A critical review of the literature to conduct a toxicity assessment for oral exposure to methyl salicylate

Figures & data

Table 1. Literature search strategy keywords.

Toxicity
Compound/products	AND	Exposure	AND	Endpoints
Methyl Salicylate OR 2-Hydroxy-benzoic acid methyl ester OR 119-36-8 OR Methyl 2 –hydroxybenzoate OR Oil of wintergreen OR Wintergreen oil		Oral OR inhalation OR chronic OR repeated OR subchronic		Toxicity OR reproductive OR developmental OR teratogenic OR teratogens OR pregnancy OR fertility OR carcinogenicity OR cancer OR genotoxicity OR mutagenicity
Mechanism of action
Methyl Salicylate OR 2-Hydroxy-benzoic acid methyl ester OR 119-36-8 OR Methyl 2 –hydroxybenzoate OR Oil of wintergreen OR Wintergreen oil	AND	Oral OR inhalation OR in vitro	AND	pharmacokinetic OR pharmacodynamic OR metabolism OR mode of action OR mechanism OR human relevance

Table 2. Inclusion and exclusion criteria.

Include/exclude	Criteria
Inclusion	• Exposure was to methyl salicylate only, as opposed to a mixture containing methyl salicylate.• Exposure was repeated and not a single acute exposure (with the exception of studies performed in vitro).• The route of exposure in in vivo studies was either via an oral or inhalation pathway.• The study was conducted in humans or a relevant mammalian species such as rats, mice, dogs, rabbits, guinea pigs, or monkeys if in vivo; and in bacterial, animal, or human cell lines if in vitro.
Exclusion	• Exposure was not to methyl salicylate only.• Exposure occurred via a single exposure or multiple exposures over a 24 hour period (with the exception of studies performed in vitro).• The route of exposure for in vivo studies was not by the oral or inhalation route.• Or for in vitro studies, conducted in a cell line that was not bacterial, animal, or human.

Table 3. Criteria from Klimisch et al. (1997).

Quality score	Requirements
1	Studies or data from the literature or reports that are carried out or generated according to generally valid and/or internationally accepted testing guidelines (preferably performed according to Good Laboratory Practices [GLP]), or studies in which the test parameters documented are based on a specific (national) testing guideline (preferably performed under GLP), or studies in which all parameters described are closely related/comparable to a guideline method
2	Studies that are not performed according to GLP or specific testing guidelines but are well documented and scientifically acceptable with thefollowing information reported: For in vivo animal studies this includes the following criteria:  • Data on the test animals including species, sex, and strain;  • The purity/composition/origin of the test substance;  • The number of animals evaluated;  • The scope of the investigation per animal (e.g. clinical chemistry, hematology, organ weights, pathology or histopathology) and description of the methods;  • Description of the changes observed;  • Control group or historical control data of the laboratory;  • Description of the test conditions;  • Description of the route and doses of administration;  • Dose/concentration relationship is possible. For in vitro studies this includes the following criteria:  • Description of the test system and test method in details;  • Purity/composition/origin of the test substance;  • Data on the dose/concentration differentiated according to the toxicity of the test substance on the test system; information on volatility;  • Data on secondary effects which may influence a result (solubility, impurities, pH shifts, influence on the osmolarity, etc.);  • Appropriate negative/positive controls as integral parts of the test;  • References on adequacy of the method should be given or generally known.
3	Studies or data from the literature which do not meet the criteria for a quality score of 2. These include studies for which the methods were not clearly defined or specified in the study, in which only one dose was administered, or small numbers of animals per group were tested. The appropriate number of animals per group was determined based on recommendations from OECD guidelines for comparable study types
4	Studies or data from the literature which do not provide sufficient experiment details and which are only listed in short abstracts or secondaryliterature (books, reviews, etc.)  • Additional criteria for in vivo animal studies:  • Single exposure;  • Inappropriate or irrelevant species tested;  • Route of exposure not relevant to humans;  • If the animals tested underwent any type of alteration or injury prior to testing.

Table 4. Literature identified from authoritative reviews and literature searching.

Toxicity studies
Abbott DD, Harrisson JWE (1978). Methyl salicylate: Studies of osseous changes in the rat, reproduction in the rat and mouse, and liver and kidney effects in the dog. Unpublished communication to the Flavor and Extract Manufacturers Association^a
Collins TF, Hansen WH, Keeler HV (1971). Effect of methyl salicylate on rat reproduction. Toxicology and Applied Pharmacology 18(4): 755–765
Cosmetic Ingredient Review (2003). Safety assessment of Salicylic Acid, Butyloctyl Salicylate, Calcium Salicylate, C12-15 Alkyl Salicylate, Capryloyl Salicylic Acid, Hexyldodecyl Salicylate, Isocetyl Salicylate, Isodecyl Salicylate, Magnesium Salicylate, MEA-Salicylate, Ethylhexyl Salicylate, Potassium Salicylate, Methyl Salicylate, Myristyl Salicylate, Sodium Salicylate, TEA-Salicylate, and Tridecyl Salicylate. International Journal of Toxicology 22(Suppl 3): 1–108
Cosmetic Ingredient Review (2008). Final report of the safety assessment of Alcohol Denat., including SD Alcohol 3-A, SD Alcohol 30, SD Alcohol 39, SD Alcohol 39-B, SD Alcohol 39-C, SD Alcohol 40, SD Alcohol 40-B, and SD Alcohol 40-C, and the denaturants, Quassin, Brucine Sulfate/Brucine, and Denatonium Benzoate. International Journal of Toxicology 27(Suppl 1): 1–43
Crossland AM (1948). Methyl salicylate poisoning. Canadian Medical Association Journal 58(1): 75–77 (FREE)
Gulati DK, Choudhury H, Chambers R, Sabharwal PS, Lamb JIV (1984). Methyl salicylate: fertility assessment in cd-1 mice when administered by gavage.NTIS (pb 85-164283, ntp 85-022)
Harrisson JWE et al. (1963). Federation Proceedings, Federation of American Societies for Experimental Biology 22: 554.^b
Hutt AJ, Caldwell J, Smith R (1986). The metabolism of aspirin in man: a population study. Xenobiotica; the fate of foreign compounds in biological systems 16(3): 239–249.
Infurna R, Beyer B, Twitty L, Koehler G, Daughtrey W (1990). Evaluation of the dermal absorption and teratogenic potential of methyl salicylate in a petroleum-based grease. Exxon Biomedical Sciences, Inc., East Millstone, New Jersey. Teratology 41: 566
LaWall and Harrisson Research Laboratories, Inc. (1964). Study of Methyl Salicylate on rats. L&H 58188. (Submitted to USFDA in response to an FOI request – 1998; 120 pages.)^a
Morrissey RE, Lamb J, Morris R, Chapin R, Gulati D, Heindel J (1989). Results and evaluations of 48 continuous breeding reproduction studies conducted in mice. Developmental and Reproductive Toxicology Group, NIEHS, NTP Program, Research Triangle Park, NC, Analytical Sciences Inc., Durham, North Carolina, Environmental Health Research and Testing, Inc., Lexington, Kentucky; Fundamental and Applied Toxicology 13: 747–777
National Toxicology Program. 1984. Abstract for RACB82104 – Methyl salicylate CAS no. 119-36-8. NTIS #RACB82104. Available at: http://ntp.niehs.nih.gov/testing/types/repro/abstracts/racb/index-8.html
Opdyke DL (1978). Acute oral toxicity: rat; acute dermal toxicity: rabbit; and primary dermal irritation:rabbit) for methyl salicylate, fragrance raw materials monographs. Food and Cosmetics Toxicology 16: 821–825
Opdyke DL (1979). Monographs on fragrance raw materials. Food and Cosmetics Toxicology 17(4): 357–390
Opdyke DL (1979). Monographs on fragrance raw materials. Food and Cosmetics Toxicology 17(Special Issue V): 695–923
Overman DO, White JA (1983). Comparative teratogenic effects of methyl salicylate applied orally or topically to hamsters. Teratology 28(3): 421–426
Packman E, Abbott D, Wager B, Harrisson J (1961). Chronic and oral toxicity of oil sweet birch (methyl salicylate). Pharmacologist 3: 62^b
Rainsford K (2004a). History and development of the salicylates. In Aspirin and Related Drugs (K Rainsford, Ed.), Taylor and Francis, New York
Rainsford K (2004b). Pharmacology and biochemistry of salicylates and related drugs. In Aspirin and Related Drugs (K Rainsford, Ed.), pp. 215–366. Taylor and Francis, New York
Rainsford K (2004c). Side effects and toxicology of the salicylates. In aspirin and related drugs (K Rainsford, Ed.), Taylor and Francis, New York
Rudolph AM (1981). Effects of aspirin and acetaminophen in pregnancy and in the newborn. Archives of Internal Medicine 141(3 Spec No): 358–363
Sharpe GL, Larsson KS, Thalme B (1975). Studies on closure of the ductus arteriosus. XII. In utero effect of indomethacin and sodium salicylate in rats and rabbits. Prostaglandins 9(4): 585–596
Ungvary G, Tatrai E, Lorincz M, Barcza G (1983). Combined embryotoxic action of toluene, a widely used industrial chemical, and acetylsalicylic acid (aspirin). Teratology 27(2): 261–269
USFDA (1966). Chronic oral toxicity of Methyl Salicylate using dogs and reproductive and developmental effects oral study of Methyl Salicylate using rats. United States Food and Drug Administration. Studies dated May. (Submitted by FDA in response to an FOI request – 1998; 33 pages.)^a
Warknay J, Takacas E (1959). Experimental production of congenital malformations in rats by salicylate poisoning. American Journal of Pathology 35: 315–331
Webb WK, Hansen WH (1963). Chronic and subacute toxicology and pathology of methyl salicylate in dogs, rats, and rabbits. Toxicology and Applied Pharmacology 5: 576–587
Mode of action studies
Battino M, Ferreiro MS, Armeni T, Politi A, Bompadre S, Massoli A, Bullon P (2005). In vitro antioxidant activities of antioxidant-enriched toothpastes. Free Radical Research 39(3): 343-350.
Cerletti C, Dell'Elba G, Manarini S, Pecce R, Di Castelnuovo A, Scorpiglione N, Feliziani V, de Gaetano G (2003). Pharmacokinetic and pharmacodynamic differences between two low dosages of aspirin may affect therapeutic outcomes. Clinical Pharmacokinetics 42(12): 1059–1070
Daston GP, Rehnberg BF, Carver B, Rogers E, Kavlock R (1988). Functional teratogens of the rat kidney. I. Colchicine, dinoseb, and methyl salicylate. Fundamental and Applied Toxicology 11(3): 381–400
Davis LE, Westfall BA (1972). Species differences in biotransformation and excretion of salicylate. American Journal of Veterinary Research 33(6): 1253–1262
Davison C, Zimmerman EF, Smith P (1961). On the metabolism and toxicity of methyl salicylate. The Journal of Pharmacology and Experimental Therapeutics 132: 207–211
Dorne JL (2004). Impact of inter-individual differences in drug metabolism and pharmacokinetics on safety evaluation. Fundamental and Clinical Pharmacology 18(6): 609–620
Gatti G, Barzaghi N, Attardo Parrinello G, Vitiello B, Perucca E (1989). Pharmacokinetics of salicylic acid following administration of aspirin tablets and three different forms of soluble aspirin in normal subjects. International Journal of Clinical Pharmacology Research 9(6): 385–389
Graham G, Roberts M, Day R, Rainsford K (2004). Pharmacokinetics and Metabolism of the Salicylates. In Aspirin and Related Drugs (K Rainsford, Ed.), Taylor and Francis, New York
Ishidate M, Sofuni T, Yoshikawa D, Hayashi M, Nohmi T, Sawada M, Matsuoka A (1984). Primary mutagenicity screening of food additives currently used in Japan. Food and Chemical Toxicology 22: 623–636
Kuboyama, N. and Fujii A. (1992). Mutagenicity of analgesics, their derivatives, and anti-inflammatory drugs with S-9 mix of several animal species. The Journal of Nihon University School of Dentistry 34(3): 183–195
Lapczynski A, Jones L, McGinty D, Bhatia S, Letizia C, Api A (2007). Fragrance material review on methyl salicylate. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 45(Suppl 1): S428–452
Levy G, Weintraub L, Matsuzawa T, Oles S (1966). Absorption, metabolism and excretion of salicylic phenolic glucuronide in rats. Journal of Pharmaceutical Sciences 55(11): 1319–1320
Montgomery PR, Berger LG, Mitenko P, Sitar D (1986). Salicylate metabolism: effects of age and sex in adults. Clinical pharmacology and therapeutics 39(5): 571–576
Mortelmans K, Haworth S, Lawlor T, Speck W, Tainer B, Zeiger E (1986). Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals. Environmental Mutagenesis 8(Suppl 7): 1–119
Murugesh N, Kumar VR, Vembar S, Damodaran C (1981). Studies on erythrocyte membrane. V. Haemolytic effect of methylsalicylate and its possible mechanism. Toxicology Letters 9(3): 225–229
Needs CJ, Brooks PM (1985). Clinical pharmacokinetics of the salicylates. Clinical Pharmacokinetics 10(2): 164–177
Patrono C, Coller B, Dalen J, Fitzgerald G, Fuster V, Gent M, Hirsh J, Roth G (2001). Platelet-active drugs: the relationships among dose, effectiveness, and side effects. Chest 119(Suppl 1): 39S–63S
Roberts LJ III, Morrow JD (2001). Analgesic-antipyretic and antiinflammatory agents and Drugs Employed in the Treatment of Gout. In Goodman & Gilman's The Pharmacological Basis of Therapeutics (JG Hardman, LE Limbird, Eds.), McGraw-Hill, New York, New York
Rotilio D, Joseph D, Hatmi M, Vargaftig B (1984). Structural requirements for preventing the aspirin- and the arachidonate-induced inactivation of platelet cyclo-oxygenase: additional evidence for distinct enzymatic sites. European Journal of Pharmacology 97(3-4): 197–208
Vane JR, Botting RM (2003). The mechanism of action of aspirin. Thrombosis Research 110(5-6): 255–258
Verstraelen S, Nelissen I, Hooyberghs J, Witters H, Schoeters G, Van Cauwenberge P, Van Den Heuvel R (2009a). Gene profiles of a human alveolar epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis. Toxicology Letters 185(1): 16–22
Verstraelen S, Nelissen I, Hooyberghs J, Witters H, Schoeters G, Van Cauwenberge P, Van Den Heuvel R (2009b). Gene profiles of a human bronchial epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis. Toxicology 255(3): 151–159
Vigano G, Garagiola U, Gaspari F (1991). Pharmacokinetic study of a new oral buffered acetylsalicylic acid (ASA) formulation in comparison with plain ASA in healthy volunteers. International Journal of Clinical Pharmacology Research 11(3): 129–135
Wolowich WR, Hadley CM, Kelley M, Walson P, Casavant M (2003). Plasma salicylate from methyl salicylate cream compared to oil of wintergreen. Journal of Toxicology. Clinical Toxicology 41(4): 355–358
Zhang Z, Jia C, Hu Y, Sun L, Jiao J, Zhao L, Zhu D, LI J, Tian Y, Bai H, Li R, Hu J (2012). The estrogenic potential of salicylate esters and their possible risks in foods and cosmetics. Toxicology Letters 209(2): 146–153

^a This study was identified through a review of the CIR (2003) and was determined to be an unpublished study that is not available publicly.

^b For this citation, only an abstract was available.

Table 5. Results of the study quality analysis.

Study	Study quality score	Rationale for study quality score
Gulati et al. (1984)	2	Study was performed according to GLP. The study did not specify that it was performed according to standard study guidelines recommended by regulatory agencies (e.g. USEPA or OECD); however, the methods are well documented and scientifically acceptable.
Morrissey et al. (1989)	2	Study was performed according to GLP. The study did not specify that it was performed according to standard study guidelines recommended by regulatory agencies (e.g. USEPA or OECD); however, the methods are well documented and scientifically acceptable.
Webb and Hansen (1963)	2	The study was performed prior to GLP. The study was not performed according to standard study guidelines recommended by regulatory agencies (e.g. USEPA or OECD); however, the methods are well documented and scientifically acceptable.
Collins et al. (1971)	3	The study was performed prior to GLP and was not performed according to standard study guidelines recommended by regulatory agencies (e.g. USEPA or OECD). The purity of the test substance was not specified.
Packman et al. (1961)	4	Only an abstract was available. Limited study details were available.
Harrisson et al. (1963)	4	This citation could not be obtained in the publically available literature. Information considered was based on the summary reported in NFSA (2012).
USFDA (1966)^a	4	This is an unpublished study and could not be obtained. Information considered was based on the summary reported in CIR (2003). Due to lack of appropriate protocol and dose response information presented in CIR (2003) the study was not considered for this assessment.
LaWall and Harrisson Research Laboratories, Inc. (1964)^a	4	This is an unpublished study and could not be obtained. Information considered was based on the summary reported in CIR (2003). Due to lack of appropriate protocol and dose response information presented in CIR (2003) the study was not considered for this assessment.
Abbot and Harrisson (1978)^a	4	This is an unpublished study and could not be obtained. Information considered was based on the summary reported in CIR (2003). Due to lack of appropriate protocol and dose response information presented in CIR (2003) the study was not considered for this assessment.
Rudolph (1981)	4	This is a review article and not a toxicity study.
Crossland (1948)	4	This is a review article and not a toxicity study.
Opdyke (1979a)	4	This is a review article and not a toxicity study.
Opdyke (1979b)	4	This is a review article and not a toxicity study.
Rainsford (2004a)	4	This is a review article and not a toxicity study.
Rainsford (2004b)	4	This is a review article and not a toxicity study.
Rainsford (2004c)	4	This is a review article and not a toxicity study.
Opdyke (1978)	4	This is a review article and not a toxicity study.
CIR (2003)	4	This is a review article and not a toxicity study.
CIR (2008)	4	This is a review article and not a toxicity study.

^a This study was identified through a review of the CIR (2003) and was determined to be an unpublished study that is not available publicly.

Table 6. Summary or studies considered for evaluation in the QRA

Study	Species	Route ofexposure	Number ofanimals/sex/group	Administered dose	Duration	NOAEL(mg/kg/d)	Endpoint	Studyqualityscore	Rationale for studyquality score
Chronic toxicity studies
Webb and Hansen (1963)	Rats	diet	25	0, 0.1, 0.5, 1, 2% (0, 50, 250, 500, 1000 mg/kg/d)^a	2 years	500	Cancellous bone in the metaphysis	2	The study was performed prior to GLP. The study was not performed according to any specific published testing guidelines; however, the study protocol was well documented and scientifically acceptable
	Dogs	capsule	2	0, 50, 150, 350 mg/kg/d	2 years	350	No adverse effects reported	2	The study was performed prior to GLP. The study was not performed according to any specific published testing guidelines; however, the study protocol was well documented and scientifically acceptable.
Reproductive/developmental studies
Morrissey et (al. 1989), Gulati et al. (1984), and NTP (1984)	Mice	Gavage	40 (control);20 (treated)	0, 25, 50, 100 mg/kg/d (NTP 1984) 0, 100, 250, 500 mg/kg/d (Gulati et al. 1984)	7 d (premating); 100 d (cohabited)	100	Decreased number of litters, number of pups per litter, proportion of pups born alive and decreased mean pup weight	2	Study was performed according to GLP. Testing guideline is a published guideline but not OECD. Testing guideline is well documented and scientifically acceptable.

^a Concentrations were converted from percent methyl salicylate in the diet to doses of mg/kg/d assuming a food factor of 0.05 for rats or 0.13 for mice (USEPA 1985). Food consumption data was not reported in the study.

Table 7. Reproductive performance of fertile pairs during continuous breeding (task 2) methyl salicylate (Gulati et al. 1984).

	Treatment group (methyl salicylate concentration)
Reproductive parameter^a	Control	100 mg/kg	250 mg/kg	500 mg/kg
Litters per pair	4.92 ± 0.044 (38)^b	4.82 ± 0.128 (17)	4.78 ± 0.129 (18)	4.50 ± 0.258 (16)*
Proportion of pups born alive	0.97 ± 0.010 (38)	0.98 ± 0.010 (17)	0.96 ± 0.017 (18)	0.91 ± 0.033 (16)*
Live pups per litter
Male	5.73 ± 0.209 (38)	5.41 ± 0.249 (17)	4.75 ± 0.384 (18)	4.32 ± 0.364 (16)**
Female	5.55 ± 0.206 (38)	5.05 ± 0.228 (17)	4.83 ± 0.313 (18)	3.46 ± 0.351 (16)**
Combined	11.29 ± 0.364 (38)	10.47 ± 0.348 (17)	9.58 ± 0.648 (18)	7.78 ± 0.635 (16)**
Live pup weight (g)
Male	1.65 ± 0.014 (38)	1.64 ± 0.020 (17)	1.64 ± 0.018 (18)	1.60 ± 0.023 (16)
Female	1.60 ± 0.014 (38)	1.59 ± 0.021 (17)	1.57 ± 0.020 (18)*	1.53 ± 0.016 (16)*
Combined	1.62 ± 0.013 (38)	1.62 ± 0.021 (17)	1.60 ± 0.017 (18)	1.57 ± 0.021 (16)*
Adjusted live pup weight (g)^c
Male	1.67 ± 0.014 (38)	1.65 ± 0.019 (17)	1.63 ± 0.019 (18)	1.57 ± 0.021 (16)**
Female	1.61 ± 0.013 (38)	1.59 ± 0.019 (17)	1.56 ± 0.018 (18)*	1.51 ± 0.021 (16)**
Combined	1.64 ± 0.013 (38)	1.62 ± 0.018 (17)	1.59 ± 0.018 (18)*	1.54 ± 0.020 (16)**

^a Mean ± SE

^b Number of pairs providing data indicated in ().

^c Means adjusted for total number of live and dead pups per litter by analysis of covariance.

* Significantly different p < .05 from control group.

** Significantly different p < .01 from control group.

Table 8. Litter data for individual mating pairs (Gulati et al. 1984).

	Number of live pups in each litter					Minimum number of dead pups in each litter					Total pups dead and alive^a
Litter number	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5
Control	12	15	11	11	10	0	0	0	0	0	12	15	11	11	10
	12	9	9	10	12	0	0	0	0	0	12	9	9	10	12
	8	11	4	14	15	0	1	0	0	1	8	12	4	14	16
	11	12	10	13	12	0	0	0	0	0	11	12	10	13	12
	11	13	11	11	11	0	0	0	1	0	11	13	11	12	11
	15	15	17	16	13	0	0	0	1	0	15	15	17	17	13
	11	12	12	11	9	0	0	1	1	0	11	12	13	12	9
	13	14	12	5	0	0	0	0	3	6	13	14	12	8	6
	11	12	10	13	12	0	0	0	0	0	11	12	10	13	12
	10	14	15	13	6	0	0	0	0	0	10	14	15	13	6
	10	9	15	16	0	1	0	0	0	3	11	9	15	16	3
	6	4	8	5	8	0	0	0	0	3	6	4	8	5	11
	9	12	6	8	14	0	0	0	0	0	9	12	6	8	14
	10	11	12	11	7	0	0	0	0	0	10	11	12	11	7
	12	13	13	11	7	0	0	0	0	0	12	13	13	11	7
	9	14	14	15	12	0	0	0	0	0	9	14	14	15	12
	7	9	2	10	11	0	0	3	0	0	7	9	5	10	11
	11	15	8	0	0	0	0	0	9	6	11	15	8	9	6
	9	12	11	10	13	0	0	0	0	0	9	12	11	10	13
	11	10	11	10	6	0	0	0	0	0	11	10	11	10	6
	12	14	13	14	8	0	1	0	0	0	12	15	13	14	8
	15	17	16	17	16	0	0	0	0	1	15	17	16	17	17
	11	14	11	13	–	0	0	0	0	–	11	14	11	13	–
	9	9	13	12	13	0	0	0	0	0	9	9	13	12	13
	10	12	8	11	–	0	0	0	0	–	10	12	8	11	–
	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	12	14	11	13	12	0	1	0	0	0	12	15	11	13	12
	9	12	12	10	10	0	0	0	0	0	9	12	12	10	10
	10	14	16	15	13	0	2	0	0	0	10	16	16	15	13
	11	12	0	0	12	0	2	2	3	0	11	14	2	3	12
	14	15	7	12	10	0	0	0	0	0	14	15	7	12	10
	10	11	11	9	11	0	0	0	0	0	10	11	11	9	11
	10	14	12	15	15	1	0	0	0	0	11	14	12	15	15
	13	13	14	16	13	0	0	0	0	0	13	13	14	16	13
	13	10	13	11	10	0	0	0	0	0	13	10	13	11	10
	10	12	12	12	14	0	0	0	0	0	10	12	12	12	14
	13	11	11	12	11	0	0	0	0	0	13	11	11	12	11
	15	16	19	16	15	0	0	0	0	0	15	16	19	16	15
	17	10	15	13	–	0	1	0	0	–	17	11	15	13	–
MEAN	11.1	12.3	11.2	11.4	10.3	0.1	0.2	0.2	0.5	0.6	11.2	12.5	11.4	11.9	10.9
SE	0.4	0.4	0.63	0.6	0.7	0	0.1	0.1	0.3	0.3	0.4	0.5	0.73	0.9	1
0.1 g/kg	15	10	4	–	–	0	0	0	–	–	15	10	4	–	–
	14	12	5	11	2	0	0	0	0	3	14	12	5	11	5
	11	12	11	9	8	0	0	0	0	0	11	12	11	9	8
	9	10	10	10	12	0	0	0	0	0	9	10	10	10	12
	10	12	11	8	10	0	0	0	0	0	10	12	11	8	10
	11	12	12	10	11	0	0	0	0	0	11	12	12	10	11
	10	13	11	10	10	0	0	0	0	0	10	13	11	10	10
	10	12	16	13	9	1	1	0	0	0	11	13	16	13	9
	6	13	10	10	13	0	0	0	0	0	6	13	10	10	13
	11	13	13	9	11	0	0	0	0	0	11	13	13	9	11
	14	12	12	11	10	0	0	0	0	1	14	12	12	11	11
	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	14	3	7	12	12	0	0	0	0	0	14	3	7	12	12
	11	14	13	14	12	0	0	0	0	0	11	14	13	14	12
	13	10	11	12	9	1	0	0	0	0	14	10	11	12	9
	11	11	10	11	–	1	0	0	0	–	12	11	10	11	0
	11	12	8	10	–	0	0	1	0	–	11	12	9	10	0
	7	15	11	13	10	1	0	0	0	0	8	15	11	13	10
	10	6	9	0	7	0	2	2	1	1	10	8	11	1	8
Mean	11	11.2	2.9	10.2	9.7	0.2	0.2	0.2	0.1	0.3	11.2	11.4	3.1	10.3	10
SE	0.6	0.7	0.7	0.7	0.7	0.1	0.1	0.1	0.1	0.2	0.7	0.8	0.8	0.8	0.9
0.25 g/kg	9	9	6	6	8	2	0	0	1	0	11	9	6	7	8
	10	7	11	10	7	0	0	0	0	0	10	7	11	10	7
	12	10	9	7	10	0	0	0	0	0	12	10	9	7	10
	11	14	13	10	9	0	0	0	0	0	11	14	13	10	9
	11	11	13	12	–	0	0	0	0	–	11	11	13	12	0
	14	12	15	12	11	0	0	0	0	0	14	12	15	12	11
	9	3	11	1	0	0	0	0	1	2	9	3	11	2	2
	11	11	11	11	10	1	0	0	0	0	12	11	11	11	10
	11	2	5	–	–	0	0	1	–	–	11	2	6	0	0
	10	–	–	–	–	0	–	–	–	–	10	0	0	0	0
	11	14	13	13	14	0	0	0	0	0	11	14	13	13	14
	6	7	7	5	9	0	0	0	2	0	6	7	7	7	9
	11	14	11	10	10	0	0	0	0	0	11	14	11	10	10
	11	15	10	14	11	0	0	0	0	0	11	15	10	14	11
	10	12	7	2	8	0	0	3	0	0	10	12	10	2	8
	10	15	–	–	–	0	0	–	–	–	10	15	0	0	0
	10	13	0	2	–	0	0	7	3	–	10	13	7	5	0
	11	11	13	15	12	0	0	0	0	0	11	11	13	15	12
	14	14	9	12	11	0	0	0	0	0	14	14	9	12	11
	6	8	7	12	–	0	1	0	0	–	6	9	7	12	0
Mean	10.4	10.6	9.5	9.1	9.3	0.2	0.1	0.6	0.4	0.1	10.6	10.7	10.1	9.5	9.4
SE	0.4	0.9	0.9	1.1	0.9	0.1	0.1	0.4	0.2	0.1	0.5	1	1.3	1.3	1
0.5 g/kg	8	9	12	7	–	0	0	0	0	–	8	9	12	7	–
	13	6	6	11	1	0	1	0	0	0	13	7	6	11	1
	10	12	13	14	13	0	0	1	0	0	10	12	14	14	13
	10	5	8	11	–	0	0	0	0	–	10	5	8	11	0
	10	11	12	11	9	0	2	0	0	0	10	13	12	11	9
	7	0	8	0	–	0	4	0	4	–	7	4	8	4	0
	2	9	11	10	–	2	2	1	0	–	4	11	12	10	0
	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	10	8	9	11	8	0	0	0	0	0	10	8	9	11	8
	9	11	11	–	–	0	0	0	–	–	9	11	11	0	0
	6	6	5	7	6	1	1	0	1	1	7	7	5	8	7
	10	–	–	–	–	0	–	–	–	–	10	–	–	–	–
	2	12	15	17	13	0	0	0	0	0	2	12	15	17	13
	8	8	11	10	0	1	0	0	0	2	9	8	11	10	2
	10	9	11	9	8	0	0	0	0	0	10	9	11	9	8
	3	8	11	0	0	8	0	0	7	2	11	8	11	7	2
	9	4	7	3	4	0	1	0	0	1	9	5	7	3	5
	10	6	12	0	–	0	2	0	5	0	10	8	12	5	0
	5	–	–	–	–	0	–	–	–	–	5	–	–	–	–
	13	12	7	7	0	0	0	0	0	2	13	12	7	7	2
Mean	8.2	8	9.9	8	5.6	0.6	0.8	0.1	1.1	0.7	8.8	8.8	10	9.1	6.3
SE	0.8	0.8	0.7	1.3	1.5	0.4	0.3	0.1	0.6	0.3	1.2	1.1	0.8	1.9	1.8

^a The total number of pups was not reported, so it was assumed the total pups equaled sum of the live and dead pups.

– indicates no litter data available.

Figure 1. (a) Graph of benchmark modeling output for number of litters per mating pair reported in Gulati et al. (1984). Linear model with BMR of 1 standard deviation for the BMD and 0.95 lower confidence limit for the BMDL. (b) Graph of benchmark modeling output for number of litters per mating pair reported in Gulati et al. (1984). Power Model with BMR of 1 standard deviation for the BMD and 0.95 lower.

Table 9. BMD results from Gulati et al. (1984).

					(Continuous – 1 STD change, extra risk)(g/kg/d)
Endpoint	Model	AIC	Chi-square or likelihoodratio goodnessof fit p value	Scaled residualat observeddose closest to BMD	BMD	BMDL
Average pup weight	Exponential2	−362.11	.664	−0.081	0.41	0.29
	Exponential3	−360.13	.373	−0.007	0.41	0.29^a
	Exponential4	−362.11	.664	−0.081	0.41	0.24
	Exponential5	−358.13	N/A	−0.007	0.41	0.29
	Hill	−358.13	N/A	−0.006	0.42	0.23
	Linear	−362.12	.667	−0.065	0.41	0.30
	Polynomial	−360.14	.374	−0.001	0.42	0.30
	Power	−360.13	.373	−0.007	0.42	0.30
Litters per mating pair	Exponential2	−44.09	N/A	−0.212	0.42	0.21
	Exponential3	−44.09	N/A	−0.212	0.42	0.21
	Exponential4	−43.44	N/A	−0.506	0.42	0.13
	Exponential5	−43.44	N/A	−0.506	0.42	0.13
	Hill	−43.73	.104	−0.547	0.43	–
	Linear	−43.96	.110	−0.222	0.43	0.22^a
	Polynomial	−43.06	.068	−0.435	0.41	0.15
	Power	−43.96	.940	−0.222	0.43	0.22^a

AIC: Akaike information criterion; BMD: benchmark dose; BMDL: lower bound on BMD.

^a Lowest BMDL with the best model fit.

N/A: not available. A p value could not be determined by BMDS. Model output indicates that other models should be considered because model fit cannot be confirmed. – Benchmark computation failed.

Table 10. Individual breeding pair BMD modeling results for Gulati et al. (1984).

Endpoint	Model	Covariate	AIC	Chi-Square P-value (bootstrap)	Average scaled residual for dose group nearest the BMD	Average ABS(scaled residual) for dose group nearest the BMD	Mean litter specific covariate type for BMD and BMDL	BMD 5 (g/kg/d)	BMDL 5 (g/kg/d)
No. dead pups/ individual breeding pair	NLogistic	Litter size	843.58	.782	−0.180	0.283	Control mean =11.5	2.71	0.77^a
	NCTR	Litter size	864.47	.935	−0.366	0.366		0.49	0.49
	Rai_and_Van_Ryzin	Litter size	836.38	–	–	–		–	–
	NLogistic	Not used	879.54	.287	−0.444	0.444		0.41	0.30
	NCTR	Not used	879.72	.303	−0.444	0.444		0.48	0.48
	Rai_and_Van_Ryzin	Not used	879.72	.321	−0.444	0.444		0.48	0.48
	NLogistic	Litter size	843.58	.763	0.279	0.760	Overall mean =10.6	1.67	0.60
	NCTR	Litter size	864.47	.920	−0.118	0.610		0.48	0.48
	Rai_and_Van_Ryzin	Litter size	836.38	–	–	–		–	–
	NLogistic	Not used	879.54	.309	−0.190	0.606		0.41	0.30
	NCTR	Not used	879.72	.322	−0.189	0.606		0.48	0.48
	Rai_and_Van_Ryzin	Not used	879.72	.315	−0.189	0.606		0.48	0.48

AIC, Akaike information criterion; BMD: benchmark dose; BMDL, lower bound on BMD. –: Benchmark computation failed.

^a Lowest BMDL with the best model fit.

Klimisch HJ, Andreae M, Tillmann U. 1997. A systematic approach for evaluating the quality of experimental toxicological and ecotoxicological data. Regul Toxicol Pharmacol. 25:1–5.

 PubMed Web of Science ®Google Scholar

Collins TF, Hansen WH, Keeler HV. 1971. Effect of methyl salicylate on rat reproduction. Toxicol Appl Pharmacol. 18:755–765.

 PubMed Web of Science ®Google Scholar

[CIR] Cosmetic Ingredient Review. 2003. Safety assessment of salicylic acid, butyloctyl salicylate, calcium salicylate, C12-15 alkyl salicylate, capryloyl salicylic acid, hexyldodecyl salicylate, isocetyl salicylate, isodecyl salicylate, magnesium salicylate, MEA- salicylate, ethylhexyl salicylate, potassium salicylate, methyl salicylate, myristyl salicylate, sodium salicylate, TEA- salicylate, and tridecyl salicylate. Int J Toxicol. 22 Suppl 3:1–108.

 Google Scholar

[CIR] Cosmetic Ingredient Review. 2008. Final report of the safety assessment of alcohol denaturants, including SD alcohol 3-A, SD alcohol 30, SD alcohol 39, SD alcohol 39-B, SD alcohol 39-C, SD alcohol 40, SD alcohol 40-B, and SD alcohol 40-C, and the denaturants, quassin, brucine sulfate/brucine, and denatonium benzoate. Int J Toxicol. 27 Suppl 1:1–43.

 Google Scholar

Crossland AM. 1948. Methyl salicylate poisoning. Can Med Assoc J. 58:75–77.

 PubMed Web of Science ®Google Scholar

Gulati DK, Choudhury H, Chambers R, Sabharwal PS, Lamb JIV. 1984. Methyl salicylate: fertility assessment in cd-1 mice when administered by gavage. National Toxicology Program, National Institutes of Environmental Health Sciences. NTP-85-022. Available from: https://www.ntis.gov/products/ntrl/.

 Google Scholar

Harrisson JWE, Abbott DD, and Packman EW. 1963. Salicylates and other hydroxybenzoates: effect upon osseous tissue of young rats. Federation Proceedings, vol. 22, p. 554.

 Google Scholar

LaWall and Harrisson Research Laboratories, Inc. 1964. Study of Methyl Salicylate on rats. L&H 58188. (Submitted by FDA in response to an FOI request – 1998; 120 pages) (As cited in CIR 2003).

 Google Scholar

[USFDA] United States Food and Drug Administration. 1966. Chronic oral toxicity study of Methyl Salicylate using dogs and reproductive and developmental effects oral study of Methyl Salicylate using rats. Studies dated May. Submitted by FDA in response to an FOI request – 1998; 33 pages (As cited in CIR 2003).

 Google Scholar

Battino M, Ferreiro MS, Armeni T, Politi A, Bompadre S, Massoli A, Bullon P. 2005. In vitro antioxidant activities of antioxidant-enriched toothpastes. Free Radic Res. 39:343–350.

 PubMed Web of Science ®Google Scholar

Daston GP, Rehnberg BF, Carver B, Rogers E, Kavlock R. 1988. Functional teratogens of the rat kidney. I. Colchicine, dinoseb, and methyl salicylate. Fundam Appl Toxicol. 11:381–400.

 PubMedGoogle Scholar

Davis LE, Westfall BA. 1972. Species differences in biotransformation and excretion of salicylate. Am J Vet Res. 33:1253–1262.

 PubMed Web of Science ®Google Scholar

Graham GG, Roberts MS, Day RO, Rainsford KD. 2004. Pharmacokinetics and metabolism of the salicylates. In: Rainsford KD, editor. Aspirin and related drugs. New York: Taylor and Francis, p. 97–155 (Chapter 4).

 Google Scholar

Ishidate M, Sofuni T, Yoshikawa D, Hayashi M, Nohmi T, Sawada M, Matsuoka A. 1984. Primary mutagenicity screening of food additives currently used in Japan. Food Chem Toxicol. 22:623–636.

 PubMed Web of Science ®Google Scholar

Kuboyama N, Fujii A. 1992. Mutagenicity of analgesics, their derivatives, and anti-inflammatory drugs with S-9 mix of several animal species. J Nihon Univ School Dentistry. 34:183–195.

 PubMedGoogle Scholar

Lapczynski A, Jones L, McGinty D, Bhatia S, Letizia C, Api A. 2007. Fragrance material review on methyl salicylate. Food Chem Toxicol. 45(Suppl 1):S428–S452.

 PubMed Web of Science ®Google Scholar

Levy G, Weintraub L, Matsuzawa T, Oles S. 1966. Absorption, metabolism and excretion of salicylic phenolic glucuronide in rats. J Pharm Sci. 55:1319–1320.

 PubMed Web of Science ®Google Scholar

Montgomery PR, Berger LG, Mitenko P, Sitar D. 1986. Salicylate metabolism: effects of age and sex in adults. Clin Pharmacol Therap. 39:571–576.

 PubMed Web of Science ®Google Scholar

Mortelmans K, Haworth S, Lawlor T, Speck W, Tainer B, Zeiger E. 1986. Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals. Environ Mutagen. 8(Suppl 7):1–119.

 PubMedGoogle Scholar

Murugesh N, Kumar VR, Vembar S, Damodaran C. 1981. Studies on erythrocyte membrane. V. Haemolytic effect of methylsalicylate and its possible mechanism. Toxicol Lett. 9:225–229.

 PubMed Web of Science ®Google Scholar

Rotilio D, Joseph D, Hatmi M, Vargaftig B. 1984. Structural requirements for preventing the aspirin- and the arachidonate-induced inactivation of platelet cyclo-oxygenase: additional evidence for distinct enzymatic sites. Eur J Pharmacol. 97:197–208.

 PubMed Web of Science ®Google Scholar

Verstraelen S, Nelissen I, Hooyberghs J, Witters H, Schoeters G, Van Cauwenberge P, Van Den Heuvel R. 2009a. Gene profiles of a human alveolar epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis. Toxicol Lett. 185:16–22.

 PubMed Web of Science ®Google Scholar

Verstraelen S, Nelissen I, Hooyberghs J, Witters H, Schoeters G, Van Cauwenberge P, Van Den Heuvel R. 2009b. Gene profiles of a human bronchial epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis. Toxicology. 255:151–159.

 PubMed Web of Science ®Google Scholar

Vigano G, Garagiola U, Gaspari F. 1991. Pharmacokinetic study of a new oral buffered acetylsalicylic acid (ASA) formulation in comparison with plain ASA in healthy volunteers. Int J Clin Pharm Res. 11:129–135.

 PubMedGoogle Scholar

Wolowich WR, Hadley CM, Kelley M, Walson P, Casavant M. 2003. Plasma salicylate from methyl salicylate cream compared to oil of wintergreen. J Toxicol-Clin Toxic. 41:355–358.

 PubMed Web of Science ®Google Scholar

Zhang Z, Jia C, Hu Y, Sun L, Jiao J, Zhao L, Zhu D, LI J, Tian Y, Bai H, et al. 2012. The estrogenic potential of salicylate esters and their possible risks in foods and cosmetics. Toxicol Lett. 209:146–153.

 PubMed Web of Science ®Google Scholar

Morrissey RE, Lamb J, Morris R, Chapin R, Gulati D, Heindel J. 1989. Results and evaluations of 48 continuous breeding reproduction studies conducted in mice. Developmental and Reproductive Toxicology Group, NIEHS, NTP Program, Research Triangle Park, North Carolina, Analytical Sciences Inc., Durham, North Carolina, Environmental Health Research and Testing, Inc., Lexington, Kentucky. Fund Appl Toxicol. 13:747–777.

 Google Scholar

Webb WK, Hansen WH. 1963. Chronic and subacute toxicology and pathology of methyl salicylate in dogs, rats, and rabbits. Toxicol Appl Pharm. 5:576–587.

 PubMed Web of Science ®Google Scholar

Packman EW, Abbott DD, Wagner BM, Harrisson JWE. 1961. Chronic oral toxicity of oil sweet birch (methyl salicylate). Pharmacologist. 3:62 (As cited in CIR 2003).

 Google Scholar

[NFSA] Norwegian Food Safety Authority. 2012. Risk profile methyl salicylate CAS no. 119-36-8.

 Google Scholar

Abbot DD, Harrisson JWE. 1978. Methyl salicylate: studies of osseous changes in the rat, reproduction in the rat and mouse, and liver and kidney effects in the dog. Unpublished communication to the Flavor and Extract Manufacturers Association (As cited in CIR 2003, NFSA 2012).

 Google Scholar

Rudolph AM. 1981. Effects of aspirin and acetaminophen in pregnancy and in the newborn. Arch Intern Med. 141:358–363.

 PubMedGoogle Scholar

Opdyke DL. 1979a. Monographs on fragrance raw materials. Food Cosmet Toxicol. 17:357–390.

 PubMedGoogle Scholar

Opdyke DL. 1979b. Monographs on fragrance raw materials. Food Cosmet Toxicol. 17:(Special Issue V):695–923.

 PubMedGoogle Scholar

Rainsford K. 2004a. History and development of the salicylates. In: Rainsford K, editor. In aspirin and related drugs. New York: Taylor and Francis; p. 1–24.

 Google Scholar

Rainsford K. 2004b. Pharmacology and biochemistry of salicylates and related drugs. In: Rainsford K, editor. In aspirin and related drugs. New York: Taylor and Francis; p. 215–366.

 Google Scholar

Rainsford K. 2004c. Side effects and toxicology of the salicylates. In: Rainsford K, editor. In aspirin and related drugs. New York: Taylor and Francis; p. 367–554.

 Google Scholar

Opdyke DL. 1978. Acute oral toxicity: rat; acute dermal toxicity: rabbit; and primary dermal irritation: rabbit for methyl salicylate, fragrance raw materials monographs. Food Cosmet Toxicol. 16:821–825.

 Google Scholar

[NRC] National Research Council of the National Academies. 2014. Review of EPA’s Integrated Risk Information (IRIS) Process. Committee to Review the IRIS Process Board on Environmental Studies and Toxicology Division on Earth and Life Studies.

 Google Scholar

[USEPA] United States Environmental Protection Agency. 1985. Reference values for risk assessment. United States Environmental Protection Agency, Washington, (DC): Environmental Criteria and Assessment Office.

 Google Scholar