Accepted Manuscript
Toxicological characteristics of edible insects in China: A historical review
Yu Gao, Di Wang, Meng-Lei Xu, Shu-Sen Shi, Jin-Feng Xiong
PII: S0278-6915(18)30218-7 DOI: 10.1016/j.fct.2018.04.016 Reference: FCT 9705
To appear in: Food and Chemical Toxicology
Received Date: 26 January 2018 Revised Date: 1 April 2018 Accepted Date: 7 April 2018
Please cite this article as: Gao, Y., Wang, D., Xu, M.-L., Shi, S.-S., Xiong, J.-F., Toxicological characteristics of edible insects in China: A historical review, Food and Chemical Toxicology (2018), doi: 10.1016/j.fct.2018.04.016.
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1
2 Toxicological Characteristics of Edible Insects in China: A historical review
3 Yu Gao a, Di Wang a, Meng-Lei Xu b*, Shu-Sen Shi a* , Jin-Feng Xiong c
4
5 a College of Agriculture, Jilin Agricultural University, Changchun, 130118, P. R.
6 China
7 b State Key Laboratory of Supramolecular Structure and Materials, Jilin University,
8 Changchun, 130000, P. R. China
9 c Changchun Institute of Biological Products Co. Ltd., Changchun, 130012, P. R.
10 China
11 12 *Co-Corresponding authors: MANUSCRIPT 13 Meng-Lei Xu, [email protected]
14 Shu-Sen Shi, [email protected]
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15 Abbreviations
16 ED 50 , median effective doses;
17 SOD, superoxide dismutase;
18 MDA, malondialdehyde;
19 MTD, maximum tolerated dose;
20 PSP, protein from silkworm pupae;
21 LD 50 , median lethal dose;
22 MNEL, maximal no-effect level;
23 ADI, acceptable daily intake.
24
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25
26 Abstract : Edible insects are ideal food sources, which contain important nutrients and
27 health-promoting compounds. With a rapid development of industrial insect farming,
28 insect-derived food is a novel and emerging food industry. Edible insects have been
29 traditionally consumed in various communities, while continuously gaining relevance
30 in today's society; however, they currently remain underutilized. Although there are a
31 large number of literatures on edible insects, these literatures primarily focus on the
32 nutritional value edible insects. The toxicity assessment data of edible insects remain
33 incomprehensive, especially for the new national standard that is currently in effect;
34 and many data and conclusions are not accurately specified/reported. Therefore, we
35 performed a literature review and summarized the data on the toxicological 36 assessment of edible insects in China. The review MANUSCRIPT first describes the research progress 37 on safety toxicological assessment, and then offers references regarding the
38 development of 34 edible insect species in China. These data can be a platform for the
39 development of future toxicological assessment strategies, which can be carried out
40 by a multidisciplinary team, possibly consisting of food engineers, agronomists,
41 farmers, and so on, to improve the acceptability of edible insects.
42
43 Keywords : ACCEPTEDEdible insects; Toxicological assessment; China; Food safety
44 ACCEPTED MANUSCRIPT
45
46 Highlights
47 1. 34 edible insect species with long history in China are reviewed based on
48 toxicology assessment.
49 2. Toxicological assessment methods and testing procedures are briefly
50 summarized.
51 3. Traditional edible insects in China are safe and can potentially be a food
52 resource.
53 TOC
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54 55 ACCEPTED ACCEPTED MANUSCRIPT
56 1. Introduction
57 Edible insects have been part of the cultural and genetic heritage in different
58 regions worldwide (Anankware et al., 2015; Belluco et al., 2015). With the increased
59 demand of animal-derived food to feed a continuously growing global population,
60 insects have great potential in dealing with the food crisis because of their diversity,
61 apparent abundance, and low environmental influence required for high-scale
62 production (Yen, 2015; Huis, 2016; Payne et al., 2016). Although edible insects are
63 currently underutilized, they are still consumed traditionally by different communities
64 while gaining relevance in today's society (Domingo, 2017). Thus, edible insects have
65 been valued and dedicated as a source of food by the Food and Agricultural
66 Organization of the United Nations (FAO) in 2013. 67 Edible insects have historically been consumedMANUSCRIPT in China for more than 2000 68 years owing to their medicinal and trophic values (Feng et al., 2018). The most
69 commonly consumed insects includes caterpillars, beetles, bee, wasps, crickets, ants,
70 termites, and flies (Zhang et al., 2008; Chen et al., 2009). In China, food safety, which
71 plays a central role in providing a guidance for the suitability of food for human
72 consumption, is one of the most challenging social issues that need to be addressed
73 (Wu and Chen, 2013; Nongonierma and Fitzgeral, 2017). However, the scientifically
74 based knowledgeACCEPTED to ensure safety of insect food processing, especially in the
75 industrial scale, is still lacking (Schlüter et al., 2017). For instance, the Chinese oak
76 tasar moth ( Antheraea pernyi ), which has traditionally been used as food as well as
77 traditional medicine in some Asian countries, has recently been listed as a novel ACCEPTED MANUSCRIPT
78 common food source by the Ministry of Health P. R. China (Zhu, 2004; Shao et al.,
79 2014). Possible hazards from insects are contaminants such as heavy metals, toxins,
80 pesticide residues, and pathogens (Huis, 2015). Currently, most edible insects have
81 not been incorporated into policy documents, while have largely been omitted from
82 the regulatory frameworks. Moreover, in some nations where there are traditions of
83 consuming edible insects, the edible insects do not explicitly appear in their dietary
84 guidelines (Halloran et al., 2015). For this reason, some organizations and researchers
85 have proposed that edible insects should be systematically evaluated by a series of
86 toxicological assessments to ensure its safety as a new available food resource (Zhou
87 and Han, 2006; Poma et al., 2017).
88 Although the nutrient components of edible insects have been largely discussed 89 in a work by Feng et al. (2016), its toxicological MANUSCRIPT data are still incomprehensive. 90 Therefore, this article reviews the toxicological characteristics of edible insects in
91 China. The insect species were selected based on toxicological data recalled from
92 memory, such as traditional knowledge and practices in the use of edible insects.
93
94 2. Toxicity Assessment methods and testing procedures
95 Toxicology assessment is a judgment process based on the data/results obtained
96 from standardACCEPTED testing procedures. The standardization of food toxicology testing and
97 assessment in China began in 1980s. The Ministry of Health has promulgated a draft
98 of 'Procedures for toxicological assessment on food safety' in 1994, while national
99 standard 'Procedures for toxicological assessment of food' (GB 15193–1994, GB ACCEPTED MANUSCRIPT
100 15193–2003) in 2003 (Ministry of Health P. R. China, 2003). After the Food Safety
101 law went into effect and the toxicology technology was developed worldwide, all
102 toxicological assessments were complied with these laws and regulations. The
103 National Health and Family Planning Commission of P. R. China
104 (http://www.nhfpc.gov.cn/) gathered experts to revise the above standards, resulting in
105 the current version of 'Procedures for toxicological assessment of food' (GB
106 15193.1–2014). This new standard not only defines the contents of tests for
107 toxicological assessment, the principle of different subjects, the purpose and results
108 determination, and the factors to be considered in the assessment, but also contains
109 improved scientificity and universality. Whereas sperm deformity test was
110 discontinued due to its non-universality, sex-linked recessive lethal test was directly 111 transferred to new version. Nineteen standards MANUSCRIPT were revised and 6 new standards were 112 developed to fill in the technological gap (Table S1). This China's food toxicology
113 assessment procedures and methods is a mandatory standard that is a particularly
114 important part of the national food safety standards.
115 The food toxicity tests set by this standard include: acute toxicity test,
116 genotoxicity test, twenty-eight days oral toxicity test, ninety days oral toxicity test,
117 teratogenicity study, reproductive toxicity test and reproductive development toxicity
118 test, toxicokinetics,ACCEPTED chronic toxicity test, carcinogenicity tests and combined chronic
119 toxicity with carcinogenicity studies (Fig. S1). The genotoxicity tests should follow
120 the principles, which combined prokaryotic and eukaryotic cells, both in vivo and in
121 vitro . According to the characteristic and the aim of the test, it is recommended as ACCEPTED MANUSCRIPT
122 follows:
123 ‹ Group one: Bacterial reverse mutation assay, mammalian erythrocyte
124 micronucleus test or mammalian bone marrow cell chromosome aberration test,
125 mice spermatogonium/spermatocyte chromosome aberration test or rodent
126 dominant lethal test.
127 ‹ Group two: Bacterial reverse mutation assay, mammalian erythrocyte
128 micronucleus test or mammalian bone marrow cell chromosome aberration test,
129 in vitro mammalian chromosome aberration test or TK gene mutation in
130 mammalian cells test in vitro . The bacterial reverse mutation assay includes two
131 different tests: (i) Salmonella typhimurium reverse mutation assay (Ames test);
132 and (ii) Escherichia coli reverse mutation assay. 133 Alternative genotoxicity tests: the sex-linked MANUSCRIPT recessive lethal test, DNA damage 134 and repair (Unscheduled DNA synthesis) test in mammalian cells in vitro , or HGPRT
135 gene mutation test.
136 Basic problems of the tests are detailed, and provisions are made specific to
137 facilitate the standardization of food toxicology assessment research in laboratory.
138 Whether it is a guideline or a mandatory standard, the objectives of food safety
139 toxicology assessment in different countries and agencies are indistinguishable: to
140 obtain comprehensiveACCEPTED toxicological information by conducting toxicological tests in
141 different combinations, so as to predict the safe exposure dose of the substance being
142 evaluated.
143 ACCEPTED MANUSCRIPT
144 3. Major groups of edible insect species
145 Toxicity of edible insects has not been given sufficient attention and/or
146 investigation in the context of modern food industry. It may not only be related to
147 dietary traditions, but also time-consuming and complex procedures/methods with
148 high costs and other unknown factors. Additionally, the basic research on the
149 toxicological assessment is presently and relatively weak. Due to the absence of
150 systematic assessment standards and/or procedures that are specific to edible insects,
151 the toxicological assessment of edible insects have generally been implemented in
152 reference to common agricultural products or animal food. Thus, only less than 34
153 insect species, belonging to Lepidoptera, Coleoptera, Orthoptera, Hemiptera,
154 Hymenoptera, Blattodea, Diptera, and Amorphosceloidea, have been assessed by 155 toxicology studies (Fig. 1). Although these studiesMANUSCRIPT are heterogenous, which is thus 156 difficult to compare, the data on the food safety of insects indicate that these insects
157 are safe for consumption (Halloran et al., 2016) (Table 1).
ACCEPTED
158
159 Fig. 1 Examples of edible insects that have been toxicologically evaluated in China ACCEPTED MANUSCRIPT
160 3.1 Lepidoptera (Moths)
161 3.1.1 Bombyx mori
162 Silkworm Bombyx mori (Lepidoptera: Bombycidae) is an economically
163 important insect primary used in silk production (Rockwood et al., 2011). The
164 cultivation and utilization of B. mori , which is originated from China, has a long
165 history of more than 5000 years. Recent toxicity tests showed that most B. mori and
166 its relevant products (such as body fluid, graine powder, and body fluid) had no acute
167 toxicity or mutagenicity in bone marrow cell micronucleus of mice or rats (Duan et al.,
168 2000). In contrast, Gu et al. (2009) reported that one mouse gavagely administered
169 with female B. mori moth powder constantly laid down and reacted slowly; it,
170 however, was able to restore its normal after 2-4 h. The other mouse appeared 171 flatulence while recovered after 2 h. MANUSCRIPT 172 The larva of B. mori parasitized by a fungus species, Beauveria bassiana is
173 called batryticated silkworm. The median effective doses (ED 50 ) of cancer cell lines
174 L1210, P388, and SNU-1 with respects to batryticated silkworm were 13.80, 19.95,
175 and 31.62 µg/kg, respectively (Huang et al., 1997). The studies also showed that
176 silkworm and its related products increased superoxide dismutase (SOD) activity
177 while decreased malondialdehyde (MDA) content, which is linked with antioxidant
178 activity. ACCEPTED
179 3.1.2 Antheraea pernyi
180 Chinese oak tasar moth, Antheraea pernyi (Lepidoptera: Satumiidae), which feed
181 on plantations of specially trimmed oak trees, Quercus mongolica (Fagaceae), is used ACCEPTED MANUSCRIPT
182 in the production of brownish silk as well as for food in the Northeastern China. In
183 recent years, the amount of pupae consumption has surpassed tasar silk yield, which
184 indicates that tasar has increasingly been utilized as food. An acute test illustrated that
185 the oral maximum tolerated dose (MTD) of protein from silkworm pupae (PSP) for
186 KM mice was > 15.0 g/kg·bw (Zhou and Han, 2006). In addition, acute toxicity was
187 not observed in rats administrated with 20.0 g/kg of tasar larvae powder (Tian et al.,
188 2012), or in KM mice administered with 18 g/kg of male tasar moth powder (Lv et al.,
189 2016). Furthermore, toxicity or death was not found in mice administered with skim
190 tasar pupae protein powder at a dose range of 2.15-21.50 g/kg·d (Zhang et al., 1991).
191 Tasar moth pupae play an extremely important role in rural society because it is a
192 readily available source of protein; thus its various products have been reported. 193 According to one acute toxicity test, tasar functio MANUSCRIPTnal food showed no toxicity (median 194 lethal dose (LD 50 ) > 10 g/kg·bw) to aged Wister rats (Zhao and Liu, 1995). The
195 toxicity test carried out using amino acids hydrolyzed from the tasar moth pupae also
196 showed LD 50 = 21.5 g/kg·bw (Chen et al., 1993). A 30-day feeding test showed that
197 death or abnormal hematology, clinical chemical and histopathological changes, and
198 clinical signs were not found in rats administrated with 0.30–1.5 g/kg of PSP (Zhou
199 and Han, 2006). Zhou et al. (2004) also conducted acute and 30-day feeding tests on
200 tasar moth pupae–ACCEPTEDLigustri lucidum (Oleaceae) compound capsule. Whilst the acute
201 test showed that the compound was non-toxic with LD50 > 15 g/kg, the 30-d feeding
202 test indicated that no hematological abnormality, and clinic chemical and
203 histopathological changes were induced; and a maximal no-effect level (MNEL) ACCEPTED MANUSCRIPT
204 was > 15 g/kg and the acceptable daily intake (ADI) was 0.90 g/60 kg. Protein from
205 silkworm pupae (PSP) and male tasar moth powder at doses of 0.5–3 and 2.5–10 g/kg,
206 respectively had no mutagenicity with negative results in genotoxicity test (Zhou and
207 Han, 2006; Lv et al., 2016). The skim tasar pupae protein powder and male moth
208 extract were also found to be non-mutagenic (non-genotoxicity) both in vitro and in
209 vivo mutation tests (Zhang et al, 1991; Zhao and Liu, 1995). The tests also showed
210 that amino acids hydrolyzed from tasar moth pupae were non-acute, non-genotoxic
211 (Chen et al., 1993). To estimate the safety of novel food with long consumption
212 history, the 90-day toxicity test was also conducted. The growth as well as parts of
213 physiological and biochemical indexes of male and female rats fed with 0–6 g/kg bw
214 of tasar moth powder for 90 d were not significantly different from control group (Lv 215 et al., 2014). Mice dietarily administered with MANUSCRIPT 1%, 3%, and 10% skim tasar pupae 216 protein powder for 90 d showed no hematological, clinic chemical and
217 histopathological changes. The results also showed that low fat albumen powder had
218 MNEL of 10 g/kg (Liu et al., 1992). According to the results of 90-day feeding test
219 reported in Chen et al. (1993), the ADI of amino acids hydrolyzed from tasar moth
220 pupae was 1.29 g/kg. The data from acute toxicity test, 30-day feeding test,
221 genotoxicity test, mutation test, and 90-day feeding test indicate that the toxicity of
222 silkworm pupaeACCEPTED and its relevant products is not definite.
223 3.1.3 Philosamia cynthia cynthia
224 Castor silkworm, Philosamia cynthia cynthia (Lepidopter: Saturniidae) is also
225 used in the production of silk. It mainly feed on Ailanthus altissima (Simaroubaceae), ACCEPTED MANUSCRIPT
226 Sapium sebiferum (Euphorbiaceae), Ricinus communis (Euphorbiaceae), and Ilex
227 chinensis (Aquifoliaceae), as well as other trees that are widely distributed in China.
228 Yan et al. (1996) have shown that castor silkworm is rich in protein, especially for
229 essential amino acids. According to acute toxicity test, death or abnormal was not
230 observed in mice administered with 18 g/kg bw of P. cynthia cynthia pupae for 7 d.
231 3.1.4 Philosamia cynthia ricini
232 Eri-silkworm (or cassava-silkworm), Philosamia cynthia ricini (Lepidopter:
233 Saturniidae) is originated from Assam, India. It was first introduced into Kaohsiung
234 city, Taiwan Province of China in 1938 and thereafter into the Mainland China in
235 1940s. Eri-silkworm is a subspecies of P. cynthia , whose appearance is highly similar
236 to castor silkworm. Acute toxicity test showed that the MTD of mouse to the pupae 237 was greater than 16 g/kg bw, which indicates MANUSCRIPT that cassava silkworm pupae is 238 non-toxic. On the other hand, genotoxicity test demonstrated that when pupae
239 concentration was higher than 0.25 g/mL, the growth rate of broad bean root tip
240 micronucleus was significantly increased compared with that of control. The pupae (at
241 doses of 5 g/kg bw in male mice and 2.5 g/kg bw in female mice) caused the growth
242 rate of mice bone marrow micronucleus significantly increase. In addition, male mice
243 exposed to 5 g/kg bw of pupae, had sperm aberration with increased distortion rates.
244 Whereas theACCEPTED acute toxicity test of pupae was negative, the genotoxicity test of
245 high-dose pupae was positive (Li et al., 2017).
246 3.1.5 Dendrolimus punctatus
247 Pine caterpillar, Dendrolimus punctatus (Lepidoptera: Lasiocampidae), is a ACCEPTED MANUSCRIPT
248 serious forestry pest in South China. Oral acute toxicity test illustrated that D.
249 punctatus had a maximum dose of 10 g/kg and higher amount of proteins were
250 extracted from its larva in 1 d; and it did not cause death or other abnormalities to
251 mice. Additionally, other mortalities were not observed after two weeks of
252 observation. Protein extracted from the larva of D. punctatus, using a buffer solution
253 method, had LD 50 > 12.05 g/kg. Therefore, these evidences indicate that D. punctatus
254 and its products are non-toxic (Liu and Wei, 2008).
255 3.1.6 Aglossa dimidiata
256 Black rice worm or Aglossa dimidiata (Lepidoptera: Pyralidae), unlike other
257 edible insects, is an insect used in tea production, in which the larval excreta is used
258 (not the insect body) (Seabrooks and Hu, 2017). The larvae feed a wide range of trees, 259 such as Malus sieboldii (Rosaceae), Machilus MANUSCRIPT chuanchienensis (Lauraceae), or 260 Machilus rehderi (Lauraceae), etc. The larval excreta was collected and processed
261 into a so-called 'insect tea' after a series of special processing techniques. Insect tea,
262 also locally known as ‘dragon-pearl tea’, is not only a popular traditional drink for the
263 ethnic minority communities in Hunan, Guizhou, and Guangxi Provinces, but also one
264 of China's exported traditional products (Xu et al., 2013; Zhou et al., 2014). When tea
265 is produced from the larval excreta of A. dimidiata that feed Malus sieboldii
266 (Rosaceae), ACCEPTED it is called ‘Sanye insect tea’. Acute toxicity test demonstrated that the
267 larval excreta was not toxic with LD 50 > 10 g/kg in rats, and Ames test using the
268 micronucleus of mouse’s bone marrow cell also showed that it had no genotoxicity
269 (Wen et al., 1996 and 2004). A 30-day feeding test was conducted using rats dietarily ACCEPTED MANUSCRIPT
270 administered with 4.0 - 8.0 g/kg water of liquid extract from larval excreta (1 g larval
271 excreta in 1 mL water extract). The results showed that the rats were not death, and
272 other hematological, clinical chemical and histopathological abnormalities, and/or
273 clinical signs were not observed (Yi et al., 2007). Wen et al. (2004) also reported that
274 the toxicity of larval excreta at a dose of 10 g/kg was not observed in mice through
275 micronucleus test, teratogenicity study, and Ames test. In addition, when tea is made
276 from the larval excreta of A. dimidiata that feed achilus chuanchienensis (Lauraceae),
277 or Machilus rehderi (Lauraceae), it is called ‘Guizhou white insect tea’. Acute toxicity
278 test the larval excreta in KM mice showed that it had no acute toxicity with LD 50 >
279 5.0 g/kg, while genotoxicity test illustrated that it had no mutagenicity (Yang and Yi,
280 2010). 281 3.1.7 Pyralis farinalis MANUSCRIPT 282 Pyralis farinalis (Lepidoptera: Pyralidae), which feeds Litsea coreana
283 (Lauraceae), is also an insect used in the production of insect tea. Acute oral test
284 indicated that it had a maximum LD 50 > 30 g/kg bw, while micronucleus test
285 illustrated that the results obtained from the treating group and the control group was
286 not remarkably different. These data demonstrate that the larval excreta, which are
287 used in the production of P. farinalis -Litsea coreana insect tea, are non-toxic and
288 non-mutagenicACCEPTED (Wang et al., 2017a).
289 3.1.8 North caterpillar fungus
290 Cultured Cordyceps militarisi (Hypocreales: Cordycipitaceae) is widely used as a
291 traditional medicine in China for the treatment of a variety of diseases owing to its ACCEPTED MANUSCRIPT
292 biological effects (Yu et al., 2006). Acute test showed that MTD of 20 g/kg was
293 observed in rats administered with C. militarisi for 14 d, in addition to LD 50 > 10 g/kg
294 bw, which indicates that the insect is non-toxic (Che, 2003). Gao et al. (2012)
295 described that north caterpillar fungus caused no acute toxicity to rats (in a 14-day test
296 period) with MTD > 10.2 g/kg bw. In addition, it was evident from the in vitro and in
297 vivo mutation tests in rats that north caterpillar fungus at doses of 1.6 - 6.7 g/kg bw
298 was non-mutagenic or non-genotoxic. And 30-day feeding test also showed that rats
299 administered with 0.63 - 2.5 g/kg bw of north caterpillar fungus were not death and
300 abnormal hematological, clinical chemical and histopathological changes, and clinical
301 signs were not found. Additionally, the toxicity to the reproductive system as well as
302 eating, growth, and development abnormalities in the rats were not observed (Che, 303 2003). Gao et al (2012) presented negative results MANUSCRIPT from 30-day feeding test, in which 304 rats were administrated with 1.13 - 4.5 g/kg bw of north caterpillar fungus.
305 Furthermore, the results of acute toxicity test, in which rats and mice were
306 administered with 1.0–10.0 g/kg bw of north caterpillar fungus, exhibited that rats’
307 and mice’s death and/or abnormality were not observed, indicating a lack of toxicity
308 of north caterpillar fungus. Genotoxicity test also indicated that north caterpillar
309 fungus was non-mutagenic. There were no hematological, clinic chemical and
310 histopathologicalACCEPTED changes found in 30-day feeding test with a MNEL of 4.0 mg/kg
311 north caterpillar fungus in mice and rats (Gong et al., 2003 and 2004), while 90-day
312 feeding test revealed that north caterpillar fungus at a high dose of 8 g/kg bw had
313 inhibitory effects on growth and development of young rats, and caused kidney injury; ACCEPTED MANUSCRIPT
314 however, no toxicity effects were observed at low dose (Xie, et al., 2007). It is evident
315 that further studies using other animal models are needed to prominently identify the
316 toxicity of North caterpillar fungus.
317 3.1.9 'Jiuzhou' caterpillar fungus
318 Clanis bilineata tsingtauica (Lepidoptera: Sphingidae) is a type of legume pest
319 that can be divided in to groups: (i) 'Jiuzhou' caterpillar fungus refers to Cordyceps
320 Kyushuensis (Hypocreales: Cordycipitaceae) that grows on larvae of Clanis bilineata
321 tsingtauica is called (Guo and Li, 2000); an (ii) 'Taishan' caterpillar fungus refers to
322 Cordyceps taishanensis (Hypocreales: Cordycipitaceae) that grows on larvae of
323 Clanis bilineata . Chen et al. (2005) conducted an experiment to test the effects of
324 'Jiuzhou' caterpillar fungus on the longevity of fruit fly (Diptera: Tephritidae). The 325 results showed that 'Jiuzhou' caterpillar fungus MANUSCRIPT at concentrations of 0.5 - 2.0% in the 326 culture medium affected the life span of fruit fly, and the effects were more prominent
327 in female fly than in male fly. Nonetheless, the toxicity of 'Jiuzhou' caterpillar fungus
328 has rarely been reported.
329 3.1.10 'Xinjiang' caterpillar fungus
330 Paecilomyces tenuipes (Clavicipitales: Clavicipitaceae), an anamorph of
331 Cordyceps takaomontana , which grows on the pupae of Hepialus spp. (Hepialidae:
332 Hepialus ), isACCEPTED called 'Xinjiang' caterpillar fungus. Pan et al. (2009) reported that acute
333 toxicity of artificial fermentation mycelium of P. tenuipes (LD 50 > 10 g/kg) was not
334 observed in both rats and mice. Furthermore, the observation showed that
335 micronucleus rate of polychromatic erythrocytes, sperm aberration rate, weight, food ACCEPTED MANUSCRIPT
336 utilization, and blood biochemical indexes of mice infected with artificial
337 fermentation mycelium of P. tenuipes were not significantly different from the control
338 group. On the other hand, Che et al. (2014) reported that subchronic oral exposure of
339 P. tenuipes at a concentration of higher than 0.5 g/kg·bw may induce kidney
340 abnormality. These evidences suggest that further studies, possibly by using other
341 animal models, are still needed to better identify the toxicity of P. tenuipes .
342 3.2 Coleoptera (Beetles)
343 3.2.1 Tenebrio molitor
344 Mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae), is a common
345 pest that infests stored food products in many regions. Owing to its high nutritional
346 value, mealworm beetle can be used as food in various forms, such as protein powder, 347 oil, chitin, or it can be consumed directly as an MANUSCRIPT edible worm (Ghaly and Alkoaik, 2009; 348 Siemianowska et al., 2013; Grau et al., 2017; Lee et al., 2017). Toxicity test in KM
349 mice revealed that T. molitor larvae had no acute toxicity or genotoxicity (Yang et al.,
350 1999). Additionally, toxicity test in SD rats orally fed freeze-dried powder of T.
351 molitor larvae for 28 d indicated that it was not mutagenic or clastogenic (Han, et al.,
352 2014). While subchronic toxicity was not observed for 90 d, secretion from
353 alimentary tract/gland indicated that it might have low toxicity (Zhou et al., 1996).
354 Another studyACCEPTED described that the viscera of T. molitor needed to be removed in order
355 to remove larvae toxin (Chen and Wang, 1997).
356 3.2.2 Massicus raddei
357 Oak longhorn beetle, Massicus raddei (Coleoptera: Cerambycidae), is widely ACCEPTED MANUSCRIPT
358 spread in the forest zone of the northeastern China. It is generally used as food, owing
359 to its high nutritional value, such as abundant protein and amino acids (Song, 2012). A
360 study carried out using oak longhorn beetle mixed with mice’s diet at 0 - 100%
361 indicated that it had no acute or subchronic toxicity, as well as genotoxicity (Li, and
362 Wang, 2011). Moreover, mice fed with diet containing 25% of oak longhorn beetle
363 had normal biochemical indexes, such as serum total cholesterol, three acylglycerol,
364 albumin, urea nitrogen, blood glucose, alanine aminotransferase, alkaline phosphatase,
365 and so on. Furthermore, the nonspecific immune functions of mice fed with oak
366 longhorn beetle powder were improved, which indicates that oak longhorn beetle
367 powder has immunomodulatory effect (Wang, 2011).
368 3.2.3 Mysore thorn borer 369 Mysore thorn borer is the larva of longhornMANUSCRIPT beetle, Anoplophora chinensis 370 (Coleoptera: Cerambycidae) or Apriona germarii, and its related species boring into
371 the stem and root of Caesalpinia decapetala . Xu (2013a) carried out acute,
372 genotoxicity, and micronucleus toxicity tests on mysore thorn borer. His results
373 showed that mysore thorn borer was not toxic to mice and rats; and as a result of in
374 vitro and in vivo mutation tests using MTD of 20.0 g/kg, it was found to be
375 non-mutagenic or non-genotoxic. Zhang (2013) also demonstrated that no toxicity or
376 death were foundACCEPTED in mice gavagely administered with 5.0 g/kg of mysore thorn borer,
377 and mice's autonomic activity were promoted compared with control group.
378 3.2.4 Palembus dermestoides
379 Palembus dermestoides (Coleoptera: Tenebrionidae) is used as a material in ACCEPTED MANUSCRIPT
380 traditional Chinese medicine, as well as in food and feedstuff. Moreover, it has been
381 used as medicine and healthcare food product in the treatment of many types of
382 diseases (Xu, 2008).Chu and Yan (2008) described the anti-aging effects of dietary
383 supplement containing P. dermestoides on the antioxidant activities in mice exposed
384 to D-Galactosamine.
385 3.2.5 Holotrichia diomphalia
386 Holotrichia diomphalia (Coleoptera: Melolonthidae) is an underground pest that
387 is seriously spread in Northern China, and is the dominant species of white grubs in
388 Northeastern China. Grubs are not only edible, but also medicinal with
389 anti-inflammatory, analgesic, and antioxidant activities (Oh et al., 2003; Liu, et al.,
390 2012). Acute toxicity test in mice illustrated that petroleum ether-extracted grubs had 391 LD 50 = 48.73 g/kg. Additionally, mice exposed MANUSCRIPT to petroleum ether-extracted grubs had 392 higher SOD activity and lower MDA content than the control group (Jin et al., 2007).
393 An in vivo test further showed that the grub extracts inhibited growth rate of H22
394 hepatocellular carcinoma, which was grafted onto healthy mice; many lymphocyte
395 infiltrations and necrosis were also observed (Yang et al., 2006). Functions of
396 different components of grubs (e.g., polysaccharide) have also been discussed: Kan et
397 al. (2009) isolated five different types of polysaccharide and demonstrated that two
398 out of the fiveACCEPTED components could significantly improved the proliferation of mice’s
399 immunocytes in vitro .
400 3.3 Orthoptera (Locusts, Grasshoppers and Mole crickets)
401 3.3.1 Oxya chinensis and Locusta migratoria migratorioides ACCEPTED MANUSCRIPT
402 Locusts, such as African migratory locust, Locusta migratoria migratorioides
403 (Orthoptera: Oedipodidae) and Chinese rice grasshopper, Oxya chinensis (Orthoptera:
404 Catantopidae), which are rich in proteins and micronutrients, are the most commonly
405 consumed insects (Osimani et al., 2017). Although these insects have historically been
406 consumed, no relevant toxicological data has been reported. Nevertheless, a number
407 of articles increasingly report that the insects can be used as functional food, which
408 can lower blood lipid while exerts anti-oxidant activity (Xu, 2013b; Liu and Duan,
409 2007).
410 3.3.2 Gryllotalpa orientalis
411 Oriental mole cricket Gryllotalpa orientalis (Orthoptera: Gryllotalpidae) is
412 commonly known and found in Asia. It is a polyphagous pest that damages crops by 413 gnawing their roots. One report suggested that MANUSCRIPT mole cricket extracts could be used as 414 anti-oxidant and/or anti-inflammatory agents (Heo et al., 2008). An in vitro test also
415 indicated its anti-tumor activity on three types of human liver cancer cells (HepG2,
416 BEL7402, and SMMC772), or three types of human cervical cancer cells (HeLa,
417 Caski, and C-33A) (Zi et al., 2017).
418 3.4 Hemiptera (Cicadas, Aphids, True bugs)
419 3.4.1 Ericerus pela
420 ChineseACCEPTED white wax scale, Ericerus pela (Hemiptera: Coccoidae) is economically
421 significant scale insect because of its role in wax production (Feng et al., 2016): in the
422 white wax producing area, its eggs are used in healthcare products. An acute test
423 illustrated that Chinese white wax scale was not toxic to rats and mice with LD 50 > 10 ACCEPTED MANUSCRIPT
424 g/kg bw. And when administrated at 0 - 5.0 g/kg bw to rats or mice, it showed no
425 mutagenicity. A 30-day feeding test also indicated that it did not induce hematological,
426 and clinic chemical and histopathological changes in rats administered with 0 - 10.0
427 g/kg bw of scale (Feng et al., 2001; Feng, 2005).
428 3.4.2 Cryptotympana atrata
429 Black cicada Cryptotympana atrata (Hemiptera: Cicadidae) is a commonly
430 consumed insect that is also used as an ingredient in the formulation of Chinese
431 medicine. The exuviae of black cicada emerged from the nymph are called ‘cicada
432 slough’. The cytotoxicity of a total extract and a fraction from boiling water of cicada
433 slough against various cancer cell lines (L1210, P388, and SNU-1) was investigated
434 in vitro . The results of the investigation showed that the cicada had high cytotoxicity
–1 435 with median effective dose (ED 50 ) of 1.48, MANUSCRIPT 2.29, and 1.29 µg·mL against L1210, 436 P388, and SNU-1, respectively (Huang et al., 1997). Another experiment also showed
437 that the larvae powder of C. atrata was able to lower blood lipid level as well as
438 postprandial blood glucose level (Sun et al., 2009).
439 3.4.3 Melaphis chinensis
440 Chinese gall is formed from Chinese aphid or Melaphis chinensis (Hemiptera:
441 Eriosomatidae) on the leaf of Chinese sumac or Rhus chinensis (Anacardiaceae)
442 (Feng et al.,ACCEPTED 2016). Huang et al. (1997) investigated the in vitro cytotoxicity of total
443 extract and ethyl acetate-extracted fraction of Chinese gall against cancer cell lines:
444 L1210, P388, and SNU-1. Their findings showed that the samples had ED50 against
445 L1210, P388, and SNU-1 of 0.55, 0.50, and 0.83 µg/kg, respectively. ACCEPTED MANUSCRIPT
446 3.4.4 Aspongopus chinensis
447 Chinese stink bug or Aspongopus chinensis (Hemiptera: Dinidoridae), which is
448 one of the insects in the Pentatomidae family, is mainly distributed in southern China.
449 The insect has been utilized in traditional Chinese medicine, which relieves pain and
450 treats nephropathy for a long period of time (Luo et al., 2012). The serum containing
451 A. chinensis inhibited the proliferation of human colon adenocarcinoma cell line
452 SW480 and improved the expression of apoptosis-associated factors (Fan et al., 2011).
453 Water-extracted A. chinensis inhibited growth of human gastric cancer cell line
454 SGC-7901 and liver cancer cell line HepG2 with half-maximal inhibitory
–1 455 concentration (IC 50 ) of 154.24 and 129.46 µg·mL , respectively (Yu et al., 2015),
456 while ethanol extracts of A. chinensis inhibited by blocking the cell cycle with IC 50 of 457 1281.62 µg·mL –1 and 582 µg·mL –1, respectively MANUSCRIPT (Hou et al., 2013). Death or toxic 458 effect was not observed in rats administrated with ethanol extracts at doses of 0.5 - 1.5
459 g/kg for 6 weeks, and the antioxidant activity in the skeletal muscle of rats was
460 improved, as indicated by longer swimming time (Ren et al., 2013). The observations
461 were explained by that the expression of genes encoding the skeletal muscle of rats
462 was up-regulated by ethanol extract of A. chinensis (Gao et al., 2015). Additionally, A.
463 chinensis could protect rats against Mn-induced reproductive system injury. An
464 experiment ACCEPTED conducted using rats administered with diet containing 5% - 20% A.
465 chinensis extracts for 8–16 weeks showed that the activities of antioxidant enzymes in
466 testis and blood were improved, which indicates possible anti-oxidant activity of A.
467 chinensis extracts; whereas the expression of Bcl-2 and Bax gene was decreased (He ACCEPTED MANUSCRIPT
468 et al., 2016a, 2016b; Fu et al., 2017; Wang et al., 2017b).
469 3.5 Hymenoptera (Bees, Wasps, and Ants)
470 3.5.1 Bee products
471 The history of bee or Apis cerana (Hymenoptera: Apidae) indicates that bee
472 products have been consumed worldwide for more than 3000 years. Other than honey,
473 which has become one among the world most popular insect product, larvae, pupae of
474 bee, propolis, royal jelly, and bee pollen are also use as nutrition in some healthcare
475 food (Giampieri et al., 2018). Various studies have examined the toxicity of propolis,
476 royal jelly, and their related products, in which the general safety of propolis extracts,
477 royal jelly complex capsule, propolis healthcare food, and propolis-based product
478 so-called bee milk soft capsule (containing 30% propolis extracts, 10% royal jelly 479 freeze-dried powder, and 60% carthamus oil) MANUSCRIPT were evaluated (Li et al., 2011; Yan et 480 al., 2005; Fu et al., 2004; Wang et al., 2009). The evaluations showed that all propolis
481 products caused no acute toxicity to mice and rats, which can be categorize as
482 non-toxic grade product. In vitro and in vivo mutation tests using gavagely
483 administered mice and rats further showed that the products were non-genotoxic.
484 3.5.2 Polyrhachis dives
485 Black ant or Polyrhachis dives (Hymenoptera: Formicidae) is mainly used for
486 medicine andACCEPTED general consumption. A scientific name, ‘ Polyrhachis vicina ’, has also
487 been widely used prior to the determination of its exact species (Tang et al., 2015).
488 Therefore, in this review, we consider that the two species are identical. There are a
489 variety of black ant products, such as extracts, powder, capsules, and so on (Wang et ACCEPTED MANUSCRIPT
490 al., 2006). Zhao et al. (1983) conducted acute toxicity test of black ant in mice, which
491 showed that all mice were survived after gastric administration with 66.7 g/kg of
492 black ant extracts. Li et al. (1995a, 1995b, and 1995c) carried out acute toxicity and
493 mutagenicity tests and demonstrated that ant powder had LD 50 of > 10 g/kg bw, which
494 can be considered as non-toxic. Negative results were obtained from genotoxicity test
495 in mice and rats, which indicates that ant powder is non-genotoxic. As a result of
496 14-week feeding test, hematological, and clinic chemical and histopathological
497 abnormalities in tested mice were not observed, and were comparable with the control
498 group. The results from one study showed that P. dives was able to decrease
499 cholesterol and triglyceride levels, while increase high density lipoprotein level in
500 sera, in addition to enhancing the DNA synthesis in testicular cells. According to a 501 180-day feeding test, in which rats were administer MANUSCRIPTed with 1, 4, and 7 g/kg powder of 502 P. dives , the behavioral, physical and physiological indicators of administered rats
503 were not significantly different from those of the control group (Cai et al., 1995).
504 3.5.3 Formica sanguinea
505 Formica sanguinea (Hymenoptera: Formicidae) is used as a medicine for the
506 treatment of rheumatic arthritis and other diseases in China. Acute toxicity test
507 conducted by Li et al. (1996) showed that at a MTD > 22.5 g/kg, mice were un-harm.
508 The results ACCEPTEDfrom 90-day and 180-day feeding tests illustrated that death or abnormal
509 hematological, clinical chemical and histopathological changes, and clinical signs
510 were not found in rats administered with 0.5–2.5 g/kg bw of powder.
511 3.5.4 Formica rufa ACCEPTED MANUSCRIPT
512 Formica rufa (Hymenoptera: Formicidae) is known as red wood ant or horse ant.
513 Nutritional ingredients of F. rufa powder (as described by the Fuhai forestry center,
514 Xinjiang Province) are abundant and reasonably constructed. Yin (2007) described
515 that the immunity of mice administrated with F. rufa powder via phagocytosis was
516 greatly enhanced, and was significantly different from the control group.
517 3.5.5 Polistes mandarinus
518 Hornet nest is built by large yellow wasp or Polistes mandarinus (Hymenoptera:
519 Vaspidae). A fraction of water-extracted hornet nest showed anti-cancer activities
520 against the cancer cell lines L1210, P388, and SNU-1 with ED 50 of 3.31, 2.00, and
521 6.61 µg/kg, respectively (Huang et al., 1997).
522 3.6 Blattodea (Termites and Cockroach) 523 3.6.1 Periplaneta americana MANUSCRIPT 524 American cockroach or Periplaneta americana (Blattodea: Blattidae) is a public
525 health pest that prefers warm, moist, and food-enriched habitats found in the tropical
526 and the subtropical areas (Ogunleye, 2010). Because it generally associates with
527 human dwelling environments and has the ability to dwell in various cracks
528 throughout the house, it can cause a serious public health problem. However, many
529 studies have recently discovered that American cockroach contains a variety of
530 medicinally ACCEPTEDactive ingredients; thus has high medicinal and edible value (Gao et al.,
531 2016; Zhang et al., 2016; Qin et al., 2017). Zhou (2008) carried out acute toxicity test
532 in mice and rats, which showed that P. americana powder was non-toxic with LD 50 >
533 10 g/kg. Furthermore, the results from 30-day feeding test conducted using rats fed ACCEPTED MANUSCRIPT
534 with 0 - 10 g/kg bw showed that P. americana had no significant effects on weight,
535 feed to weight gain ratio, and organ to body weight ratio, as well as hematological and
536 biochemical indexes of rats. While P. americana has positive effects on its other
537 clinical applications, its anti-tumor activity has increasingly been studied (Zhao et al.,
538 2017).
539 3.6.2 Opisthoplata orientalis
540 Li et al. (2013) used acute toxicity test in mice to study Opisthoplata orientalis
541 (Blattodea: Blattellidae) extracted with water, alcohol, and ether leaching method. The
542 observations showed that mice gavagely administered with the three extracts were
543 survived without signs of toxicity. Additionally, all extracts had LD 50 of > 30 g/kg bw,
544 which indicates that they are non-toxic. 545 3.6.3 Macrotermes barneyi MANUSCRIPT 546 Termite is a social cockroach widely used in traditional medicine and as food
547 resources in China (Figueirêdo et al., 2015). Termite in its adult and larvae forms, as
548 well as its nest have high edibility as well as medicinal value, which have a long
549 history of over 3000 years in China (Yan et al., 2008; Igwe et al., 2011). Wu and
550 Wang (1999) conducted acute toxicity test by using homogenized termite to feed mice
551 and rats, and reported that termite had no acute toxicity or mutagenicity with LD 50 >
552 10 g/kg, whichACCEPTED indicates that it is non-toxic. They also found that M. barneyi had
553 anti-fatigue ability, and could delay senescence, reduce the function of serum
554 cholesterol, and improve the rate of lymphocyte transformation in mice.
555 3.6.4 Odontotermes formosanus ACCEPTED MANUSCRIPT
556 Song et al. (1995) reported the results of acute and mutagenesis tests of ant
557 alcohol-extracts, which showed that it had no acute toxicity with LD 50 > 10 g/kg.
558 Additionally, its toxicity was negative in Ames test and micronucleus sperm deformity
559 test using NIH mice and SD rats. Furthermore, Zhang (1999) reported medicinal
560 effects of O. formosanus capsule, which indicate that it could cure the liver and
561 kidney deficiency in mice, in addition to its significant social and economical
562 benefits.
563 3.6.5 Coptotermes formosanus
564 The results from acute and mutagenicity test demonstrated that death was not
565 observed in mice administered with 200 mL/kg of prepared C. formosanus copond
566 (the dose was 400 times higher than that for human). Furthermore, the prepared C. 567 formosanus copond increased SOD and glutathineMANUSCRIPT peroxidase activities, while 568 decreased lipid and lipofuscin content in rats. It was also able to resist to the reduction
569 of leucocyte and speen weight by cyclophosphamidum in mice, while strengthened
570 the immunity and anti-aging ability (Wang et al., 1995).
571 3.6.6 Eupolyphaga sinensis
572 Dried body of Eupolyphaga sinensis (Blattodea: Polyphagidae), which is native
573 to Western China, is used in traditional Chinese medicine as well as a novel food
574 source (Ge ACCEPTEDet al., 2012). It could improve growth rate of rats, especially for female
575 rats. Acute toxicity test indicated that it had LD 50 > 10 g/kg bw in SD rats, while
576 exhibited no toxicity effects. The results from 30-day feeding test showed that adverse
577 effect, as a result of the administration to rats at 4.0 g/kg bw, were not observed (Chen ACCEPTED MANUSCRIPT
578 et al., 2007). Tian (2006) reported that as determined by acute toxicity test in mice,
579 alkaloids from E. sinensis had LD 50 of 0.29 g/kg, which was approximately 528 times
580 higher than the intake dose for human adults. Thus, alkaloids from E. sinensis have
581 moderate toxicity; they are safe and harmless when use at the recommended dose.
582 3.7 Diptera (Flies)
583 Larvae of housefly Musca domestica (Diptera: Muscidae) is an excellent source
584 of high-quality protein, unsaturated fats, polysaccharides, and other nutrients that can
585 be used as human food and/or animal feed (Ren and Shi, 2002). Larvae powder is a
586 potential drug, which can be used to treat lipopolysaccharide-induced atherosclerosis
587 pro-inflammatory responses. The results from various tests, including acute toxicity
588 test, bone marrow cell micronucleus test, mice sperm abnormality test, teratogenicity 589 study, and chronic toxicity test in male and femaleMANUSCRIPT rats, indicated that there were no 590 abnormalities during or after the tests, and larvae powder at doses of > 10 g/kg bw
591 could cause adverse effects (Li et al. 2010).
592 Qin et al. (2009) carried out acute toxicity test of flyblow chitosan in mice, and
593 found that it is non-toxic found with MTD of 2.7 g/kg bw, which was 67.5 times
594 higher than the dose for human. They also recommend that 2 g/kg of housefly is a
595 safe intake dose for human. Ai et al. (2008) showed that chitosan had scavenging
596 activity againstACCEPTED hydroxyl and superoxide radicals, which are similar to ascorbic acid
597 radicals. In addition, low concentration of chitosan also exhibited excellent antifungal
598 activity, while significantly inhibited the growth of Rhizopus stolonifer (Mucorales:
599 Mucoraceae) (Ai et al., 2012; Gou and Min, 2016). ACCEPTED MANUSCRIPT
600 Protein-enriched fraction from the larvae of housefly showed antiviral activity
601 against avian influenza virus: H 1N1 and H 9N2, and had virucidal effect against
602 multicapsid nucleopolyhedrovirus of alfalfa looper (Ai, 2013; Wang et al., 2013).
603 Moreover, it could modulate immune function in mice and possessed excellent
604 scavenging activity toward 1,1-diphenyl-2-picrylhydrazyl radical and superoxide
605 anion radical. Wang et al. (2007) discovered that protein-enriched fraction isolated
606 from the maggots of housefly could protect against carbon tetrachloride
607 (CCl 4)-induced acute hepatic damage in rats. They also showed that the peptide
608 fraction from larvae could act as antitumor agent, possessing immunomodulatory
609 activity. Sun et al. (2014) reported that peptide fraction from larvae could inhibit the
610 growth of S180 sarcoma-transplanted mice by turning on Th1-based protective 611 cell-mediated immunity in mice. Zhu et al. (2013) MANUSCRIPT isolated polypeptides from larva of 612 housefly using common protease, and reported its protective effect against hydrogen
613 peroxide-induced oxidative injury in cells through its ability to decrease intracellular
614 reactive oxygen species and elevate activities of antioxidant enzymes.
615 3.8 Amorphosceloidea (Mantis)
616 Praying mantis is generally known as a natural enemy of pests in agriculture and
617 forestry. A variety of egg sheath produced by the mantis is called ‘mantis egg-case’ or
618 Tenodera sinensisACCEPTED (Amorphosceloidea: Manteidae). It is used as a material in
619 traditional Chinese medicine and healthcare food. Jia et al. (2016) confirmed that both
620 unprocessed and processed mantis egg-case could strengthen the immunologic and
621 oxidation functions in mice; however, the unprocessed mantis egg-case had higher ACCEPTED MANUSCRIPT
622 effect than the processed one.
623
624 4. Future Perspectives
625 Traditional edible insects in China can potentially be a great food resource. The
626 toxicological assessment, which was conducted by acute test, genotoxicity test, and 30
627 days feeding study (according to the 2003 version of national standard in China, Table
628 S1), indicates that most of these insects are non-toxic. The revised 2014 version, in
629 which some tests were directly transferred from the 2003 version, and others were
630 added or discontinued, has improved scientificity and universality. According to the
631 2014 version, edible insects with some negative results from acute toxicity test,
632 genotoxicity test, and 28-day oral toxicity test can be considered safe. In this version, 633 the MTD method in acute toxicity test, the spermMANUSCRIPT deformity test in genotoxicity test, 634 and 30-day feeding study were discontinued. Nonetheless, the basic framework of
635 both versions remains identical. Many tests can be chosen from genotoxicity test
636 except for sperm deformity test. The use of edible insects has a long history in China;
637 a comprehensive review of their toxicological assessment indicates that the traditional
638 edible insects in China are safe and have great potential as novel food resources.
639 However, these edible insects need a more complete toxicological assessment
640 according toACCEPTED the new standard so that they can become more favorable internationally.
641 Currently, the edible insect industry has become the global craze known as 'sun
642 industry', in which the insects are used in the production of so-called novel and
643 healthy food; the industry has broad prospects for development. However, edible ACCEPTED MANUSCRIPT
644 insects remain unpopular worldwide, while are only popular among less developed
645 countries, and this situation may be linked to traditional consumption habits, which
646 are highly regional. Although the consumption of insects has no adverse effects, as
647 future food resources, it is necessary to strengthen their toxicological assessment. The
648 use of pesticide may be reduced, and more efficient pest protection methods may be
649 developed, which may include the use of some insect species, such as locusts, corn
650 borers and pine caterpillars. Furthermore, most edible insects in China have been
651 proven to be a safe food resource. In order to provide a more complete toxicological
652 assessment data for edible insects that have long consumption history, the
653 development of future strategies is needed; this can be carried out by a
654 multidisciplinary team, consisting of food engineers, agronomists, farmers, and so on. 655 With more science-based data on edible insects, MANUSCRIPT they may increasingly be approved 656 and exploited.
657
658 Conflict of Interest
659 The authors declare that there are no conflicts of interest.
660
661 Acknowledgements
662 This workACCEPTED was partially supported by Modern Agricultural Industry Technology
663 System (Grant No. CARS-04), the Open Research Fund of State Key Laboratory of
664 Integrated Pest Management on Crops in Northeast China (Grant No.
665 DB201505KF03), and the Initial Postdoctoral Scientific Research Fund of Jilin ACCEPTED MANUSCRIPT
666 University.
667
668 Supporting information
669 The supporting information provides comparison of national standard (2003 and
670 2014 version) and procedures for toxicological assessment of food in China.
671
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Table 1 Toxicological data of Edible Insects in China
Edible tolerated Common Dose Species stage/ Samples Animals/strains Duration type of test dose Results Ref. name (g·kg -1·bw -1) body parts (g·kg -1·bw -1)
Body fluid KM mice 10 >10 Acute toxicity test (MTD) and bone No acute toxicity, no significant mice Duan, et al., Pupae body fluid KM mice 10 >10 marrow cell micronucleus test micronucleus. 2000 Bombyx mori Silkworm Larvae Graine powder KM mice 0.68 ～17 17 Female moth ICR mice 83 7 d >83 No death in mice or rats, no Acute toxicity test (MTD) Gu, 2009 powder SD rats 34.4 7 d >34.4 abnormalities in organs KM mice 15.0 14 d Acute toxicity test 15 No acute toxicity TA97, TA98, 200 ～5000 µg/plate Ames test No genotoxicity Protein of silkworm TA100, TA102 Zhou and pupae (PSP) KM mice 0.50 ～5.00 5 d Sperm deformity test No sperm malformation Han, 2006 KM mice 0.50 ～3.00 30 d No significant hematological, clinic 30 days feeding study SD rats 0.30 ～1.50 30 d 10 chemical and histopathological changes Tian et al., Tasar larvae powder ICR mice 20.0 14 d Acute toxicity test >20.0 no acute toxicity 2012 KM mice 20.0 g 14 d Acute toxicity test >15.0 no acute toxicity TA97, TA98, 16 ～5000 µg /plate Ames test No genotoxicity Lv et al., TA100, TA102 Male tasar moth 2016 KM mice 2.5 ～10.0 twice, 24 h apart Bone marrow cell micronucleus test no significant mice micronucleus powder KM mice 2.5 ～10.0 35 d Sperm deformity test No sperm malformation Chinese MANUSCRIPT Lv et al., Antheraea (oak) Wistar rats 1.5 ～6.0 90 d 90 days feeding study No subchronic toxicity Pupae 2014 pernyi tussar KM mice 2.15 ～21.5 7 d Acute toxicity test >21.5 No acute toxicity moth TA97, TA98, 1～1000 µg /plate Ames test No genotoxicity Zhang et al., TA100 Skim tasar pupae 1991 KM mice 0.625 ～5 once Bone marrow cell micronucleus test >5 no significant mice micronucleus. protein powder KM mice 1.25 ～5 Sperm deformity test >5 No sperm malformation Liu et al., SD rats 1% ～10% diet 90 d 90 days feeding study No subchronic toxicity 1992 Acute toxicity test, bone marrow cell No acute toxicity, genotoxicity, no Male tasar Zhao and Aged Wistar rats 5g each mouse 6 w micronucleus test and sperm >10 significant in mice micronucleus or functional food Liu, 1995 deformity test sperm malformation tests Tasar moth KM mice 0.25 ml/10 g 14 d Acute toxicity test >15.0 No acute toxicity pupa–Ligustri ACCEPTED Zhou et al., lucidum (Oleaceae) Wistar rats 0.30 ～1.5 30 d 30 days feeding study >15.0 No sub-acute toxicity 2004 compound capsule Philosamia Castor Yan et al., cynthia Pupae P. cynthia pupae mice 18 7 d Acute toxicity test >18 No acute toxicity silkworm 1996 cynthia Philosamia Cassava Pupae P. cynthia ricini KM mice 16 14 d Acute toxicity test (MTD) >16 No acute toxicity Li et al., ACCEPTED MANUSCRIPT
Edible tolerated Common Dose Species stage/ Samples Animals/strains Duration type of test dose Results Ref. name (g·kg -1·bw -1) body parts (g·kg -1·bw -1) cynthia ricini Silkwor pupae Vicia faba root tip Micronucleus rate increased significantl 2017 0.1 ～0.5 g/mL cell micronucleus test m cells when dose higher than 250 mg/mL Male and female mice bone marrow micronucleus rate dose increase KM mice 0.04 ～10 Bone marrow cell micronucleus test significantly when dose higher than 5 g·kg -1·bw -1 and 2.5 g·kg -1·bw -1 respectively Odds of male mice sperm aberration KM mice 0.04 ～10 Sperm deformity test increase significantly in 5 g·kg -1·bw -1 Dendrolimus Pine Proteins Extraction Liu and Wei, Larvae mice 4.005 ～12.0 Once Acute toxicity test >12.05 No acute toxicity punctatus caterpillar from Larva 2008 Wistar rats 10 Acute toxicity test >10 No acute toxicity TA97, TA98, Wen et al., 5～5000 µg/plate Ames test No genotoxicity TA100, TA102 1996 KM mice 0.652 ～2.5 twice, 24 h apart Bone marrow cell micronucleus test No significant mice micronucleus No significant hematological, clinic Yi et al., Larval excreta SD rats 4～8 30 d 30 days feeding study >8.0 "Sanye" chemical and histopathological changes 2007 (Sanye Insect Tea) or Wistar rats 2.15 ～21.5 14 d Acute toxicity test >10 No acute toxicity Aglossa Larval Wen et al., "Laoying TA97, TA98, dimidiata excreta 50 ～5000 µg /plate MANUSCRIPT Ames test No genotoxicity 2004 " insect TA100, TA102 tea KM mice 0.652 ～2.5 5d Bone marrow cell micronucleus test No significant mice micronucleus
KM mice 4.3 ～21.5 5 d Sperm deformity test No sperm malformation KM mice 5.0 14 d Acute toxicity test >5.0 No acute toxicity Larval excreta TA97, TA98, Yang and (Guizhou White 8～5000 µg /plate Ames test No genotoxicity TA100, TA102 Yi, 2010 Insect Tea) KM mice 1～4 twice, 24 h apart Bone marrow cell micronucleus test No significant mice micronucleus Larval excreta KM mice 30 14 d Acute toxicity test >30 No acute toxicity Pyralis "Baicha" Larval (Pyralis Wang et al., farinalis insect tea excreta Farinalis-Litsea KM mice 2.5 ～10 twice, 24 h apart Bone marrow cell micronucleus test No significant mice micronucleus 2017a Coreana Insect Tea) KM mice 20 14 d Acute toxicity test >20.0 no acute toxicity Complex no significant hematological, clinic Wistar rats 0.63 ～2.5 30 days 30 days feeding study Che, 2003 of insect ACCEPTED chemical and histopathological changes Bombyx mori North and fugi Wistar rats 0.3 ～1.25 20 d Teratogenicity study No teratogenicity or Antheraea caterpillar (Cordycep Cordyceps militarisi ICR mice 1～10 14 d Acute toxicity test >10.2 No acute toxicity pernyi fungus s militaris TA97, TA98, 8～5000 µg /plate Ames test No genotoxicity Gao et al., parasitize TA100, TA102 2012 pupae) ICR mice 1.6 ～6.7 2 d Bone marrow cell micronucleus test No significant mice micronucleus ICR mice 1.68 ～6.7 5 d Sperm deformity test No sperm malformation ACCEPTED MANUSCRIPT
Edible tolerated Common Dose Species stage/ Samples Animals/strains Duration type of test dose Results Ref. name (g·kg -1·bw -1) body parts (g·kg -1·bw -1)
No significant hematological, clinic Wistar rats 1.13 ～4.5 30 d 30 days feeding study chemical and histopathological changes Inhibition effects on the growth and Xie, et al., SD rats 2～8 91 d 90 days feeding study development of young rats, and kidney 2007 injury in high dose KM mice 1～10 14 d Acute toxicity test >10 No acute toxicity SD rats 1～10 14 d Acute toxicity test >10 No acute toxicity TA97, TA98, 8～5000 µg /plate Ames test No genotoxicity Gong et al., TA100, TA102 2003 KM mice 2.5 ～10 twice, 24 h apart Bone marrow cell micronucleus test No significant mice micronucleus KM mice 2.5 ～10 5 d Sperm deformity test No sperm malformation SD rats 1～4 30 d 30 days feeding study No subchronic toxicity KM mice 10 14 d Acute toxicity test >10 No acute toxicity Wistar rats 10 14 d Acute toxicity test >10 No acute toxicity TA97, TA98, 8.8 ～5500 µg /plate Ames test No genotoxicity TA100, TA102 Pan et al., KM mice 0.833 ～3.333 twice Bone marrow cell micronucleus test No significant mice micronucleus 2009 KM mice 0.833 ～3.333 twice Sperm deformity test No sperm malformation No significant hematological, clinic 'Xinjiang' caterpillar Wistar rats 0.833 ～3.333 30 d MANUSCRIPT 30 days feeding study chemical and histopathological changes fungus SD rats 20 14 d Acute toxicity test No acute toxicity ICR mice 20 14 d Acute toxicity test >2 No acute toxicity May induce kidney abnormality at high SD rats 0.5 ～2 13 w Chronic toxicity study Che et al., concentration 2014 TA98, TA100, 312.5 ～5000 µg TA102, TA1535, Ames test genotoxicity at high dose /plate TA1537 KM mice 0.39 ～25 7 d Acute toxicity test >25 g/kg No acute toxicity Yang et al., larvae of T. molitor Anti-genotoxicity induced by KM mice 30 d 30 days feeding study 1999 Cyclophosphamide Tenebrio Breadwor SD rats 0.3 ～3 28 d Acute toxicity test No acute toxicity Larvae Freeze-dried Powder Han et al., molitor ms TA98, TA100, of T. molitor Larvae 8～5000 µg /plate No genotoxicity 2014 TA1535, TA1537ACCEPTED No significant hematological, clinic Zhou et al., Hanxia flour SD rats 1.25 ～10 g/kg 90 d 90 days feeding study chemical and histopathological changes 1996 KM mice 12.5 ～100% diet 28 d Acute toxicity test No acute toxicity Massicus Hachong Larvae M. raddei powder KM mice 1.25 ～5% diet Once Bone marrow cell micronucleus test No significant mice micronucleus Song, 2012 raddei KM mice 60 d One Massicus raddei each cage/d No genetic or chronic toxicity ACCEPTED MANUSCRIPT
Edible tolerated Common Dose Species stage/ Samples Animals/strains Duration type of test dose Results Ref. name (g·kg -1·bw -1) body parts (g·kg -1·bw -1)
Anoplophora Longhorn Larvae Mysorethorn borer ICR mice 5 7 d Acute toxicity test >5 g/kg no acute toxicity Zhang, 2013 sp. ed beetle Nine Wistar rats 156.25 mg/mL 21 d Palembus P. dermestoides Water extracts and ether extracts have dragon Larvae KM mice 225.2 mg/mL 7 d Acute toxicity test Xu, 2008 dermestoides extracts better anti-inflammatory effects ． worms KM mice 225.2 mg/mL ICR mice 20 14 d Acute toxicity test >20.0 No acute toxicity Liu, 2014 Northeast Holotrichia H. diomphalia The grub extract belongs to low toxicity black Larvae 46.43 ～ Jin et al., diomphalia extracts KM mice 3～77 14 d Acute toxicity test Chinese medicine with significantly grub 51.14 2007 anti-tumor and antioxidant effect. Chinese ICR mice 0.1 ～0.5 4 w 30 days feeding study Lowering blood lipid Oxya rice Adult, Liu and O. chinensis extract chinensis grasshopp nymphae ICR mice 0.05 ～0.2 4 w 30 days feeding study Anti-oxidant activities Duan, 2007 er SD rats fourth, apart 2 h Acute toxicity test >10 No acute toxicity ICR mice twice, 2 h apart Acute toxicity test >10 No acute toxicity TA97, TA98, 78 ～5 000 µg /plate Ames test No genotoxicity TA100, TA102 White Ericerus pela Adult, E. pela Chavannes ICR mice 1.25 ～5 twice, 24 h apart Bone marrow cell micronucleus test No significant mice micronucleus wax Feng, 2005 Chavannes nymphae eggs ICR mice 1.25 ～5 g/kg Sperm deformity test No sperm malformation insect MANUSCRIPT SD rats 0.31 ～5 g/kg Acute toxicity test No acute toxicity No significant hematological, SD rats 2.5 ～10 g/kg 30 d 30 days feeding study clinic chemical and histopathological changes Cryptotympa Black C. atrata Fabricius Lowering blood lipid and reduce Sun et al., Nymphae KM mice 3% diet 4 w 30 days feeding study na atrata cicadas larvae powder postprandia blood glucose levels 2009 TA97, TA98, 5～5000 µg /plate Acute toxicity test No genotoxicity TA100,TA102 Li et al., Propolis extraction KM mice 2.5 ～10 twice, 24 h apart Bone marrow cell micronucleus test No significant mice micronucleus 2011 KM mice 2.5 ～10 35 days Sperm deformity test No sperm malformation ICR mice 10 7 d Acute toxicity test >10 No acute toxicity TA97a, TA98, 100 ～5000 µg Asian Larvae, Ames test No genotoxicity Apis cerana TA100,TA102 /plate honey bee Honey Royal Jelly Yan et al., ICR mice ACCEPTED2.5 ～10 twice, 24 h apart Bone marrow cell micronucleus test No significant mice micronucleus Complex Capsule 2005 ICR mice 2.5 ～10 35 days Sperm deformity test No sperm malformation No significant hematological, clinic ICR mice 1.35 ～2.7 30 d 30 days feeding study chemical and histopathological changes SD rats 14 d Acute toxicity test >10 No acute toxicity Fu et al., Propolis health food KM mice 14 d Acute toxicity test >10 No acute toxicity 2004 ACCEPTED MANUSCRIPT
Edible tolerated Common Dose Species stage/ Samples Animals/strains Duration type of test dose Results Ref. name (g·kg -1·bw -1) body parts (g·kg -1·bw -1)
No significant hematological, clinic SD rats 1.12 ～4.5 30 d 30 days feeding study chemical and histopathological changes TA97, TA98, 8～5000 µg /plate 48 h Ames test No genotoxicity TA100,TA102 KM mice 2.5 ～10 Bone marrow cell micronucleus test no significant mice micronucleus KM mice 2.5 ～10 Sperm deformity test No sperm malformation KM mice 21.5 14 d Acute toxicity test >21.5 no acute toxicity SD rats 21.5 14 d Acute toxicity test >21.5 no acute toxicity Bee milk soft TA97, TA98, Wang et al., 8～5000 µg /plate Ames test No genotoxicity capsule TA100,TA102 2009 KM mice 0.064 ～8 2 d Bone marrow cell micronucleus test No significant mice micronucleus KM mice 0.32 ～8 Sperm deformity test No sperm malformation KM mice Acute toxicity test >21.5 No acute toxicity 1000 ～4000 µg Ames test No genotoxicity /plate Wang et al., Ant powder KM mice 5～20 Bone marrow cell micronucleus test No significant mice micronucleus 2006 KM mice 5～20 Sperm deformity test No sperm malformation No significant hematological, clinic SD rats 0.42 ～1.67 30 d 30 days feeding study MANUSCRIPT chemical and histopathological changes KM mice 4.64 ～46.4 48 h Acute toxicity test >10 No acute toxicity Adult, TA97, TA98, Polyrhachis Black 2～50000 µg /plate Ames test No genotoxicity larvae, TA100,TA102 Li et al., dives thorn ants Eggs KM mice 0.5 ～5 Bone marrow cell micronucleus test No significant mice micronucleus 1995a, KM mice 0.5 ～5 Sperm deformity test No sperm malformation 1995b, Ant powder Wistar rats 1.875 ～7.5 Teratogenicity study >10 g/kg No teratogenicity 1995c No significant hematological, clinic Wistar rats 0.5625 ～2.25 14 w 90 days feeding study chemical and histopathological changes Cai et al., Wistar rats 1～7 6 m Chronic toxicity study No chronic toxicity 1995 Zhao et al., Ethanol Extracts mice 66.7 Once Acute toxicity test >66.7 No acute toxicity 1983 Adult, ICR mice 10 7 d Acute toxicity test >22.5 No acute toxicity Formica blood-red F. sanguinea Li et al., larvae, SD rats ACCEPTED 0.5, 2.5 90 d 90 days feeding study No significant hematological, clinic sanguinea ant powder 1996 Eggs SD rats 0.5, 2.5 180 d Chronic toxicity study chemical and histopathological changes ICR mice 10 7 dt Acute toxicity test >10 No acute toxicity American Periplaneta Adult, P. americana SD rats 10 7 d Acute toxicity test >10 No acute toxicity cockroac Zhou, 2008 americana nymphae powder No significant hematological, clinic h SD rats 2.5 ～10 30 d 30 days feeding study chemical and histopathological changes ACCEPTED MANUSCRIPT
Edible tolerated Common Dose Species stage/ Samples Animals/strains Duration type of test dose Results Ref. name (g·kg -1·bw -1) body parts (g·kg -1·bw -1)
Opisthoplata Water, alcohol, Li et al., KM mice 16 ～31.25g 14 d Acute toxicity test >3 g/kg No acute toxicity, weights all increased orientalis ether Extracts 2013
Macrotermes Termite, Adult, M. barneyi and O. Wu and barneyi white ant nymphae formosanus body KM mice 0.39 ～25 7 d Acute toxicity test >10 g/kg No acute toxicity Wang, 1999 homogenate liquid
NIH mice 10 d Acute toxicity test >10 No acute toxicity SD rats 10 d Acute toxicity test >10 no acute toxicity Odontoterme Termite, Adult,nym TA97, TA98, 625 ～5000 µg Song et al., s formosanus white ant phae Ethanol Extracts Ames test No genotoxicity TA100, TA102 /plate 1995 NIH mice 5～5 twice, 24 h apart Bone marrow cell micronucleus test No significant mice micronucleus NIH mice 2.5 ～10 35 d Sperm deformity test No sperm malformation Coptotermes Termite, Adult, Wang et al., Preparation copond KM mice 0.2 7 d Acute toxicity test No acute toxicity formosanus white ant nymphae 1995 Alkaloids from E. Chinese KM mice 0.17613 ～0.43 Acute toxicity test >0.17613 Medium toxic grade Tian, 2006 Eupolyphaga Adult,nym sinensis ground sinensis phae Chen et al. beetle E. sinensis powder SD rats 10 g/kg 14 d Acute toxicity test >10 No acute toxicity 2007 KM mice 8.3 ～33 2 w Acute toxicity test >10 No acute toxicity KM mice 2.8 ～11 twice, 24 h apartMANUSCRIPT Bone marrow cell micronucleus test No significant mice micronucleus M. domestica KM mice 2.8 ～11 5 d Sperm deformity test No sperm malformation Li, 2010 Musca Larvaes powder Housefly Larvae SD rats 13% ～40% 21 d Teratogenicity study No genotoxicity domestica SD rats 14.5% ～40% 106 d Chronic toxicity study No chronic toxicity KM mice 2.7 14 d Acute toxicity test (MTD) >2.7 No acute toxicity Qin et al., Flyblow chitosan KM mice 0.9 14 d Acute toxicity test >0.9 No acute toxicity 2009
ACCEPTED