean

8.672

8.000

9.76

9.34

0.4598

0.4264

53.04

53.32 

1.126

1.168

21.23

21.91

1.936

1.696

765.8

747.4

24.14

23.70*

SD

0.213

0.322

0.21

0.15

0.0086

0.0132

1.18

1.07

0.018

0.031

0.30

0.46

0.390

0.227

30.7

75.0

2.33

0.33

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

[g] - Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[g1] - Kruskal-Wallis & Dunnett on Ranks

 

 

Clinical chemistry– Male (m) | Female (f)

0

mg/m3

ALP (U/L)

ASAT (U/L)

ALAT (U/L)

GGT (U/L)

Bilirub Total (umol/L)

Creatinine (umol/L)

Total Protein (g/L)

Albumin (g/L)

Albumin / Globulin

Glucose Plasma (mmmol/L)

Statistics

(g)

(g)

(g1)

(g)

(g)

(g)

(g1)

(g)

(g1)

(g)

(g)

(g)

(g1)

(g)

(g1)

(g)

(g)

(g2)

(g)

(g)

Sex

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

(m)

(f)

Mean

160.2

74.8

90.6

77.4

53.8

37.0

0.08

0.08

1.38

1.14

32.6

30.4

55.2

57.2

29.6

31.4

1.158

1.218

7.102

5.966

SD

37.4

19.8

3.6

11.8

8.5

7.1

0.18

0.18

0.18

0.34

2.6

4.2

0.8

1.5

0.5

0.9

0.051

0.039

0.642

0.810

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

10 mg/m3

ALP (U/L)

ASAT (U/L)

ALAT (U/L)

GGT (U/L)

Bilirub Total (umol/L)

Creatinine (umol/L)

Total Protein (g/L)

Albumin (g/L)

Albumin / Globulin

Glucose Plasma (mmmol/L)

Mean

149.2

76.4

80.8

70.0

54.6

36.0

0.12

0.10

1.44

1.28

31.8

31.0

55.4

57.2

29.8

32.2

1.166

1.290

6.992

5.750

SD

41.6 

15.7

5.5

7.0

5.6

7.3

0.27

0.17

0.36

0.62

5.4

3.2

1.9

2.6

0.8

1.1

0.051

0.048

1.253

0.854

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

50 mg/m3

ALP (U/L)

ASAT (U/L)

ALAT (U/L)

GGT (U/L)

Bilirub Total (umol/L)

Creatinine (umol/L)

Total Protein (g/L)

Albumin (g/L)

Albumin / Globulin

Glucose Plasma (mmmol/L)

Mean

129.8

94.0

83.8

74.8

46.8

35.6

0.02

0.24

1.12

1.62

30.8

31.0

55.4

56.0

29.6

31.0

1.150

1.244

7.004

5.180

SD

18.6

27.0

8.8

9.3

6.6

5.5

0.04

0.36

0.65

0.38

4.9

3.2

1.5

2.4

0.5

1.4

0.060

0.087

0.645

0.614

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

250 mg/m3

ALP (U/L)

ASAT (U/L)

ALAT (U/L)

GGT (U/L)

Bilirub Total (umol/L)

Creatinine (umol/L)

Total Protein (g/L)

Albumin (g/L)

Albumin / Globulin

Glucose Plasma (mmmol/L)

Mean

148.8

124.2*

 97.8

85.4

53.6

46.4

0.00

0.00

1.18

1.64

32.8

31.6

53.4

54.2

29.6

30.2

1.245*

1.261

7.010

4.872

SD

52.3

33.8

12.5

8.6

12.7

7.6

0.00

0.00

0.30

0.36

3.8

1.7

1.1

2.9

0.5

1.3

0.042

0.058

0.747

0.491

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

[g] - Anova & Dunnett: * = p < 0.05

[g1] - Kruskal-Wallis & Dunnett on Ranks

 

 

 

 

Conclusions:
Under the conditions of the current study, inhalation exposure to hexyl salicylate up to a concentration of 249 mg/m3 was tolerated well by the animals and did not result in any adverse exposure-related changes.
Executive summary:

The toxicity of hexyl salicylate following repeated inhalation exposure was investigated in a sub-acute (28-day) toxicity study in rats. Four groups of 5 male and 5 female rats each were exposed nose-only to concentrations of 0 (controls), 10.9, 52.3 or 249 mg/m3 hexyl salicylate for 6 hours/day, 5 days/week over a 28 -day period, with a total number of 20 exposure days. The average particle size (MMAD) of the aerosol was 3.3 μm (gsd of 1.7), 2.9 μm (gsd of 1.8) and 2.6 μm (gsd of 1.9) for the low, mid and high concentration test atmospheres, respectively. Animals were sacrificed on the day after the last exposure. The target concentration levels for this sub-acute study were selected on the basis of the results of a preceding 7 -day range finding study. Exposure was well tolerated. Exposure to 250 mg/m3 resulted in slightly decreased growth and food consumption in male rats. No exposure-related changes were observed at 10 and 50 mg/m3. No treatment-related clinical abnormalities were observed. During the last week of the exposure the average body weight of male animals of the high concentration group was slightly, but statistically significantly below controls. Since the relative difference with controls was small (about 5% at most), this finding – although likely to be treatment-related - was considered to be of little, if any, toxicological significance. The few slight, but statistically significant differences in clinical pathology parameters in females of the high concentration group (increased plasma ALP activity and prothrombin time) were considered chance findings – and therefore not treatment related – because they were observed in one sex only, the difference with controls was small, and no changes were observed in any of the associated parameters investigated. No changes were observed in absolute and relative organ weights. No macroscopic pathological changes were observed at necropsy, and treatment related histopathological abnormalities were not observed. A NOAEC of 249 mg/m3 is therefore derived for this study, in the absence of any adverse treatment-related findings at the highest exposure level.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
not documented
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Female rats were exposed for 7 hours daily via whole body inhalation chamber to 700 mg/m3, 120 ppm, for a period of 20 days.
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
other: Alderley Park
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Specified pathogen free colony
- Age at study initiation: Not documented
- Weight at study initiation: Average 200g
- Fasting period before study: Not documented
- Housing: cages
- Diet (e.g. ad libitum): ad libitum in housing cages
- Water (e.g. ad libitum): ad libitum in housing cages
- Acclimation period: Not documented

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not documented
- Humidity (%): Not documented
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): Not documented

IN-LIFE DATES: Not documented
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Not determined
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
A nearly saturated vapour obtained by passing air through a liquid contained in a bubbler with a sintered glass air-distributor disc. The volume of the liquid was usually 10-20 ml and, if the size of the sample available permitted, it was replaced daily. The bubbler was maintained in a water-bath at room temperature, about 20°C.

TEST ATMOSPHERE
- Brief description of analytical method used: The nearly saturated concentration prepared by method was estimated by weighing the sample before and after the day's run, and relating the weight loss to the volume of air passing. This concentration, expressed in milligrammes per litre, was converted to parts per million on the assumption that the sample was pure.
- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not documented
Duration of treatment / exposure:
7 hour exposure, once daily, for 20 days
Frequency of treatment:
7 hour exposure, once daily, for 20 days
Dose / conc.:
700 mg/m³ air
Remarks:
120 ppm, nominal
No. of animals per sex per dose:
4 females
Control animals:
not specified
Details on study design:
Four female rats were exposed for 7 hours daily via whole body inhalation chamber to 700 mg/m3, 120 ppm methyl salicylate, for a period of 20 days.
Dose selection rationale: The concentration was selected to produce, if possible, acute effects after short exposures and the exposure period was extended until the animals could survive 6-hour exposures, for up to four weeks.
Positive control:
No information provided
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: throughout the exposure period
- Cage side observations checked: conditions and behaviour

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: each morning

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: No
- How many animals: All
- Parameters checked: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked: No data

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No additional information.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The rats were anaesthetized with halothane and partially exsanguinated by heart puncture for haematological tests. After a gross examination of the organs, the lungs were inflated with formol-saline and immersed in the same fixative.

HISTOPATHOLOGY: Yes
The following organs were also taken for microscopical examination after fixation in formol-corrosive: lungs, liver, kidneys, spleen, and adrenals; and occasionally heart, jejunum, ileum, and thymus.
Other examinations:
No additional information
Statistics:
No information provided
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No effects, no further details given.

BODY WEIGHT AND WEIGHT GAIN
No effects, no further details given.

FOOD CONSUMPTION
No data

FOOD EFFICIENCY
No data

WATER CONSUMPTION
No data

OPHTHALMOSCOPIC EXAMINATION
No data

HAEMATOLOGY
No effects, no further details given.

CLINICAL CHEMISTRY
No data

URINALYSIS
No effects, no further details given.

NEUROBEHAVIOUR
No data

ORGAN WEIGHTS
No effects, no further details given.

GROSS PATHOLOGY
No effects, no further details given.

HISTOPATHOLOGY: NON-NEOPLASTIC
No effects, no further details given.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No data

HISTORICAL CONTROL DATA (if applicable)
No information provided

OTHER FINDINGS
No further data
Key result
Dose descriptor:
NOAEL
Remarks:
(>120 ppm)
Effect level:
700 mg/m³ air (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no toxic signs: autopsy, organs normal

No additional information

Conclusions:
A NOAEC of 700 mg/m3 can be determined for this study, in the absence of any treatment-related findings at the single exposure concentration.
Executive summary:

In this study, a group of four female rats was exposed (whole body) to an atmosphere containing methyl salicylate at a concentration of 700 mg/m2 (120 ppm) for 7 hours/day on 20 consecutive days. Observations included conditions and behaviour, body weight, haematology, urinalysis, gross pathology and histopathological examinations. No signs of toxicity were observed throughout the duration of the study and at autopsy, organs appeared normal. A NOAEC of 700 mg/m3 can therefore be determined for this study, in the absence of any treatment-related findings.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
249 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
A modern, guideline-compliant study is available for hexyl salicylate and is supported by a 20-day study with methyl salicylate.

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

Repeated dose oral toxicity

No repeated dose oral toxicity data are available for hexyl salicylate; however read-across data are available for the read-across (category) substances methyl salicylate (17-week rat study, 2-year rat study, 8-week dog study, 2-year dog study) and isoamyl salicylate (90-day rat study). The data are relatively old but together provide a consistent weight of evidence.

In a 90-day rat study with (Drake, 1974), isoamyl salicylate was administered to male and female Wistar rats via oral administration in the diet at dose levels of 0, 50, 500 and 5000 ppm. 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 5000 ppm. 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 (~50 mg/kg bw/d).

In a range-finding study with methyl salicylate (Webb, 1963), groups of 10 male and 10 female Osborne-Mendel rats were fed methyl salicylate at three dietary concentrations (0%, 0.1% or 1.0%), equivalent to intakes of 0, 50 and 500 mg/kg bw/d in the diet for 17 weeks. Bodyweight gain and selected organ weight and pathology were assessed. The high dose (1.0%) was associated with reduced bodyweight gain but had no effect on organ weight or histopathology. No effects were reported in the other dose groups. The results of this study support a NOAEL value of 0.1% in the diet, equivalent to 50 mg/kg bw/d.

Webb (1963) also reports a 2-year rat study in which methyl salicylate was administered to groups of male and female Osborne-Mendel rats at concentrations of 0, 0.1%, 0.5%, 1.0% or 2.0% in the diet providing doses of approximately 0, 50, 250, 500, and 1000 mg/kg bw/d for two years. All rats in the 1000 mg/kg bw/d group died by the 49th week. Body weight of both sexes was significantly decreased in the 500 and 1000 mg/kg bw/d dose groups. An increased amount of cancellous bone was present in the metaphyses in rats treated at either 500 or 1000 mg/kg bw/d, with a more marked effect at the highest dose level. The relative testis weight of males was significantly increased as were the relative weights of the heart and kidneys of females in the 500 mg/kg bw/d group. Gross pituitary gland lesions were increased at 250 but not at 500 mg/kg bw/d compared to controls. Based on this study, the NOAEL is 50 mg/kg bw/d.

Webb (1963) reports a range-finding study in the dog, in which groups of two dogs were given doses of 50 to 1200 mg/kg bw/d methyl salicylate orally in capsule form daily, 6 days per week for up to 59 days. All dogs receiving 500 mg/kg bw/d or more of methyl salicylate lost weight and died or were killed due to moribund condition within 1-month of study initiation. One animal given 800 mg/kg bw/d and both given 1200 mg/kg bw/d vomited for 3 -4 hours following each dose.  Microscopically, moderate to marked fatty changes were observed in the liver of one animal at 800 mg/kg bw/d and both at 1200 mg/kg bw/d. Animals given 500 mg/kg bw/d had diarrhoea and weakness during their last 3 days. No adverse effects were observed in animals given 50, 150 and 250 mg/kg bw/d. A NOAEL of 250 mg/kg bw/day was identified for this study.

In a subsequent 2-year dog study (Webb, 1963), groups of two male and two female beagles were administered methyl salicylate in capsules at dose levels of 0, 50, 150, or 350 mg/kg bw/d on 6 days/week for 2 years. One high-dose animal died of hepatitis apparently unrelated to methyl salicylate. Haematological analyses at 1, 3, 6, 12 and 24 months and complete necropsy examination were normal. Dogs treated at 150 and 350 mg/kg bw/d had enlarged livers, seen microscopically as enlarged hepatic cells. No other pathology was reported in any of the animals. Reduced body weight was reported in the 350 and 150 mg/kg bw/d groups. Based on this chronic oral study, the NOAEL value is 50 mg/kg bw/d.

Repeated exposure inhalation toxicity

A modern, guideline-compliant study is available for hexyl salicylate and is supported by a 20-day study with the read-across (category) substance methyl salicylate.

In the 28 -day toxicity study with hexyl salicylate, four groups of 5 male and 5 female rats each were exposed nose-only to concentrations of 0 (controls), 10.9, 52.3 or 249 mg/m3 hexyl salicylate for 6 hours/day, 5 days/week over a 28 -day period, with a total number of 20 exposure days. The average particle size (MMAD) of the aerosol was 3.3 μm (gsd of 1.7), 2.9 μm (gsd of 1.8) and 2.6 μm (gsd of 1.9) for the low, mid and high concentration test atmospheres, respectively. Animals were sacrificed on the day after the last exposure. The target concentration levels for this sub-acute study were selected on the basis of the results of a preceding 7 -day range finding study. Exposure was well tolerated. Exposure to 250 mg/m3 resulted in slightly decreased growth and food consumption in male rats. No exposure-related changes were observed at 10 and 50 mg/m3. No treatment-related clinical abnormalities were observed. During the last week of the exposure the average body weight of male animals of the high concentration group was slightly, but statistically significantly below controls. Since the relative difference with controls was small (about 5% at most), this finding – although likely to be treatment-related - was considered to be of little, if any, toxicological significance. The few slight, but statistically significant differences in clinical pathology parameters in females of the high concentration group (increased plasma ALP activity and prothrombin time) were considered chance findings – and therefore not treatment related – because they were observed in one sex only, the difference with controls was small, and no changes were observed in any of the associated parameters investigated. No changes were observed in absolute and relative organ weights. No macroscopic pathological changes were observed at necropsy, and treatment related histopathological abnormalities were not observed. A NOAEC of 249 mg/m3 is therefore derived for this study, in the absence of any adverse treatment-related findings at the highest exposure level.

In the 20 -day study with methyl salicylate, a group of four female rats was exposed (whole body) to an atmosphere containing methyl salicylate at a concentration of 700 mg/m2 (120 ppm) for 7 hours/day on 20 consecutive days. Observations included conditions and behaviour, body weight, haematology, urinalysis, gross pathology and histopathological examinations. No signs of toxicity were observed throughout the duration of the study and at autopsy, organs appeared normal. A NOAEC of 700 mg/m3 can therefore be determined for this study, in the absence of any treatment-related findings

Justification for classification or non-classification

Based on the results of available studies, hexyl salicylate does not require classification for repeated exposure (STOT-RE) according to Regulation EC No. 1272/2008.