3.2. Low level inhaled carbon dioxide toxicity in animal studies

One principle of toxicological consideration of the risk of exposure to noxious agents to humans is the use of evidence from animal studies. Therefore, the most important animal studies on carbon dioxide respiration at low concentrations are presented. They provide information on possible mask effects. It should be mentioned that in a great work of toxicology [57] following statement on page 156 can be found: "Small laboratory animals (mice) cannot serve well as indicators for dioxide as they do for carbon monoxide, since they are much less sensitive to it than humans". Therefore, in an appropriate risk assessment it is necessary to apply an inter-species uncertainty factor.

3.2.1. Teratogenicity and stillbirth

From decades of studies on the toxicity of carbon dioxide it is known that just 0.5% carbon dioxide for a few minutes to an hour per day is capable of inducing stillbirth and teratogenic birth defects in guinea pigs [58] (Page 14 of the referred FDA document). People in positions of responsibility in the US Navy have been aware that this level of 0.5% carbon dioxide in submarines is often exceeded. They therefore set up a study in pregnant rats, the details of which have been published [59,60]. In rats the first signs of toxicity to pups were observed at a level of 3% carbon dioxide exposure for the pregnant dam with no signs of toxicity at 2.5% exposure. In the 3% CO2 exposure group the findings were a statistically significant mean litter proportion of post-implantation loss (resorptions occurring in the early phase of pregnancy) and a corresponding statistically significant lower mean litter proportion of viable fetuses. Moreover, they found one fetus that had gastroschisis (stomach, several loops of the intestine and liver protruding through an opening in the ventral midline) and localised fetal edema was noted in 2 fetuses: one for hind limbs and the other for neck and thorax. With a safety factor between animals and humans of about three, the US Navy toxicity experts then set the exposure limits for submarines carrying female crews to 0.8% carbon dioxide as well as emergency exposure with a limit of 24 hours [59,60].
The exact mechanism of low level CO2 toxicity for unborn life is not known in detail. Maternal and fetal mechanisms have to be taken into account. With regard to the adverse maternal changes an increased CO2 and acidity in the blood (pH changes) trigger various compensatory mechanisms. These include pH buffering systems in the blood, increased breathing to reduce excess CO2 in the bloodstream, increased excretion of acid by the kidneys to restore pH balance and nervous system stimulation due to changes of heart contractibility and vasodilation [61,62]. During respiratory acidosis the kidneys retain bicarbonate helping to normalize the pH of the blood. With prolonged CO2 stress a metabolic acidosis occurs and the kidneys no longer respond in producing bicarbonate [63]. Thereafter –with further prolonged CO2 burden – the body uses the bones to regulate the acid levels in the blood: Bicarbonate and a positive ion (Ca2+, K+, Na+) are exchanged for H+. The kidney tubule recovers filtered bicarbonate or secretes bicarbonate into the urine to help maintain the pH balance in the blood, which involves the Carbonic Anhydrase (CA) enzyme [64]. CA enzymes participate in metabolic reactions that convert CO2 and result in the precipitation of calcium carbonate [65-67]. CA is involved in the calcification of human tissues including bone and soft-tissue calcification [65]. Carbon dioxide conversion by the CA enzyme provides bicarbonate and hydrogen ions that fuel the uptake of ionised calcium, which is then deposited in the body tissues as calcium carbonate. Increased CO2 in the blood caused by breathing elevated levels of the gas could lower the pH enough to increase the activity of CA thereby potentially increasing calcium carbonate deposits [67]. Significant tissue calcification has been observed in animals after a 2-week exposure to 1% CO2 or an 8-week exposure to 0.5% CO2 with only slight reductions in pH [68]. This would occur by CA activity where tissues connect with plasma, e.g. arteries, kidneys or even the placenta. A placenta calcification is associated with a higher risk of adverse pregnancy outcomes [69-71]. This mechanism appears plausible as the final damaging step in the maternal body.
In addition, carbon dioxide is also known to play a role in oxidative stress caused by reactive oxygen species (ROS) [72]. This would impede fetal body development. In particular, oxidative damage to cellular DNA can lead to mutations [16,72].
Moreover, inflammation is a serious illness that is known to be caused by low-level CO2 exposure in humans and animals [16,73-76]. CO2 increases the result in higher levels of pro-inflammatory Interleukin-1β, a protein involved in regulating immune responses, which causes inflammation and vascular damage [73]. In this case, both fetal as well as maternal vascular damages are to be expected.

3.2.2. Neurotoxicity

To figure out the negative impact of poor indoor air quality on early brain development a research study exposed pregnant rats [77] to carbon dioxide levels of 0.1 to 0.3 %, which is unfortunately commonplace in poorly ventilated closed buildings [15]. At an exposure of 0.3% carbon dioxide for the pregnant rats the pups demonstrated reduced spatial learning and memory at the age of approx. 6 weeks [77]. This reduced spatial learning and memory was attributed to histologically proven damaged neurons in a part of the brain called the hippocampus [77]. This damage is irreversible and it affects mental health in the long term. When the pregnant rats were exposed to just 0.1% CO2 the pups demonstrated increased anxiety [77], which is even more pronounced when the dams were exposed to 0.3% CO2.
Carbon dioxide exposure, depending on its duration and intensity can cause oxidative stress [78]. Oxidative stress mediates apoptosis by forming lipid hydroperoxides that are highly toxic and cause DNA fragmentation [79]. This condition causes mitochondrial damage, which can lead to a release of Cytochrome C, Caspase activation and finally cell death [80].
Low indoor air quality in classrooms is well known to be associated with a negative impact on the learning capacity of school children [15,16,76]. To establish whether this only indicates a short-term effect or possible substantial damage to brain function, a study in mice was performed and published [81]. Adolescent mice were exposed 24 hours a day for 7 weeks to a level of 0.3% carbon dioxide, but with normal atmospheric levels of oxygen [81]. At the end of the study a so-called water maze exercise was performed. Here the mice have to find a life-saving platform in a water basin. This test distinguishes between impact on physical function and on mental function. Mice were tested on four consecutive days. On the first test day mice in all groups (carbon dioxide exposed and normal air exposed) typically needed around 40 seconds to find the platform. Healthy mice exposed to normal air learned to find the platform more quickly and after four days the healthy mice finally only needed 20 seconds to find the platform, whereas the carbon dioxide exposed mice were unable to learn the shortest way to the platform. Although the carbon dioxide exposed mice were able to swim as quickly as their healthy controls, they were not able to learn the shortest route. They swam around in a very disoriented manner day after day of the four test days. Histology tests demonstrated apoptosis of brainstem neurons in those 0.3% carbon dioxide exposed mice [81]. This is a very disturbing finding because this CO2-induced loss of neurons is irreversible.
When exposure to low level CO2 is prolonged (several hours to one week) the organism depletes its buffer systems [81-84]. The number of cells in the brain of adolescents is a result of the equilibrium of cell proliferation and apoptosis. External factors can affect both cell proliferation and death. In the case of prolonged low-level CO2-exposure the latter occurs, especially under exercise or stress [85-88]. Blood carbon dioxide concentration exerts an important influence on intra- and extracellular pH, CO2 passes quickly through the cell membranes to form carbonic acid with H2O, which releases H+ ions and, in excess, causes acidosis [89-91]. Acidosis decreases transmembrane Ca+2 conductivity and decreases the excitability of neurons [92,93]. Calcium overload causes excitotoxicity and apoptosis during hypoxia [94].

3.2.3. Male reproductive toxicity

As a rise in carbon dioxide when wearing a mask is scientifically proven (Tables 1 and 2) [18-27,29-44], further information about the phenomenon of the toxicological influence of elevated carbon dioxide of inhaled air on male fertility needs to be discussed. The toxic effects of low level carbon dioxide exposure on male fertility have been studied extensively in animal experiments. The exposure of adolescent rats to a carbon dioxide level of 2.5% once for four hours induced pathological signs of diminished fertility in rat testes [95]. A correct estimation of an exposure limit from animal toxicity studies to humans requires implementation of a safety factor [59,60,96]. One has to consider that small laboratory animals, evolutionarily adapted to living in burrows and caves, are limited as indicators for carbon dioxide, since they are much less sensitive to it than humans [57]. As aforementioned, the US Navy was using a safety factor of 3 from a level with no adverse effects on rat pregnancies [59,60]. In the study referred to on rat testicular function of carbon dioxide no so-called NOAEL (No-Observed-Adverse-Effect-Level) was observed [95]. Using the 2.5% level with marked damage to testes function and a minimum safety factor of 5, an exposure limit for adolescent males needs to be set at 0.5% for a maximum of 4 hours a day [59,60,95,96].
The damaging mechanism of CO2 affecting testicular tissues is based on the conditions of oxidative stress and acidosis with increased inflammation and apoptosis as described above [72,73-76,78,79]. Testes metabolism and cell respiration have been shown to be increasingly inhibited by rising levels of CO2 [95]. It has to be pointed out here that this data on the toxicity of carbon dioxide on reproduction has been known for 60 years. Exposure limits have therefore typically been set at 0.5% CO2 in working environments, e.g. according to a Safety Data Sheet by Linde Company on Exposure Limits [97]. These limits are based on EU Indicative Exposure Limit Values in Directives 91/322/EEC, 2000/39/EC, 2006/15/EC, 2009/161/EU, 2017/164/EU. An 8-hour exposure limit of 0.5% CO2 has been defined in the NIOSH regulations [17]. Looking at the potential damage to the reproduction function by subacute or chronic carbon dioxide exposure proven in animal experiments makes it very clear why these limits exist.
Table 3 sums up the significant toxicity of inhaled carbon dioxide at low levels in animal studies.