*3.3. Parenteral Toxicity*

Data regarding parenteral ricin intoxication derive mainly from animal studies. By injection, mice had an LD50 of 3 to 5 μg/kg by intravenous and 22 μg/kg by subcutaneous route [82], rabbits had LD50 0.5 μg/kg by the intravenous route and 0.1 μg/kg by the intramuscular route, while guinea pigs had LD50 <1.1 μg/kg by the intravenous route and 0.8 μg/kg by the intramuscular route [83]. Human data only derives from the few cases of suicide or murder, or their attempt; the most known episode is the assassination of the Bulgarian dissident Georgi Markov, who in 1978 died three days after possibly being stabbed with an umbrella loaded with a ricin-containing pellet (Figure 1) [84].

By parenteral administration, immediate local pain at the injection site is reported, followed by general weakness within five hours. The following symptoms, that are general and maybe similar to sepsis (fever, headache, dizziness, anorexia, nausea, vomiting, hypotension, abdominal pain), can be delayed for as much as 10 to 12 h, even with high doses. Usually local tissue damage at the site of the injection is observed. Laboratory abnormalities included elevated liver transaminases, amylase and creatinine kinase, hyperbilirubinemia, myoglobinuria, and renal impairment. The clinical course may progress to multisystem organ failure. Preterminal complications included gastrointestinal hemorrhage, hypovolemic shock, and renal insu fficiency [78,84].

## **4. Bioterrorism and Environmental Toxicity**

Ricin is currently monitored as Schedule 1A of the Chemical Weapons Convention (CWC) and is a Category B substance under the Biological and Toxins Weapons Convention (BTWC) [80]. Despite its toxicity, ricin is less potent than other agents, such as botulinum neurotoxin or anthrax. It has been estimated that eight tons of ricin would have to be aerosolized over a 100 km<sup>2</sup> area to achieve about 50% casualty, whereas only a kilogram of anthrax spores would cause the same e ffect [85]. Thus, deploying an agen<sup>t</sup> such as ricin over a wide area, although possible, becomes impractical from a logistics standpoint. However, the availability of castor beans and the quite simple procedure for rough ricin purification have attracted criminal and terrorist interest for small scale biocrimes or to cause collective media-driven alarm [80].

From castor seeds, a nontoxic oil can be extracted that has multitude of uses in many sectors, including cosmetic, pharmaceutic, mechanical, and chemical industry. Castor oil production is increasing worldwide because of its versatile application, low cost, availability, and biodegradability. In addition, the oil-free seed pulp can be used in agriculture as a natural fertilizer [86], although the processing of castor seeds requires grea<sup>t</sup> caution due to the high allergenicity [87,88] and extreme toxicity [76] of their protein fraction, represented, above all, by ricin. World production of castor oil increased from 0.8 million tons in 2000 [89] to 1.21 million tons in 2014 [90], with a castor seed production of 1.49 million tons in 2017 [91]. Leading producing countries are India, with over 80% of the global yield, Mozambique, China, Brazil, Myanmar, Ethiopia, Paraguay, and Vietnam [92]. The oil makes up about 50% of the weight of the seeds and is mostly constituted of ricinoleic acid (90%), with minor amounts of dihydroxystearic, linoleic, oleic, and stearic acids. Ricin isoforms and the alkaloid ricinine, are not transferred to the oil fraction during extraction, which can be performed by cold or warm pressing, but remain in the seed cake [93,94].

Castor bean meal press cake or other residues of the castor oil production have been employed as a protein source for feed or fertilizer, but their use is very limited due to ricin toxicity [76]. In 2008, the European Food Safety Agency defined ricin as an undesirable substance in animal feed. Ricinus derived material should be appropriately inactivated through physical and/or chemical methods to guarantee animal and human health [95]. Nevertheless, many accidental poisonings are still reported for animals eating improperly detoxified fertilizer or other agricultural products containing castor derived material [76,94].

In order to block the toxic action of ricin, di fferent strategies have been evaluated: Vaccines, inhibitors, and passive immunity. Vaccines against ricin with the consequent production of neutralizing antibodies did not give satisfactory results in vivo (reviewed in [96]). Inhibitors of ricin can block the active site or work as a substrate analogue; however, the available data are limited to in vitro experiments [97]. More recently, inhibitors of cell routing have been developed, sometimes giving promising results, also in vivo [98,99]. To date, passive immunity has proven to be the only e ffective strategy for treating intoxication caused by ricin. The delay in the appearance of signs of intoxication makes confirmation of exposure, diagnosis of intoxication, and the subsequent medical response technically and logistically challenging. The development of anti-ricin sera or antibodies, e ffective even when used several hours after toxin exposure, represents a step forward in treatment of ricin intoxication, as it increases the time window of intervention (WOO, window of opportunity). Many authors described e ffective post-exposure treatment of ricin intoxication with specific antibodies, but with a limited WOO (~8 h) [100–103]. Other authors reported a survival between 50% and 89% of mice treated with anti-sera 24 h after intoxication [104–106]. Once internalized into the cells, ricin cannot be neutralized by antibodies, thus limiting the therapeutic window. However, Whitfield et al. in 2017 reported 100% protection in aerosolized ricin-treated mice with a single administration of a F(ab')2

polyclonal ovine antitoxin given 24 h post-exposure [69]. Even when performed in the same animal species, comparison between diverse experiments is often di fficult, due to the di fferent toxin dose and route of administration utilized. Moreover, there are few data about correlation between the antitoxin dose required for protection and the WOO.
