Methyl salicylate

Endpoint summary

Description of key information

The toxicity of MeS on repeated exposure has been studied primarily by the oral route, with data acceptable for human risk assessment from studies of up to two years. Studies of shorter duration and lower reliability have been carried out by the inhalation and dermal routes. A set of studies was conducted by Webb & Hansen (1963), consisting of oral feeding studies of duration 17 weeks and 2 years in rats and studies in dogs by capsule administration of duration 59 days and 2 years.  These studies did not examine all the parameters recommended in the current OECD guidelines 409 and 452, however they were conducted according to good scientific principles by US FDA and assigned Rel. 2.  The chronic studies in rat and dog are therefore proposed as key studies. The same authors also conducted a 96-day dermal study in rabbits providing only limited data for assessment (Rel. 3).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data are available

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 hematological studies, gross pathology, and limited histopathological examinations of key organs and tissues.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

MeS was blended with diet and adminstered daily for a period of 2 years.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Osborne-Mendel

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: weanling
- Weight at study initiation: ~ 50 g
- Fasting period before study: no data
- Housing: no data
- Diet: ad libitum
- Water: no data
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod: no data


IN-LIFE DATES: no data

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): every other week
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: all diets were stored in sealed cans under refrigeration
- Other: 10% additional methyl salicylate was added at time of mixing to compensate for evaporation

VEHICLE: none

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

none

Duration of treatment / exposure:

2 years

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 0.1, 0.5, 1 and 2% (0, 50, 250, 500, and 1000 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

25/sex/dose (except for 24 males, 26 females in 2% group)

Control animals:

yes

Details on study design:

- Dose selection rationale: the data reported in the subchronic study in rats by the same authors (methyl salicylate/ dietary administration for 17 weeks in rats).

Positive control:

no data are available

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



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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

none

Statistics:

not reported

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

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

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

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.
HAEMATOLOGY
no hematological effects were observed.ORGAN WEIGHTSAverage 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% groups 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
The data are limited:Malignant pituitary tumors occurred in 1 male and 2 female rats on the 0.5% diet. Mammary tumors occurred in females rats on
all diets.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see below

Critical effects observed:

not specified

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% MeS in the diet. Based on this study, the NOAEL /oral/rat is 50 mg/kg body weight/day.

Executive summary:

Webb and Hansen (1963) administered methyl salicylate in the diet 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/day for two years. All rats in the 1000 mg/kg group died by the 49th week. Body weight of both sexes was significantly decreased in the 500 and 1000 mg/kg body weight/day groups.

An increased amount of cancellous bone was present in the metaphyses in rats treated at either 500 or 1000 mg/kg body weight/day, 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 body weight/day group. Gross pituitary gland lesions were increased at 250 but not 500 mg/kg bw/day compared to controls.

Based on this study, the NOAEL value is 50 mg/kg body weight/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Single concentration tested. No data on substance purity.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

no

Species:

rat

Strain:

other: Alderley Park

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Alderley Park
- Age at study initiation: no data
- Weight at study initiation: mean 200g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Not applicable, vapour

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

7 hours per day, 5 days per week

Remarks:

Doses / Concentrations:
700 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

4

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION: - No data

FOOD EFFICIENCY:- No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight after last exposure
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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 examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional 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:

not examined

Dose descriptor:

NOEC

Effect level:

700 mg/m³ air

Sex:

female

Basis for effect level:

other: overall effects no adverse effects

Critical effects observed:

not specified

No toxic signs and no macroscopic or microscopic effects were seen at necropsy following exposure to a saturated atmosphere for 7 hours per day, 5 days per week for 4 weeks.

Conclusions:

Methyl salicylate showed no evidence of toxicity by inhalation when tested at an effectively saturated concentration for 7 hours per day for four wekks.

Executive summary:

4 female Alderley Park rats weighing an average of 200 g were exposed to a dynamic atmosphere (atmosphere continuously generated and passed through the exposure chamber) containing a saturated atmosphere (700 mg/m3) of methyl salicylate for 7 hours per day, 5 days per week for 4 weeks. Food and water were available ad libitum. Animals were weighed each day, and their condition and behaviour were recorded throughout the exposure period. Urine was collected overnight after the last exposure day for biochemical testing. Gross necropsy was conducted as well as microscopic examination of organs. Methyl salicylate at 700 mg/m3 (120 ppm), the maximum achievable atmosphere, did not cause any adverse effects (Gage, 1970).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

700 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

35

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Single concentration tested. No data on substance purity.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

no

Species:

rat

Strain:

other: Alderley Park

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Alderley Park
- Age at study initiation: no data
- Weight at study initiation: mean 200g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Not applicable, vapour

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

7 hours per day, 5 days per week

Remarks:

Doses / Concentrations:
700 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

4

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION: - No data

FOOD EFFICIENCY:- No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight after last exposure
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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 examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional 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:

not examined

Dose descriptor:

NOEC

Effect level:

700 mg/m³ air

Sex:

female

Basis for effect level:

other: overall effects no adverse effects

Critical effects observed:

not specified

No toxic signs and no macroscopic or microscopic effects were seen at necropsy following exposure to a saturated atmosphere for 7 hours per day, 5 days per week for 4 weeks.

Conclusions:

Methyl salicylate showed no evidence of toxicity by inhalation when tested at an effectively saturated concentration for 7 hours per day for four wekks.

Executive summary:

4 female Alderley Park rats weighing an average of 200 g were exposed to a dynamic atmosphere (atmosphere continuously generated and passed through the exposure chamber) containing a saturated atmosphere (700 mg/m3) of methyl salicylate for 7 hours per day, 5 days per week for 4 weeks. Food and water were available ad libitum. Animals were weighed each day, and their condition and behaviour were recorded throughout the exposure period. Urine was collected overnight after the last exposure day for biochemical testing. Gross necropsy was conducted as well as microscopic examination of organs. Methyl salicylate at 700 mg/m3 (120 ppm), the maximum achievable atmosphere, did not cause any adverse effects (Gage, 1970).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

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

Chronic toxicity studies:

Chronic toxicity studies have been conducted on MeS in rats and in dogs for two years (Webb and Hansen, 1963). Although the studies are relatively old and limited in endpoints evaluated, the protocol and results were reported in adequate detail and included haematological studies (reliability: 2)

Webb and Hansen (1963) administered methyl salicylate in the diet 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/day for two years. All rats in the 1000 mg/kg group died by the 49th week. Body weight of both sexes was significantly decreased in the 500 and 1000 mg/kg body weight/day groups. An increased amount of cancellous bone was present in the metaphyses in rats treated at either 500 or 1000 mg/kg body weight/day, 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 body weight/day group. Gross pituitary gland lesions were found in 10 rats at 250 mg/kg bw/day compared to 4 animals in the control groups. Based on this study, the NOAEL is 50 mg/kg body weight/day. This NOAEL is obtained from 28 days to 2 years study and can be due to a constant hydrolysis rate.

The same authors fed MeS in capsule form to beagle dogs at doses of 0, 50, 150, or 350 mg/kg body weight/day, 6 days/week for 2 years. One high-dose animal died of hepatitis apparently unrelated to methyl salicylate. Hematological analyses at 1, 3, 6, 12 and 24 months and complete necropsy examination were normal, except that dogs treated at 150 and 350 mg/kg body weight/day 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 body weight/day groups. Based on this chronic oral study, the NOAEL is 50 mg/kg body weight/day.

NB:

Retrospective studies of children receiving salicylate therapy in the management of juvenile rheumatoid arthritis did not uncover any cases in which bone lesions or hepatomegaly (as seen in rats and dogs respectively) could be associated with massive daily doses of salicylate over prolonged periods of time. The reviews of human case histories (secondary reference, Abbott and Harrisson, 1978) suggest that the salicylate that the salicylate produced bone lesion in rats and hepatomegaly in dogs are not relevant to humans.

 

Subchronic toxicity studies:

1) Oral

Subchronic toxicity oral studies have been conducted on MeS in rats and dogs.

Rats:

In a 17 -week study in Osborn-Mendel rats (Webb & Hansen, 1963), male and female Osborne-Mendel rats were fed MeS 0%, 0.1% or 1.0% (0, 50 and 500 mg/kg bw/day) in the diet for 17 weeks. The high dose 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/day.

A set of six studies of 6 to12 week duration examined the effects of MeS on bone metabolism and growth (Abbott & Harrisson, 1978) in SD rats fed a diet containing 0.2%, 0.36%, 0.63%, 1.13%, or 2.0% (equivalent to 100, 180, 320, 560 and 1000 mg/kg/day ) MeS. Bone lesions and growth retardation were observed in rats fed MeS at 1% and 2% in diet. These studies indicated a NOAEL of 180 mg/kg/bw/day.

Dogs:

In a subchronic study (Webb and Hansen, 1963), groups of two beagle dogs, one male and one female, were given 50 to 1200 mg/kg of MeS orally in capsule form daily 6 days per week for up to 59 days. All dogs receiving 500 mg/kg/day or more lost weight and died or were killed due to moribund condition within 1- month of study initiation. Moderate to marked fatty changes were observed in the liver of one animal at 800 mg/kg and both at 1200 mg/kg. Animals given 500 mg/kg had diarrhea and weakness during their last 3 days. No adverse effects were observed in animals given 50, 150 and 250 mg/kg/day MeS. A NOAEL of 250 mg/kg bw/day was identified.

In the second of two studies (Abbott & Harrisson, 1978) MeS was administered via capsule at 50, 100 or 167 mg/kg/day, 6 days/week, to groups of male and female beagles for 6 months. Two high-dose and control dogs of each sex were allowed a 2 -month recovery period. There were no treatment-related effects on body weight, liver or kidney weights, or on the results of hematological and clinical chemistry evaluations. The NOAEL was 167 mg/kg/day, the highest dose tested.

2) Dermal

Webb and Hansen (1963), applied MeS dermally to groups of three rabbits of mixed sex at 0.5, 1.0, 2.0 and 5.0 mL/kg (590, 1180, 2360, and 4720 mg/kg) body weight/day, 5 days per week for up to 96 days. All 3 high-dose animals had died by day 28 of exposure, following weight loss and depressed activity. In one of these animals, local effects on the skin and lesions in a number of organs were reported. The only effect reported in the other high dose animal examined was a suggestion of an effect on the distal portion of nephrons. At 2.0 ml/kg (2360 mg/kg), slight sloughing of epidermal scales was observed in 2/3 rabbits. No dermal effects were noted in rabbits exposed to 0.5 or 1.0 ml/kg (590 and 1180 mg/kg body weight/day). The NOAEL for local effects was 1.0 ml/kg/day (1180 mg/kg bw/day). No clear NOAEL was determined for systemic effects due to the reported increased incidence in spontaneous nephritis and mild hepatitis over that in rabbits in other experiments. This dermal study at very high doses was limited in design and in reporting detail and is not useful for risk assessment for systemic toxicity.. It is not possible to determine whether the effects reported on kidney and other organs in one high dose animal represent systemic toxicity or a secondary effect due to skin and muscle damage.

Subacute toxicity studies

Inhalation

4 femalerats were exposed to a saturated atmosphere (700 mg/m3) of MeS for 7 hours per day, 5 days per week for 4 weeks. Animals were weighed each day, and their condition and behaviour were recorded throughout the exposure period. Urine was collected overnight after the last exposure day for biochemical testing. Gross necropsy and microscopic examination of organs was carried out. MeS at 700 mg/m3 (120 ppm), the maximum achievable atmosphere, did not cause any adverse effects (Gage, 1970).

Conclusion:

From the chronic studies in rats and dogs, a systemic NOAEL of 50 mg/kg bw/day can be used for quantitative human health risk assessment of the use of MeS. The target organ being bone, not seen in children (secondary reference, Abbott and Harrisson, 1978.).

Repeated dose toxicity: via oral route - systemic effects (target organ) other: bone

Justification for classification or non-classification

The guidance values for classification of a substance as harmful on repeated exposure by the oral route according to the criteria of Annex VI Directive 67/748/(R48/22) relate to severe effects reported at 50 mg/kg bw/day or below in a 90-day study. The equivalent guidance values for classification according to EU/GHS criteria (STOT Category 2) relate to effects reported at 10-100 mg/kg bw/day in a 90-day study.

For MeS, the no-effect level in both 17-week and 2-year oral dietary studies (Webb & Hansen, 1963) was 50 mg/kg bw/day. Slight effects on bone density were reported at the LOAEL of 500 mg/kg bw/day in the subchronic study.

MeS is not classified for repeat-dose toxicity according to the criteria of Annex VI Directive 67/748/EECor UN/EU GHS.