Tris(2-butoxyethyl) phosphate

Neurotoxicity

Description of key information

A number of organophosphorus compounds have been found to show neurotoxic effects by several mechanisms resulting either in high acute toxicity due to acetylcholinesterase (AChE) inhibition, organophosphate induced delayed neurotoxicity (OPIDN) after acute exposure or in neuropathy from chronic exposure. TBEP has been assessed against these various endpoints.
Two reliability 2 studies, an in vitro assay (Dramlia, 1981) and an in vivo study (in hen (Carrington, 1989), have been chosen as key studies for acute AChE inhibition. One acute neurotoxicity study (Laham, 1985) is used as a supporting study. These studies demonstrate the low potential of TBEP for cholinergic toxicity, while a number of acute toxicity studies reported in section 7.2 also support this conclusion. Carrington (1989) has also been chosen as key study for OPIDN, demonstrating that TBEP does not have potential to cause OPIDN. For chronic neurotoxicity, two reliability 2 oral studies of duration 18 weeks have been chosen. One used the gavage route (Laham, 1984b) while the other (Reyna, 1987) was a dietary study. These studies support the conclusion that TBEP demonstrates neurotoxicity only at high dose levels on repeated exposure.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion

Dose descriptor:

NOAEL

204 mg/kg bw/day

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Additional information

Acute and repeated dose studies on TBEP indicate that the test substance exhibits low toxicity, with limited potential to cause neurotoxicity at high doses. Clinical signs in both acute and repeat dose studies resemble those of cholinesterase inhibition and may be related to electrochemical disturbance associated with high doses. Dietary studies in rats of 18 weeks duration showed only minor electrophysiological effects from daily intake of doses at approximately the threshold for toxicity in acute studies. It is considered that these effects and those from equivalent doses by gavage are not likely to be relevant to repeated low level human exposure. Studies in hens have shown TBEP to have no potential for organophosphate-induced delayed neuropathy.

 

Acute Neurotoxicity

In vitroNeurotoxicity Studies

TBEP was tested for potential to inhibit acetylcholinesterase (AChE) in an in vitro assay using the Ellman method of colorimetric analysis. Assays at 10, 100 and 1000 ppm gave percentage AChE inhibition of 11.94, 14.34 and 10.78% respectively. There was therefore no dose-response and no significant inhibition of brain AChE. TBEP was concluded not to show neurotoxicity in this test system (Dramlia, 1981).

In vivoNeurotoxicity Studies

TBEP was tested for potential to inhibit neuropathy target enzyme (NTE), AChE and plasma butyryl cholinesterase (BuChE) following a single dose of 5000 mg/kg TBEP to hens. 24 hours after treatment there were no clinical signs of neurotoxicity. The hens were sacrificed and brain homogenates tested for esterase inhibition by the method of Ellman. At this high dose there was no effect onNTE. Brain AChE was inhibited by 45% and serum BuChE by 87%. The correlation between the degree of brain AChE inhibition and appearance of cholinergic signs of toxicity (absent) was considered poor (Carrington, 1989).

 

Astudy was undertaken to investigate the physiological and morphological changes induced in rat peripheral nerves following administration of a single oral dose of TBEP to rats (Laham, 1985). Three weeks after administration of a single dose of TBEP at doses of 1000 to 3200 mg/kg for females and 1000 to 9000 mg/kg for males, measurements of nerve conduction velocity (NCV), relative refractory period (RRP) and absolute refractory period (ARP) were conducted in the caudal nerve. Mortality was not formally recorded, but was high for females of 1.75 to 3.2 g/kg groups. Mortality in males was lower. In medium to high dose animals, clinical cholinergic signs were reported during the first week post-dosing, progressively disappeared during the second week in survivors. Dose related reductions in caudal NCV were seen in both sexes and a significant increase in refractory period (both RRP and ARP) were recorded in the two highest dosed male groups. No morphological changes were seen in the sciatic nerves of low dose rats. At higher doses some changes were recorded, including sciatic nerve section degenerative changes in some myelinated and unmyelinated fibres.

The NOAEL for this study was 1500 mg/kg, with LOAELs of 3200 mg/kg for males and and 1750 mg/kg for females.

This study indicated some potential for neurotoxicity from TBEP, but only at dose levels high enough to cause lethality.

 

 

Data from Acute Toxicity Studies

 

The acute toxicity of TBEP has been studied by all routes relevant to occupational exposure, as summarised in theESSfor acute toxicity, Section 7.2. Acute oral LD50 values range from 3000 to 13,000 mg/kg for rat, guinea pig and hen. Some, though not all, studies show females as more sensitive to the effects of TBEP than males. Acute dermal LD50 values in rabbits have been reported to be greater than 5000 mg/kg. Inhalation LC50 values of 30 mg/l for a 1 hour exposure and greater than 6.4 mg/l for a 4 hour exposure have been reported for rats. TBEP therefore exhibits only low acute toxicity by all routes. The signs of toxicity most frequently reported in acute toxicity studies including diarrhoea, tremor, depression and laboured breathing were consistent with cholinesterase inhibition. The lowest level at which minimal degrees of such effects have been reported is 500 mg/kg, but severe effects were observed only at lethal dose levels of 3000 mg/kg upwards. TBEP is therefore considered to be only a weak inhibitor of cholinesterases on acute exposurein vivo.

 

OPIDN

Organophosphate induced delayed neurotoxicity (OPIDN) after acute exposure is characterised by a delay period of 6 to 14 days after exposure, followed by ataxia and paralysis. It is believed to result from the inhibition of neuropathy target enzyme (NTE) by at least 70%. The ageing ofNTEappears also to be necessary for the development of OPIDN. In an acute delayed neurotoxicity study in hens (the standard test for OPIDN), two doses of TBEP given 21 days apart at 5000 mg/kg did not result in any clinical signs of toxicity, or cause damage to peripheral nerves, spinal cord or brain. There was no inhibition ofNTEactivity. It was therefore concluded that TBEP does not have the potential to cause OPIDN, or other any effects related toNTEinhibition (Carrington, 1989).

 

Repeated Dose Neurotoxicity

 

Several repeat dose neurotoxicity studies have been carried out on TBEP at high doses in rats to investigate potential for neurotoxicity from repeated exposure.

 

Subacute studies

 

In a 14 day gavage study in rats, at doses of 0.8 and 1.12 ml/kg bw (814 and 1142 mg/kg) for females and at 0.8 and 2.24 ml/kg bw (814 and 2285 mg/kg) for males, no clinical signs of neurotoxicity were reported. Significant reduction in caudal nerve conduction velocity was observed in high dose females and dose-related increases of refractory (relative and absolute) periods were also observed in all animals immediately after cessation of exposure. After 15 days recovery increases in ARP and RRP remained only in high dose females (Laham 1984a).

The NOAEL in this study was 814 mg/kg bw/day), with LOAEL for females 1142 mg/kg bw/day), based on electrophysiological changes still present after the recovery period.

 

Subchronic Studies

 

TBEP was administered by gavage to rats for 5 days per week for 18 weeks at 0.25 ml/kg (255 mg/kg) or 0.50 ml/kg (510 mg/kg) (Laham 1984b). During the first half of the studies a minority of high dose females showed temporary muscular weakness & ataxia. During the second half of the exposure period, some animals showed cholinergic effects such as breathing difficulties, ataxia, piloerection, lacrimation, increased urination, with more severe effects evident in females than males. Significant reduction in caudal nerve conduction velocity was observed in all groups. Increases of refractory (relative and absolute) periods were also observed in all animals. Both changes were dose related. Sciatic nerve sections showed dose-related morphological changes including degeneration of myelin sheaths, swelling of axons and clumping of electrofilaments.

No NOAEL was established in this study. The LOAEL was 0.25 ml/kg bw/day (255 mg/kg bw/day).

 

In a study to investigate the neurotoxicity reported by Laham et al, rats were exposed to TBEP via the diet for 18 weeks followed by a recovery period of 8 weeks (Reyna et al, 1987)

In this dietary study doses were equivalent to those used by Laham et al (300, 3000 and 10000 ppm, approximately equivalent to 0.02, 0.20 and 0.60 ml/kg bw/day, i.e. 20.4, 204 and 612 mg/kg bw/day). No clinical signs of neurotoxicity were seen, there were no morphological effects reported after the 8-week recovery period and electrophysiological effects immediately following the last exposure were limited to a small but statistically significant reduction in NCV in high dose females. The biological significance of these changes in the absence of morphological or clinical changes was considered questionable.

The NOAEL in this study was reported to be 3000 ppm (equivalent to approximately 204 mg/kg bw/day), with LOAEL for females 9900 ppm (equivalent to approximately 612 mg/kg bw/day)

 

The difference in the results of these two studies of the same duration at similar dose levels was considered likely to be due to the different dosing method. In the Reyna et al dietary study where the rats would have consumed the diet over several hours, the peak serum concentration of TBEP is likely to have been lower than in the Laham et al gavage study. The dietary study is considered to represent more closely the pattern of human exposure.

 

Data on Neurotoxicity from other Repeat Dose Toxicity Studies

 

Several subacute and subchronic studies are reported in section 7.5. The results of these support the conclusion that TBEP is not neurotoxic.

 

In a 14-day study in rats at 1, 10 and 100 mg/kg bw/day TBEP did not demonstrate any neurotoxic effects (Komsta, 1989). In a 28-day study in rats at using diets containing 500, 2000, 7500 and 15000 ppm TBEP (approximately 50, 750 and 1500 mg/kg bw/day) reported by Monsanto (1985a), no clinical signs of neurotoxicity were reported and no TBEP-related effects were observed at necropsy. Saitoh et al (1994) exposed rats to TBEP in the diet at 0.03, 0.3 or 3.0% (approximately 30, 300 or 3000 mg/kg bw/day)for 5 or 14 weeks. Serum cholinesterase activity was significantly decreased in both sexes in the 0.3 and 3.0% groups. The target organ in this study was the liver. No clinical or pathological signs of neurotoxicity were reported. In a 21 day dermal toxicity study in rabbits, application of 10, 100 or 1000 mg/kg TBEP per day 5 days per week resulted in signs of local irritation, but no systemic toxicity or neurotoxicity (Monsanto, 1985b).

 

Taken together, the neurotoxicty studies indicate that TBEP has some limited potential to cause neurotoxicity at high doses. The clinical signs seen in some repeat dose studies resemble those of cholinesterase inhibition, not of characteristic organophosphate poisoning and may have been related to electrochemical disturbance associated with the high dose levels. The dietary studies showing only minor electrophysiological effects at daily doses at approximately the threshold for toxicity in the acute studies, indicate that the findings are not likely to be relevant to repeated low level human exposure.

 

The NOAEL of 204 mg/kg bw/day for 18-weeks dietary exposure (Reyna et al, 1987) has been taken for repeat exposure risk assessment for this endpoint.

Justification for classification or non-classification

The guidance values for classification of a substance with specific target organ toxicity for single exposure according to EU/GHS criteria (STOT Category 2) relate to significant non-lethal acute effects reported at300-2000mg/kg bw/day. Acute studies of TBEP indicated some potential for cholinergic neurotoxicity, but only at lethal doses generally in excess of 3000 mg/kg bw. TBEP is therefore not classified for single-dose neurotoxicity according to the criteria of UN/EU GHS.

The guidance values for classification of a substance as harmful on repeated exposure by the oral route according to the criteria of Annex VI Directive 67/748/EEC(R48/22) relate to effects reported at 50 mg/kg bw/day or below in a 90-day study. The equivalent guidance values for classification according to EU/GHS criteria (STOT Category 2) relate to effects reported at 10-100 mg/kg bw/day in a 90-day study. For TBEP, the no-effect level for neurotoxicity in an 18-week oral dietary study (Reyna et al, 1987) was 3000 ppm (approximately equivalent to 204 mg/kg bw/day). In an oral gavage study of the same length, no NOAEL was established but the LOAEL was 0.25 ml/kg bw/day (255 mg/kg bw/day). Neither these nor any other repeat dose study indicates neurotoxic potential which would lead to classification.TBEP is not classified for repeat-dose neurotoxicity according to the criteria of Annex VI Directive 67/748/EEC or UN/EU GHS.