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In studies performed in vivo, the same signs of toxicity seen in acute toxicity studies were recorded. Thrombosis were observed in various locations (tail, dental pulp, pulmonary, ocular) sometimes leading to necrosis due to an infarction mechanism. Mechanistic studies have shown that 2 -butoxyethanol (2BE) causes haematotoxicity in vivo in rats and that butoxyacetic acid (BAA) causes the same effects in vitro at very low concentration. When metabolic pathways leading to the formation of BAA were blocked, no effects were seen on red blood cells (RBCs). It can be concluded that the metabolite BAA is responsible of hematotoxicity in vivo and not 2BE itself.

Some species are very sensitive to 2BE- or BAA-induced haemolysis (rat, mouse, hamster, baboon, rabbit) whereas other species are resistant to these effects (dog, guinea pig, pig, cat, humans). Humans are 30 x less sensitive than rats. In animal studies, an increased sensitivity to haemolysis was seen in old animals and in females in vitro and in vivo with BAA, showing that the differences of metabolism between male and female could not explain totally sex difference.

In one study, it was demonstrated that the type of analyser was important to detect early changes in blood parameters (increase of HCT and MCV). An impedance based analyser was more sensitive than a laser based analyser.

In vivo or in vitro, haemolysis was due to a decrease of erythrocyte deformability due to erythrocyte swelling. This also explains the formation of thrombosis. Newly formed erythrocytes were more resistant than old ones. It was also showed that 2BE pre-treatment gave a relative “protection” against higher doses administered later through a shift to younger average erythrocyte age. The data demonstrates an adaptive mechanism of “protection” when animals have a period of recovery time before a re-exposure to EGBE.

The mechanism leading to erythrocyte swelling and loss of deformability is for the moment unclear. There is no evidence of oxidative mechanism on erythrocyte membrane. There is data to support the theory that showed that the mode of action of BAA is via a colloid osmotic lysis of the rat red blood cell and that BAA causes sodium and calcium to enter the cell; that calcium initially has a protective effect via the calcium activated potassium channel which facilitates the loss of potassium thereby, compensating for the osmotic effect of increased cell sodium; that calcium subsequently may have other deleterious effects through activation of proteases and externalization of phosphatidylserine in the exterior leaflet of the membrane.

In humans, slight effects were seen with doses of 8 mM and 4 mM of BAA in vitro. The dog appears to be unusual in that it is sensitive to 2BE but not BAA which suggests that another mechanism is involved for this species.

In conclusion, whilst haemolysis is a critical effect for the common laboratory species of rat and mouse, it is an effect that humans are markedly resistant to. A conservative assessment is that humans are at resistant to at least a factor of 10x the plasma concentration of 2 -butoxyethanol (or its metabolite BAA) and that a toxicodynamic factor of 0.1 is appropriate when carrying out interspecies extrapolations for effects that are mediated by haemolysis.

Sensory irritation

Sensory irritation was evaluated using an in vivo method with mice who were exposed whole body to a series of chemicals (including 2 -butoxyethanol). The measured response was the maximum percent decrease in respiratory rate, averaged over 4 mice, simultaneously exposed for 10 minutes. The responses obtained for various concentrations of solvents were utilized to develop a concentration-response relationship, for this, the concentration associated with a 50 % decrease in respiratory rate (RD50) was calculated. The response obtained within the 150 -1500ppm concentration range tested was less than a 50 % decrease in respiratory rate, ie a relatively shallow dose response curve. The RD50 calculated by extrapolation was 2825 ppm, a value well in excess of the saturated vapour pressure. This was in excellent correlation with a QSAR model for RD50 generated at a later date, which predicts a value of 2818ppm. When compared with the author's criteria of evaluation, 0.01 RD50 (about 28 ppm) would cause minimal or no sensory irritation whereas 0.1 RD50 (about 280 ppm) would cause definite but tolerable sensory irritation. Sensory irritation is not an important property of 2 -butoxyethanol.

Occular toxicity

A study designed specifically to examine the occular toxicity of 2 -butoxyethanol in female rats subjected them to 3 daily doses of 250mg/kg prior to sacrifice and examination of the eyes histopathologically. Following treatment, petechial hemorrhages were noted on the sclera as well as hemorrhages localised in the posterior layers of the retina. Thrombi were identified in ciliary processes and limbal blood vessels as well as disseminated thrombosis, necrosis and infarction of other organs. It is likely however that these changes are secondary to haemolysis.

Liver pathology (mode of action of liver tumours in mice)

The available studies demonstrate that rat hepatocytes in culture are markedly less susceptible to oxidative stress than are mouse hepatocytes. In vivo there appears to be a greater antioxidant reserve in rat liver than in mouse liver, so that reduction in -tocopherol levels observed in both species after 2 -butoxyethanol treatment resulted in levels in rats that always remained higher than even the untreated control level in mice. Also in vivo, male mouse liver is marginally more susceptible than female mouse liver to oxidative damage, as demonstrated by the higher background rate of hemangiosarcoma lesions. In addition, it has been shown that ferrous sulphate can induce DNA damage as well as morphological transformation in SHE cells and these effects can be reduced or abolished by antioxidants. Ferrous sulphate and hemolysed red blood cells can also activate mouse liver endothelial cells whereas 2 -butoxyethanol and its metabolites fail to achieve this.

Forestomach pathology (mode of action of forestomach tumours in mice)

In rodents, including the mouse, rat, and Syrian hamster, the stomach is divided into two parts by the mucoepidermoid junction separating squamous from glandular epithelium. The proximal part, or forestomach, is non-glandular, continuous with the oesophagus, and lined by keratinised, stratified squamous epithelium. The distal part, or glandular stomach, empties into the duodenum and is lined by a specialized glandular epithelium. The forestomach is separated from the glandular stomach by a grossly visible, elevated fold, the limiting ridge. This contrasts with the anatomy of the human stomach, the lining of which is entirely glandular, the human mucoepidermoid junction occurring where the oesophagus joins the stomach. There is thus no forestomach in human beings. There is a histological similarity between the rodent forestomach and the human oesophagus, but the physiological functions of these two organs are quite different. While the forestomach is a storage organ, where ingested material may reside for several hours before its transfer to the glandular stomach, the oesophagus is not, ingested material passing through in a matter of seconds. Exposure of the human (or rodent) oesophagus to ingested material is, therefore, brief and it is to be noted that the histology of the rodent oesophagus is not unlike that of the human organ, yet the oesophagus of the rats and mice exposed to 2 -butoxyethanol in studies described here does not respond in the same way as the forestomach. There is good evidence to suggest that 2 -butoxyethanol can reach the forestomach by routes of intake other than oral ingestion and that it and its principle metabolite 2 -butoxyacetic acid (BAA) are particularly persistent in this organ. There is evidence to suggest that BAA causes persistent irritation and that the metabolic enzymes that create it have an activity and distribution in the mouse forestomach that make it particularly sensitive in comparison to the mouse glandular stomach or the stomachs of the rat and human. The evidence clearly suggests that the findings in mice will not be relevant to humans.