The Toxicity of Nakiri Ayame’s Horns through Human Consumption
ヒトの摂食における
百鬼あやめの角の毒性

An exploration of desire
欲望の探求

Chimatta, Peko↑→Dynamics, NekoMikuri, Kuratius, Isaac, kurapan, heresy, Rast

Abstract | 要旨        2

Preface        2

Introduction        2

Authenticity of the Horns        3

Classification of Cow Horns        3

Possibility of Regrowth        4

Horn Composition        6

Consumption of Ayame’s Horns        6

Quantity of Horns to Meet Minimum Toxicity        9

Afterward        9

Bibliography        13

Fixed Bibliography        15

Postscript        17

Abstract | 要旨

EN

In this research paper, the oral toxicity of Nakiri Ayame’s horns was explored. The type, possibility of regrowth, and composition of Ayame’s horns were determined through a combination of empirical and scientific evidence. The feasibility of horn edibility was considered by analyzing amino acid toxicity based on an assumption that her horn is composed primarily of α-keratin. Finally, mathematical models were used to determine the lethal dose of Ayame’s horns. Based on this study, we suggest that consuming 50 Ayame horns would be lethal to the average adult male.

JP

本論文では、百鬼あやめの角の経口毒性を調査する。角の種類、再生能力、そして構成成分を実証的および科学的証拠を用いて推測する。角は主にα-ケラチンで構成されていると仮定し、α−ケラチンを構成するアミノ酸の毒性の分析による角の可食性を検討する。致死量に達するまでの角の摂食数を、数学的モデルを用いて数量化する。本研究の結果から、あやめ氏の角を50本以上摂取した場合、成人男性は致死量に達すると考えられる。

Preface

In 2020, June 14th, at 12:41 PM PDT, a “theorem” was posted regarding the possibilities and analysis of eating Ayame Nakiri’s horns. This will be referred to as the “original theorem.” In this paper, This report will address the validity and clarity of statements made by previous persons, supported with clear research and citations.

Introduction

The question this report is addressing is ultimately can we eat Ayame’s horns and if so, how many?

While they are referred to as horns they also bear visual similarity to antlers. As horns and antlers have different construction, the distinction between which Ayame has is critical to the accuracy of any further conclusions.

Antlers shed and are male exclusive... Horns may be used for copulation, territory defending, or also, to radiate temperature. Considering the specimen is a female demon, they are not antlers and it is reasonable to assume the last reason is why she utilizes horns. This entire theorem is referenced in the bibliography. [23]

Authenticity of the Horns

Before considering the type of horn it is important to first consider what Ayame is: an oni (鬼). While oni is often translated as ogre in English, the oni is a Japanese mythological creature (youkai). [1] As such the western ogre does not accurately portray all aspects of the Japanese oni. Reider explains oni as "often depicted with one or more horns atop their heads, wearing only a loincloth or tiger skin." [2, pg 135] Female oni also exist in Japanese folklore. Furthermore, she explains that oni horns were derived from an ox’s horns. [2, pg 153] This was also the answer to a Yahoo Japan Chiebukuro question, where it was asked where the oni horn(s) originated from. [3]

Considering the accepted origin of the horns it can be concluded that cow horns (As oxen are the male version of cattle) are the most analogous to Ayame’s horns. Seeking confirmation of horn composition or if they do mold from Ayame directly has not yielded any concrete information.^[1]

Classification of Cow Horns

Horns of the Bos taurus, commonly known as the cow, are distinct from the antlers of other creatures in the Cervidae family, such as the deer.

Antlers are only present in males except for the caribou. They often grow as the animal matures, regulated by hormones associated with mating. Contrary to popular belief, antlers are not made of just bone, but have high water and protein content. They may even be covered with skin with soft hair, called velvet, carrying blood vessels and nerves. [4]

Horns, present in the family Bovidae, are present in both sexes. Unlike the antler, horns are never branched. Horns never go through shedding, many species never stop growing their horns, and they do not grow back if broken. [4] Due to this, there is a process called "dehorning" in commercial livestock management. [7] According to arguments against such practices, the claim that horns "may be used for copulation, territory defending, or also, to radiate temperature" seems to be accurate. [7,9] "A Greener World" has a Technical Advice Fact Sheet about the relationship between horns and thermoregulation. In this document, Bassett describes the horn's role in thermoregulation of various cattle breeds as being a key thermoregulating and autonomic function. In other words, it cannot be actively controlled.

This indicates that, if they are functionally identical to cow horns, Ayame’s horns would be unable to regrow once lost. This would render the entire topic of the quantity of consumption meaningless and futile as the maximum quantity would always be limited to less than or equal to two.^[2]

Possibility of Regrowth

On August 5th, 2019 a relevant twitter conversation of utmost importance occurred between a surprising pair: Ayame Nakiri and Houshou Marine. [11]

Fig. 1: The tweet in question^[3]

Translation:

This indicates two important pieces of information:

1. Senchou is still horny^[4]
2. Senchou seems to be suggesting that Ayame’s horns can “fall off.”

It is unclear if Senchou is aware that Ayame’s horns should not fall off or not. As there was no reply after this tweet we have two possibilities. Either Ayame was finally disgusted by the boomer captain’s advances or was too embarrassed to correct her or Ayame is able to safely lose and regrow her horns. As the first option leaves us at a similar logical end as we found previously we will pursue the reasoning that Senchou is correct about the ability for them to regrow. Further sections will operate under this assumption.

Horn Composition

With previous assumptions stated the question itself can be addressed: How many Ayame horns can we eat?

To answer this requires that the following be verified:

Although nutritional data of horns are unavailable, we can judge that it would be entirely calcium (as bone marrow is not shedded.)

See picture [6] for a diagram of an animal horn. Contrary to popular belief, the “true horn”, a class of horn-like appendages found on cows, are not mostly bone. Instead, it mostly consists of a material called keratin. Horns have a live bony core covered with layers of keratin, called a “keratin sheath.” [5] A 2009 paper by Tombolato et al. further explains that horns do “not have a mineralized component and [are] composed primarily of α-keratin.” [12] Along with chitin, keratin is often regarded as one of the mechanically strongest biomaterials other than bone. As stated, horns primarily consist of structural and fibrous proteins. This material, keratin, is the same as that which composes human hair and nails.

The 2009 paper notes that two amino acids, glycine and alanine, make up the α-keratin complex in high concentrations. A slight presence of cysteine, another amino acid, forms important bonding features that make up the structural integrity of α-keratin. These amino acids form “coiled coils” that create a tightly wound structure. When collected en masse, it results in a strong composite material. [13] The horn in particular is usually classified as a “hard” α-keratin in contrary to other “soft” α-keratins. [14] One source states that the highest composition of amino acids in cattle horns are glycine (9.8%), arginine (8.6%), and cystine (8.2%). Even in the types of hard α-keratins, there exists a large variation. [15]

Considering the variation and composition of the closest horns we have information on leads to the viability of consuming the constituent components. Practically this will be viewed as eating a combination of such amino acids, rather than calcium.

Consumption of Ayame’s Horns

Now that a reasonable estimate analogous to Ayame’s horns has been established, toxicity and consumption can be considered. Specific mechanical horn preparation, such as powdering, is not addressed. Some degree of mechanical reduction into human ingestible portions will be assumed to have been completed.

Amino acids are ubiquitous and necessary in human intake. Amino acids make up proteins of all types, and aside from energy and essential molecules like water, make up much of the “food” we eat in order to survive. Within the types of amino acids, there are three categories:

1. Essential amino acids: These amino acids cannot be synthesized de novo by humans and must be supplied through diet.
2. Conditionally essential amino acids: These amino acids can be synthesized under normal conditions, but may become limited under some physiological conditions.
3. Non-essential amino acids: These amino acids are synthesized in sufficient amounts in the human body.

Cysteine, glycine, and arginine are all considered conditionally essential amino acids. Despite being essential molecules, in the last few decades there has been a toxicity level associated with a substantial enough imbalance of amino acids, leading to physiological damage. [16,17] A true quantitative evaluation of how this imbalance may lead to toxicity would require physical experiments, which is beyond the capabilities of the researchers. Fortunately, there are prior studies done that explore this matter. In 2004, Garlick noted the rise in popularity of amino acid supplements, allowing individuals to intake large pure forms of amino acids, allowing for specific amino acids to be ingested at elevated levels. In the review, Garlick presents some possibilities for simple “amino acid toxicity” below:

Cysteine: “In studies on humans, 5–10g doses of cysteine induced nausea, lightheadedness, and dissociation. Also, in healthy subjects given increasing doses up to 20 g of cysteine, fatigue, dizziness, nausea, and insomnia, which were dose dependent, were reported.”

Glycine: There appear to be no studies of glycine toxicity in humans, but no serious side effects were noted when up to 31 g/d of glycine was given in classic studies of amino acid requirements. Also, glycine is often given as an irrigant during transurethral prostate resection, and nausea, transient blindness, and visual impairment have been reported. The visual impairment was reported to occur at a plasma glycine concentration >4mM, whereas  central nervous system symptoms occurred when 0.5 g/kg of glycine was absorbed. In children, i.v. infusion of 7.5 g of glycine was proposed as an ‘‘innocuous’’ procedure to detect growth-hormone deficiency.

Arginine: In humans, several studies investigating possible immunity enhancement and wound-healing improvement by up to 30 g/d of arginine hydrochloride reported no adverse effects except nausea and diarrhea. Moreover, i.v. infusion of patients with 30 g of arginine to evaluate pituitary hormone secretion was well tolerated except for increased plasma K+ in subjects with diabetes.

Garlick concludes (emphasis added): “Moreover, no general rule or mechanism appears to account for the effects of all amino acids or even groups of amino acids… The most toxic amino acids for both animals and humans appear to be methionine, cysteine, and histidine. Not only do these amino acids have acute adverse effects, but evidence exists that they can cause tissue damage and... may  be associated with chronic diseases if taken over long periods of time. However, in general, there is little evidence of serious adverse effects in humans from most amino acid supplements.” [18]

This conclusion seems to suggest that out of all the primary amino acids within Ayame horns, we may want to specifically consider cysteine as a lethal element via ingestion. In the medical field, there is a term called the LD50 (LD-50) and the LDLO (lowest lethal dose), which describe the dosage which kills 50% of test animals under controlled conditions, and the minimum dosage which has killed humans, respectively. [19] A paper by Dilger et al. in 2007 explores this through a motivation of establishing a clear recommended dosage amount for amino acid supplements. In their study, excessive amounts of cysteine were fed to several animal models, such as rats, pigs, and chicks. Dosage of 40g/kg cysteine, toxic to chicks, resulted in no mortality for pigs. However, in multiple animals, excessive cysteine dosage resulted in a rapid loss of weight. As a conclusion, they note that there is a non-trivial adverse effect of excessive cysteine, albeit at tremendously high excessive levels. Thus, they urge that more research is needed to determine consequences in humans. Unfortunately, it appears no such research has been done.

We will look at this problem from a different angle. Cysteine in supplement form usually comes as a “prodrug” called N-acetyl cysteine (NAC), also known as acetylcysteine. Prodrugs are a chemical that metabolizes in the body into the drug of interest (in this case, cysteine). NAC is also considered to be a useful drug that is used to treat acetaminophen overdose and loosen thick mucus linings in respiratory conditions such as cystic fibrosis. Thus, it is considered to be one of the WHO’s “essential medicines.” [20] Can such a medicine have a LDLO? Oftentimes yes, but for such medicines, it may be too high to be of significance.

The FDA’s data sheet on NAC describes a study testing the adverse effects of i.v. infusion of NAC. In a group of 180 individuals, 17% had an allergic shock (anaphylactoid reaction) which can be deadly if not treated. While it describes the recommended rate of infusion, it does not specify a LDLO. [21]

A 2015 case of NAC poisoning was reported by a hospital, where a nurse incorrectly gave 10 times the dosage of NAC (incorrect dosage of 100 g) to a 65kg female patient suffering from acetaminophen poisoning. This error caused NAC poisoning instead. This lead to death 12 days later and as such can be considered a lethal dose. This took less than half the time, 12 days instead of 30 or 60 days, required for establishing an LD50.[22]

Due to how rare this situation is, we must resign to the fact that the best we can get to a possible toxicity level of cysteine is from one case of NAC overdose. Thus, we will estimate that 100g/65kg (1.54g/kg) of acute ingestion of cysteine will result in a high chance of mortality. Since we have assumed that approximately 8.2% of the composition of a cow horn is cysteine, this means that an individual weighing 82kg (approximately 180lbs) would need to ingest over 126.15g of cysteine. To consume this level from horns alone would require 1.54 kg of Ayame horn to reach the acute toxicity of cysteine for humans.

Quantity of Horns to Meet Minimum Toxicity

With a reasonable estimate of lethality via toxicity we can move to How many Ayame horns does it take to reach the necessary toxicity level? In order to answer this question, we must first determine the characteristics and volume present in Ayame’s horns. In order to figure out the scale and size of the horn, we can use her official height and statistics as given by Hololive. Ayame’s height is officially listed at 152 cm. [24] Her official 3D model shows a height of 3288 pixels. [26] To obtain pixels per centimetre, we can calculate the value of  in , resulting in an  of 21.6315789 px/cm. Unfortunately, in much of her official art, there is variation in her height due to her leg position. The average of those heights is 22 pixels per 1 centimetre. We can use this value to reasonably approximate the dimensions of Ayame’s horns.

From here, we can imagine the horn as a three dimensional imperfect cone. To calculate its volume, we can use our previous calculation of pixels per centimetre value () to calculate the cone base area. By examining the official 3D model, we determined the cone base diameter to be 63 pixels. Using the circle area formula and , we were able to calculate that the cone base area is 6.4 cm². From there, using this formula:  (see Appendix for the reasoning behind this) we calculated that the entire horn volume is 23.8 cm3.

With the density of keratin being around 1.283 g/cm3, [25] we can conclude that a single horn weighs around 30.54 grams. Assuming the toxic dose to be 1.54 kg, we are required to eat about 50 Ayame horns.

Afterward

After a draft of the initial manuscript was completed, the draft along with an explanation was sent via email correspondence to the canonical creator of Nakiri Ayame, Nana Kagura-sensei. This email was sent on September 24, 2020.

https://media.discordapp.net/attachments/792676623450243073/793211176976121866/Screenshot_20201229-041741_Discord.jpg 

Bibliography

[1] https://en.wikipedia.org/wiki/Oni

[2] Reider, Noriko; "Transformation of the Oni: From the Frightening and Diabolical to the Cute and Sexy" https://warp.da.ndl.go.jp/info:ndljp/pid/8753502/nirc.nanzan-u.ac.jp/nfile/591

[3] https://detail.chiebukuro.yahoo.co.jp/qa/question_detail/q1456477414 "鬼にツノがあるのは鬼のモデルが牛と虎だからです。" "The horns of an oni are due to the model being the oni character being a fusion of the ox and the tiger." "鬼のツノは牛のツノと同じになります" "An oni's horns are the same as an ox's horns."

[4] https://animaldiversity.org/collections/mammal_anatomy/horns_and_antlers/

[5] https://en.wikipedia.org/wiki/Horn_(anatomy)

[6] Picture: Anatomy and physiology of an animal's horn; https://upload.wikimedia.org/wikipedia/commons/thumb/9/9a/Anatomy_and_physiology_of_animals_A_horn.jpg/220px-Anatomy_and_physiology_of_animals_A_horn.jpg

[7] https://en.wikipedia.org/wiki/Livestock_dehorning

[8]  Bassett, Anna ; " Greener World Technical Advice Fact Sheet No. 11: Horns and Thermoregulation" https://agreenerworld.org/wp-content/uploads/2018/05/TAFS-11-Horns-and-Thermoregulation-v1.pdf

[9] https://earthhaven.ca/why-cows-need-horns-c553.php 

[10] One such example of this footage is clipped here: https://www.youtube.com/watch?v=yVV-74YHxOY 

[11] https://twitter.com/houshoumarine/status/1158342666129530880

[12] Tombolato, L., Novitskaya, E. E., Chen, P.-Y., Sheppard, F. A., & McKittrick, J. (2010). Microstructure, elastic properties and deformation mechanisms of horn keratin. Acta Biomaterialia, 6(2), 319–330. doi:10.1016/j.actbio.2009.06.033

[13] https://en.wikipedia.org/wiki/Alpha-keratin

[14] Gillespie, J. M. (1990). The Proteins of Hair and Other Hard α-Keratins. Cellular and Molecular Biology of Intermediate Filaments, 95–128. doi:10.1007/978-1-4757-9604-9_4

[15] Florkin, Marcel; Chapter 7: Structural and Chemical Properties of Keratin-Forming Tissues, Comparative Biochemistry V4: A Comprehensive Treatise.

[16] ”Imbalance and Toxicity of Amino Acids”; Nutrition Reviews, Volume 26, Issue 4, April 1968, Pages 115–118, https://doi.org/10.1111/j.1753-4887.1968.tb00883.x

[17] Munro, H. N. (1978). Nutritional Consequences of Excess Amino Acid Intake. Advances in Experimental Medicine and Biology, 119–129. doi:10.1007/978-1-4684-3366-1_8

[18]  Garlick, Peter J.(2004). “The Nature of Human Hazards Associated with Excessive Intake of Amino Acids.” The Journal of Nutrition, Volume 134, Issue 6, June 2004, Pages 1633S–1639S, https://doi.org/10.1093/jn/134.6.1633S 

[19] Ryan N. Dilger, Sakino Toue, Takeshi Kimura, Ryosei Sakai, David H. Baker, Excess Dietary l-Cysteine, but Not l-Cystine, Is Lethal for Chicks but Not for Rats or Pigs, The Journal of Nutrition, Volume 137, Issue 2, February 2007, Pages 331–338, https://doi.org/10.1093/jn/137.2.331

[20]  https://en.wikipedia.org/wiki/Acetylcysteine

[21] https://www.accessdata.fda.gov/drugsatfda_docs/label/2006/021539s004lbl.pdf 

[22] Astaraki, P., Mahmoudi, G. A., Zafar Mohtashami, A., & Ahadi, M. (2015). N-acetylcysteine overdose after acetaminophen poisoning. International Medical Case Reports Journal, 65. doi:10.2147/imcrj.s74563

[23]

[24] https://vignette.wikia.nocookie.net/virtualyoutuber/images/d/d8/Hololive_VTuber_Height_Difference.jpg/revision/latest?cb=20190605202706

[25] https://www.nature.com/articles/197179a0 

[26] https://user-images.strikinglycdn.com/res/hrscywv4p/image/upload/c_limit,fl_lossy,h_9000,w_1200,f_auto,q_auto/1369026/455796_889408.png 

Fixed Bibliography

1.220px-Anatomy_and_physiology_of_animals_A_horn.jpg (JPEG Image, 220 × 281 pixels). https://upload.wikimedia.org/wikipedia/commons/thumb/9/9a/Anatomy_and_physiology_of_animals_A_horn.jpg/220px-Anatomy_and_physiology_of_animals_A_horn.jpg.

2.455796_889408.png (WEBP Image, 308 × 1000 pixels) - Scaled (99%). https://user-images.strikinglycdn.com/res/hrscywv4p/image/upload/c_limit,fl_lossy,h_9000,w_1200,f_auto,q_auto/1369026/455796_889408.png.

3.FDA. Acetadote® (acetylcysteine) Injection Package Insert. https://www.accessdata.fda.gov/drugsatfda_docs/label/2006/021539s004lbl.pdf (2006).

4.Acetylcysteine. Wikipedia (2020).

5.ADW: Horns and Antlers. https://animaldiversity.org/collections/mammal_anatomy/horns_and_antlers/.

6.Alpha-keratin. Wikipedia (2020).

7.Mason, P. Density and Structure of Alpha-Keratin. Nature 197, 179–180 (1963).

8.Earth Haven Farm - Why Cows Need Horns? | Earth Haven Farm. https://earthhaven.ca/why-cows-need-horns-c553.php.

9.Dilger, R. N., Toue, S., Kimura, T., Sakai, R. & Baker, D. H. Excess Dietary l-Cysteine, but Not l-Cystine, Is Lethal for Chicks but Not for Rats or Pigs. The Journal of Nutrition 137, 331–338 (2007).

10.Hololive_VTuber_Height_Difference.jpg (JPEG Image, 2048 × 1536 pixels) - Scaled (64%). https://vignette.wikia.nocookie.net/virtualyoutuber/images/d/d8/Hololive_VTuber_Height_Difference.jpg/revision/latest?cb=20190605202706.

11.Horn (anatomy). Wikipedia (2020).

12.Anna Bassett. Horns and  Thermoregulation. https://agreenerworld.org/wp-content/uploads/2018/05/TAFS-11-Horns-and-Thermoregulation-v1.pdf (2010).

13.IMBALANCE AND TOXICITY OF AMINO ACIDS. Nutrition Reviews 26, 115–118 (2009).

14.Livestock dehorning. Wikipedia (2020).

15.Tombolato, L., Novitskaya, E. E., Chen, P.-Y., Sheppard, F. A. & McKittrick, J. Microstructure, elastic properties and deformation mechanisms of horn keratin. Acta Biomaterialia 6, 319–330 (2010).

16.Astaraki, P., Mahmoudi, G. A., Zafar Mohtashami, A. & Ahadi, M. N-acetylcysteine overdose after acetaminophen poisoning. IMCRJ 65 (2015) doi:10.2147/IMCRJ.S74563.

17.Munro, H. N. Nutritional Consequences of Excess Amino Acid Intake. in Nutritional Improvement of Food and Feed Proteins (ed. Friedman, M.) vol. 105 119–129 (Springer US, 1978).

18.Oni - Wikipedia. https://en.wikipedia.org/wiki/Oni.

19.Matoltsy, A. G. Structural and Chemical Properties of Keratin-Forming Tissues. in Comparative Biochemistry 343–369 (Elsevier, 1962). doi:10.1016/B978-0-12-395545-6.50014-6.

20.Garlick, P. J. The Nature of Human Hazards Associated with Excessive Intake of Amino Acids. The Journal of Nutrition 134, 1633S-1639S (2004).

21.Gillespie, J. M. The Proteins of Hair and Other Hard α-Keratins. in Cellular and Molecular Biology of Intermediate Filaments (eds. Goldman, R. D. & Steinert, P. M.) 95–128 (Springer US, 1990). doi:10.1007/978-1-4757-9604-9_4.

22.Noriko T Reider. Transformation of the Oni: From the Frightening and Diabolical to the Cute and Sexy. Asian Folklore Studies 62, 133–157 (2003).

23.宝鐘マリン🏴‍☠️＠ホロライブ3期生さんはTwitterを使っています. Twitter https://twitter.com/houshoumarine/status/1158342666129530880.

24.JAPAN Y. 鬼にはなぜつのがあるのですか？ツノとはなんですか？骨？ - 鬼にツノがあるのは鬼のモデルが牛と虎だからです。これは、鬼の出入りす... Yahoo!知恵袋 https://detail.chiebukuro.yahoo.co.jp/qa/question_detail/q1456477414fr=smp_ogp_other.

Postscript

Chimatta: Wow what the hell did we do? But honestly though, I haven’t done real research or written technical material in a while, so this was really refreshing. Learning about cow horns and amino acid toxicology was very cool. You could say it was very horny research ( ¬‿¬)( ¬‿¬)( ¬‿¬).