Overview

Safety first is the best policy. —— Benjamin Franklin

The safety of the people shall be the highest law. —— Cicero

Our project aims to use engineered S. cerevisiae to produce economical fragrance compounds such as limonene, ocimene, cineole, myrcene, borneol, nerol and nerolidol for Odor signals. We aim to develop a material that, when integrated into sanitary pads and laundry detergents, releases specific scents when in contact with menstrual blood or secretions. These scent signals will allow visually impaired women to autonomously track their menstrual cycle, ensuring dignity and confidence in their day-to-day lives. This page will discuss the safety measurements we took in every aspect.

Chassis Safety

We utilized E.coli DH5α, E.coli TOP10 and Saccharomyces cerevisiae strain BY4741 to ensure the safety of our project. No other organisms will be utilized in our project, and none of our components pose any hazard, either individually or within the context of our work. Our engineered organisms and parts are designed to prevent environmental spread. Following fermentation, the supernatant is collected via centrifugation, effectively removing the yeast cells. The target product is then obtained through a series of processing treatments. For yeast and E. coli, we will employ 84 disinfectants or autoclave sterilization methods before proper disposal, ensuring biosafety protocols are strictly followed.

Parts Safety

To produce the different fragrance molecules, we introduced 9 genes into Saccharomyces cerevisiae. Before building the plasmids, we checked the origin of the genes we wanted to use. The genes all belong to the white list provided by iGEM since they all come from the genome of plants or microbe.

Genes	Source
tLS_Cl	Citrus
CcOCS	C.camphora
AmMYS	Antirrhinum majus
PgfB	Penicillium griseofulvum
AcNES1	Actinidia chinensis
CbTPS1	Cinnamomum burmanni
HpyCins	Hypoxylon
StrCins	Streptomyces clavuligerus ATCC27064
SfCins	Salvia fruticosa

Although the fragrance molecules we aim to produce in Saccharomyces cerevisiae (such as limonene, ocimene, cineole, myrcene, borneol, nerol, and nerolidol) carry some risk, they are commonly found in various essential oils and are widely used in the perfume industry, indicating that they do not pose significant risks to humans when consumed in low quantities. Furthermore, the BY4741 yeast strain is incapable of producing these fragrance molecules in amounts sufficient to pose serious risks.

Chemicals Used

In our lab, we use 4S Green Plus Nucleic Acid Stain, dodecane, and ethyl acetate, all of which may pose safety and environmental hazards. The 4S Green Plus Nucleic Acid Stain is essential for electrophoresis, while dodecane and ethyl acetate are crucial for the extraction and detection of limonene, ocimene, cineole, nerol, and nerolidol.

To minimize risks associated with these chemicals, we follow strict safety protocols. The 4S Green Plus Nucleic Acid Stain is confined to a designated contaminated area, as outlined in our laboratory safety guidelines. When handling this stain, we wear plastic gloves over rubber gloves to provide an additional layer of protection. For dodecane and ethyl acetate, we conduct all procedures within a chemical hood to ensure safety and maintain distance from any fire sources. Additionally, these processes are carried out under the supervision of our instructor to further enhance safety. These measures are critical for managing potential hazards while performing our research effectively.

Laboratory safety

Laboratory safety practices are an essential aspect of responsible scientific conduct. Safety precautions are necessary to ensure that unnecessary harm is not caused to people, animals, or the environment. This page includes our considerations for safety during the project. It also contains information about our safety training, laboratory facility safety, and biosafety. All team members must undergo comprehensive laboratory rules and safety training before entering the lab. We have jointly established laboratory safety conventions and strictly adhere to these rules. All experimental procedures are carried out under the supervision of a mentor to ensure the safety of the experiments. We equally value the safety of products, whether it is the safety standards of research sanitary pads or the assessment of the safe dosage of terpenoid compounds for human use.

Safety Training

On June 21st， the team engaged in online learning sessions via Tencent Meeting, which were mandatory for all members before entering the laboratory. These sessions included comprehensive training on biosafety, covering topics such as disinfection, sterilization, emergency procedures, and waste management. By the end of June, we initiated our wetlab activities. To ensure the safety of our laboratory environment, we have established the following laboratory safety protocols:

• Protective Equipment (PPE):It is mandatory for all team members to wear appropriate PPE at all times, including lab coats, gloves, and safety goggles.
• Disinfection and Sterilization: Regular disinfection of work surfaces and equipment is essential. All materials and equipment entering or leaving the lab must undergo strict sterilization procedures.
• Emergency Procedures: All team members are trained to respond effectively to emergencies, including chemical spills, fires, and exposure to hazardous materials.
• Waste Disposal: Adherence to proper waste disposal protocols is crucial. Hazardous waste must be segregated and disposed of in accordance with local regulations.
• Safety Inspections: The lab will undergo regular safety inspections to ensure that all equipment is in good working order and that safety protocols are being strictly followed.
• Continuous Training: Ongoing training and education on safety practices will be provided to keep all team members updated with the latest safety guidelines and procedures.
• Supervision: All experiments will be conducted under the supervision of experienced personnel to ensure compliance with safety protocols.
• Communication: An open line of communication regarding safety concerns is encouraged. Any issues or near-miss incidents must be reported promptly.
• Access Control: Access to the laboratory will be restricted to authorized personnel only to prevent unauthorized access and potential safety hazards.
• Documentation: All safety-related activities, including training, inspections, and incidents, will be meticulously documented for record-keeping and continuous improvement.

By upholding these protocols, we are committed to maintaining a safe and secure laboratory environment for all team members.

On July 9th, to deepen our understanding of laboratory safety and prevent accidents, we visited East China Normal University (ECNU) to attend a laboratory safety lecture and hands-on training. The professor emphasized the importance of carefully weighing terpenoid compounds' ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties, just as one would with pharmaceuticals. He stressed the importance of strictly adhering to safety rules and warned us of the serious consequences of neglecting laboratory safety. He also instructed us on the correct use of instruments in the laboratory, such as alcohol burners or fume hoods.

On July 24th, we were fortunate to have the esteemed Laboratory Management Manager from the BGI Research Institute, Teacher Wen Ni, to provide a detailed explanation of laboratory safety knowledge. Starting with the current safety landscape, Teacher Wen emphasized the significance of laboratory safety, then sequentially discussed common laboratory safety accidents, on-site safety management requirements, and emergency response measures. In conclusion, Teacher Wen offered us this advice for the laboratory: "Do not harm others, do not harm oneself, and do not be harmed by others. When someone else is injured, provide timely rescue; when others violate safety rules, intervene promptly." To better internalize this knowledge, students wrote summaries and reflections after the meeting.

Laboratory Facilities

Throughout the project, we have been working in a Biosafety Level 1 (BSL-1) laboratory. Our work is conducted on laboratory benches and under chemical fume hoods.

BSL-1 is the lowest level of biosafety precautions required for work with agents not known to consistently cause disease in healthy adults. Here are some of the typical safety measures associated with BSL-1 laboratories:

• Protective Equipment (PPE): Lab coats, gloves, and safety goggles are used as basic PPE.
• Hand Hygiene: Regular hand washing with soap and water is practiced.
• Work Surfaces: Benches are cleaned and disinfected regularly.
• Chemical Fume Hoods: These are used to protect personnel from inhalation of toxic gases or vapors when working with volatile chemicals.
• Sharps Safety: Proper disposal of needles and other sharp objects to prevent injury.
• Waste Disposal: Biological waste is disposed of in designated containers.
• Training: Personnel are trained in basic biosafety procedures.
• Access Control: Access to the laboratory is limited to authorized personnel.
• Emergency Procedures: There are established protocols for handling spills and other accidents.

By adhering to these guidelines, we ensure a safe working environment suitable for the type of research we are conducting.

Safety Considerations

In our project, due to the involvement of plasmid extraction, we frequently use high-speed centrifuges. When the centrifuge operates at high speeds, adhering to safety procedures, such as balancing the loads in centrifuge tubes or rotors, is crucial to ensure the safety and efficiency of the process.

Firstly, an unbalanced load during centrifugation can lead to damage or malfunction of the centrifuge and may even cause personal injury. Therefore, it is essential to always ensure the balance of the centrifuge tubes. This is typically achieved by adding opposite samples or balancing liquid to the centrifuge tubes to ensure the stability of the rotor during high-speed rotation.

Secondly, the medium prepared for the experiment needs to be sterilized in an autoclave before it can be used. Autoclaves are considered special equipment, and operating such equipment requires strict adherence to relevant safety regulations.

According to Article 14 of the "Special Equipment Safety Law of the People's Republic of China," safety management and operation personnel of special equipment must obtain the corresponding qualifications. This means that only professionals holding the "Special Equipment Safety Management and Operation Personnel Certificate" are allowed to operate autoclaves.

Therefore, to ensure the safety of the experiment, all personnel operating autoclaves should have received professional training and hold the appropriate operation certificates. This not only helps to prevent accidents caused by improper equipment operation but also ensures the sterilization effect of experimental materials, thereby ensuring the safety and reliability of the entire experimental process.

By following these safety measures, we can ensure the safety of the laboratory environment and also protect the health of researchers and the accuracy of the experiment.

Bioethics

The development of humanity, particularly its long-term sustainable development, must be based on a scientific understanding of the ethics related to life and the correct handling of ethical issues.In our project, we strictly adhere to the wishes of our interviewees by providing them with the interview outline in advance, ensuring they have ample time to prepare. Throughout the interview process, we will respect the privacy and dignity of each participant, engaging in communication with a mindset of equality and understanding. This approach not only aligns with the relevant provisions of the Civil Code of the People's Republic of China regarding personal information protection, but also reflects our commitment to honoring the fundamental rights of our interviewees and upholding ethical principles.

On July 25th, we were honored to have Teacher Weili Zhoufrom the Ethics Committee of BGI Technology to conduct a bioethics training session for us. We learned that the basic principles of bioethics are beneficence, non-maleficence, respect, and justice. In line with the principle of non-maleficence in bioethics and the iGEM safety policy, we are not allowed to conduct human experiments. When researching the safety of our products, we must carefully assess the safety of terpenoid compounds and their potential impact on human health at different concentrations. Terpenoid compounds may also cause allergies in some individuals, so we have also taken these issues into consideration. Studies have shown that a small number of people are sensitive to terpenoid compounds at normal levels. We will also strictly control the amount of terpenoid compounds in our products. They acknowledged the feasibility and positive impact of our project and reminded us to remember the 3R principles: Reduction, Replacement, and Refinement.

In our project, there are numerous issues related to bioethics. For instance, how do we test the safety of our products? We should delve into the biosafety and volatility of terpenoid compounds. While it is not difficult to synthesize synthetic compounds in the laboratory, there may be many risks when they are applied to the human body, such as allergic reactions, the combination with sanitary pads, and the spread of odors. Therefore, we should conduct simulated tests at the laboratory stage, avoiding direct contact with the human body, and perform safety inspections. In summary, the teacher affirmed our project and reminded us to always remember the "3R principles" during the experimental process

Material safety

In terms of safety, we have received a lot of advice from Teacher Tang in materials science on how to bring our products to market. The main suggestions are to strictly control the concentration of terpenoid compounds within a safe and effective range to ensure that the products are harmless to the human body. We should also study the quality requirements and certification standards for personal hygiene products in different countries (especially developed countries), such as the IPCC and FDA. At the same time, we should pay attention to the possibility of domestic SQ certification aligning with international standards to ensure that our products meet the safety standards of multiple countries and enhance our international competitiveness. As consumers increasingly focus on product safety, meeting food-grade standards will become one of the key competitive strengths of our products. This will not only improve product safety but also expand its application fields, such as baby clothing and underwear, which require higher safety standards

The U.S. Food and Drug Administration (FDA) has a series of safety standards and regulatory requirements for menstrual products such as sanitary pads. Menstrual Tampons and Pads: Information for Premarket Notification Submissions (510(k)s)

To ensure that the quality and safety performance of our products always meet the standard requirements, we will adopt advanced monitoring and control measures, and design a complete product closed-loop and full-life cycle management system. From product research and development, production, sales, to customer feedback, every step will be incorporated into a strict quality control system. By regularly testing products and conducting customer surveys to collect data and conduct analysis and evaluation, we can identify and resolve potential issues in a timely manner. At the same time, we need to ensure that the reverse osmosis membrane does not allow the intrusion of external contaminants, thereby ensuring the purity and safety of the product.

In accordance with the General Data Protection Regulation (GDPR), we have implemented stringent measures to ensure the secure handling of personal data, which are detailed as follows:

• No collection of sensitive personal data: We do not collect any personally identifiable information in our project, including personal IDs, religious beliefs, health conditions, etc. This policy aims to minimize the risk to participants' privacy.
• Transparency of information: Before interacting with participants, we provide our interview outline in advance and clearly inform them that the answers they provide will be used for our project. Through such transparent communication, participants can understand the purpose of data use, thereby enhancing trust.
• Informed consent process: Respecting the wishes of participants, we proactively ask interviewees for their consent to publish the videos on Wiki and public accounts before conducting person interviews and creating videos. We explain in detail the purpose of the filming, the way data will be used, and the rights of the participants, including the right to withdraw consent at any time.
• Privacy protection: We strictly follow the policy to ensure that photos without personal consent will not be used or uploaded to Wiki. This approach is based on respect for the privacy rights of participants.
• Compliance and auditing: We regularly review and evaluate our data processing procedures to ensure compliance with GDPR regulations and update our policies in a timely manner to adapt to the changing legal environment.

Through these measures, we pledge to strictly adhere to the requirements of the GDPR at every stage of the project implementation to ensure the effective protection of the privacy and rights of participants.

In summary, we are committed to strictly adhering to safety and ethical standards at every stage of the project implementation to ensure the effective protection of the privacy and rights of participants. Our project has received affirmation from teachers and experts, and we will continue to follow best practices to ensure the safety and ethics of our research.

Terpene dosage safety

We synthesized nine terpenoid synthase genes for seven fragrance compounds and successfully constructed 12 out of 19 plasmids, using different promoter and terminator combinations. Using GC-MS analysis, we successfully produced 4 terpenoids: ocimene, cineole, nerolidol, and nerol. The safe dosage amount for each of them is taken under consideration for product design.

1. Ocimene

High ocimene strains tend to have sweet, fruity aromas and flavors complemented by spice. Ocimene also displays energizing, uplifting effects and is more abundant in stimulating Sativa strains than in the more relaxing varieties. Cannabis strains’ therapeutic value and sensory experience results from a sophisticated interaction between its chemical components, known as “the Entourage Effect.” As a result of “the Entourage Effect”, which describes the process where cannabinoids and terpenes blend in the human body to modify and enhance one another’s mechanism action, ocimene makes strains more stimulating while contributing to their anti-inflammatory and antioxidant properties. Besides, it has antiviral characters, as indicated by a 2008 Chemistry & Biodiversity report, suggesting that the oil of plants containing high levels of ocimene, such as bay laurel, inhibits the herpes simplex virus (HSV-1) and the SARS virus, which causes severe acute respiratory syndrome. There may be concerns regarding addiction to ocimene. But there’s no need to, as isolated terpenes like ocimene are not psychoactive and will not get you high. However, terpenes are a crucial part of the entourage effect and can directly affect the cannabis experience.

2. Cineole

Eucalyptus oil is an essential oil obtained from the Eucalyptus globus tree and contains up to 70% eucalyptol (also known as cineole). It has anti-inflammatory, analgesic (pain relief), decongestant, antibacterial, and antiseptic (germicidal) properties. Its many medical uses include acting as a treatment for colds, cough, asthma, sinus and seasonal allergies, muscle pain and sprains, joint pain and arthritis, skin conditions (such as wounds, cuts, burns, insect bites and acne), headache, stress release, and improved energy and focus. Depending on the indication, eucalyptus oil is usually inhaled directly, by steam inhalation, or by a diffuser. These applications indicate examined safety of cieole on human health when used in an adequate amount. Noteably, you can put eucalyptus oil on your skin but not in your eyes, so diffusion of cineole in sanitary products would be fine. However, we must be careful as if it is undiluted or not thoroughly diluted. It can irritate the respiratory tract, resulting in nose and throat irritation, coughing, and difficulty breathing. It is not recommended to use cineole oil during pregnancy or when breast- or chest-feeding, and it should never be ingested since a teaspoonful or less of undiluted cineole may be fatal .

To further demonstrate the safe amount, according to a study on acute and subacute toxicity study of 1,8-cineole in mice (Xu et. al, 2014), 21.38 and 64.15 mg/kg/day doses of 1,8-cineole had no or mild damages on liver and kidney, while 192.45 mg/kg/day dose of 1,8-cineole had serious damages on liver and kidney, with main lesions of granular and vacuolar degeneration and vascular congestion.

3. Nerolidol

Nerolidol is a sesquiterpene alcohol that naturally occurs in essential oils of several plants including ginger, jasmine, neroli, tea tree, lavender, and cannabis. It has been widely used in cosmetics (e.g., shampoos and perfumes) and in non-cosmetic products (e.g., detergents and cleansers). In fact, U.S. Food and Drug Administration (FDA) has also permitted the use of nerolidol as a food flavoring agent. To date, various pharmacological and biological activities of nerolidol have been reported such as anti-microbial, anti-biofilm, anti-oxidant, anti-parasitic, skin-penetration enhancer, skin-repellent, anti-nociceptive, anti-inflammatory and anti-cancer. In fact, it has been demonstrated that nerolidol exhibits potent antioxidant properties in counterbalancing the effect of ROS by protecting the cells against oxidative damage to lipids, proteins and DNA. For example, in a study, cis-nerolidol was found to possess higher scavenging activity towards hydroxyl radicals with IC50 measured at 1.48 mM. Besides, an in vivo study by Nogueira Neto et al. demonstrated the neuroprotective effects of nerolidol (a mixture of cis- and trans-nerolidol) in adult male Swiss albino mice hippocampus against neuronal damages induced by oxidative stress. The study demonstrated that significant decrease in MDA and nitrite levels were observed for the nerolidol group at doses of 25, 50 and 75 mg/kg when compared to saline, the negative control.

The European Chemicals Agency described reproductive/developmental toxicity tests in male and female rats orally dosed (up to 1000 mg/kg) with nerolidol daily during 2 weeks of cohabitation and for up to 58 days resulting in changes in body weights and developmental effects in the offspring (ECHA, 2006). Rather than reproductive toxicity, another study has described the potential of nerolidol (200 or 400 mg/kg/day for 14 days) to reverse cyclophosphamide-induced reduced sperm count, sperm motility, and testosterone levels in Swiss Albino mice. This was accompanied by protection against epididymal, seminal, and spermatozoal toxicities (Iqubal et al., 2020). These studies also suggest that while nerolidol may induce developmental effects in offspring of animals treated with high doses of the compounds, lower doses could be protective against gonadal toxicity in male rodents.

Nerolidol also acts as chemo-radiation therapy sensitizers in cancers, with some studies underlining the combination of NER and DOX suppressing the migration ability of breast cancer cells and leading to the accumulation of DOX within the cancer cells. In another study, the combination of b-caryophyllene (CRY) and CRYO were found to be effective in sensitizing human colorectal adenocarcinoma and leukemia cells to DOX by synergistically increasing the cytotoxicity of doxorubicin. [5]

To conclude, a safe amount of nerolidol would already have great pharmaseudical qualities and the dosage shall be strictly limited to a fair amount to avoid negative consequences. For aromatherapy, a 1-3% concentration in a carrier oil is typically recommended. For perfumes and fragrances, a 5-15% concentration may be used.

4. Nerol

Natural nerol is found in various plants, including orange leaves, rose, lavender, lemon, and grapefruit. It is recognized as an edible flavoring permitted under China's GB 2760-2014 standards. Nerol can effectively inhibit the growth of microorganisms, whether used as a component of essential oils or on its own, and is regarded as safe for consumption.

Reference:

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5. Jiao Xu and others, ‘Acute and Subacute Toxicity Study of 1,8-Cineole in Mice’, International Journal of Clinical and Experimental Pathology, 7.4 (2014), pp. 1495–1501.

6. Weng-Keong Chan and others, ‘Nerolidol: A Sesquiterpene Alcohol with Multi-Faceted Pharmacological and Biological Activities’, Molecules, 21.5 (2016), p. 529, doi:10.3390/molecules21050529.

7. James Akingbasote and others, ‘Chapter 24 - Safety of Cannabis- and Hemp-Derived Constituents in Reproduction and Development’, in Reproductive and Developmental Toxicology (Third Edition), ed. by Ramesh C. Gupta (Academic Press, 2022), pp. 455–87, doi:10.1016/B978-0-323-89773-0.00024-2.

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