Santicizer 2148

Neurotoxicity

Description of key information

Delayed Neurotoxicity: does not produce delayed neurotoxicity in hens.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-01-23 to 1990-03-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 418 (Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

hen

Strain:

other: gallus gallus domesticus

Sex:

female

Details on test animals or test system and environmental conditions:

41 healthy young adult white leghorn hens (Gallus gallus domesticus), 17 months old, and weighting a mean of 1.53 kg (range 1.40 – 1.70 kg, Feather Down Farm, Raleigh, NC) were used.

Hens were vaccinated against Marek’s disease at hatching, and for Newcastle-Bronchitis at 10 days of age, along with a booster at 8 weeks of age, and were vaccinated at 18 weeks of age against Laryngotracheitis.

The hens were uniquely numbered (685-690 and 701-716) with metal leg tags. the hens were pre-designated to treatment groups by using randomisation tables.
Groups of 4 hens were placed in 3×3×3 ft stainless-steel cages in a humidity and temperature controlled room with a 12 hour artificial light, 12 hour dark cycle before and during the study.

Birds were provided with a fee supply of feed (Layena Poultry Feedm Ralston Purina Co., St. Louis, MO) and water

Route of administration:

oral: unspecified

Vehicle:

unchanged (no vehicle)

Details on exposure:

A group of 6 hens was given a single oral dose of 5000 mg/Kg/bw of XP-2563 without a vehicle.
A group of 5 hens was similarly treated with a single oral dose of 750 mg/Kg/bw of TOCP and served as a positive control for delayed neurotoxicity.
A third group of 5 hens was not treated and used as a control.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Single oral dose

Dose / conc.:

5 000 mg/kg bw/day

No. of animals per sex per dose:

A group of 6 hens was given a single oral dose of 5000 mg/Kg bw of XP-2563 without a vehicle.
A group of 5 hens was similarly treated with a single oral dose of 750 mg/Kg bw of TOCP and served as a positive control for delayed neurotoxicity.
A third group of 5 hens was not treated and used as a control.

Control animals:

yes, concurrent no treatment

Details on study design:

XP-2563 was orally administrated at a dose level of 5000 mg/kg/bw to 5 unprotected hens without atropine sulphate or pyridine-2-aldoxime dimethyl-chloride (2-PAM) against anticholinesterase effects. Treated hens were kept for observation for 14 days prior to termination.

Dose level of 5000 mg/kg/bw was used to study the effect of XP-2563 on hen brain NTE and AChE and plasma BuChE.

24 hours after treatments, treated and control hens was anesthetised with carbon dioxide and killed by heart exsanguination followed by decapitation and dissection of the brain.

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: Yes
Brain NTE was determined by the differential assay of phenyl valerate hydrolyzing activity using paraoxon and mipafox as selective inhibitors. In two tubes a 50 µL aliquot of the 10% brain homogenate was incubated with a 40 µM paraoxon and 50 mM Tris-HCl buffer containing 0.1 mM disodium EDTA, pH 8.0, in a total volume of 2 mL at 37C for 20 min. Two similar tubes that contained an additional 50 µM Mipafox were incubated for 20 mins at 37C.

Following the initial incubation period, a 2 mL substrate solution consisting of 1.4 mM phenyl valerate in 0.03% Triton X-100 an 3.3% of dimethyl formamide was added and incubated for another 15 min at 37C. The enzymatic reaction was stopped with 2 mL of stopping solution containing 1% sodium dodecyl sulfate and 0,025% 4-aminoantipyrine. Finally, a 0.5 mL of 0.8% potassium ferricyanide solution was added, followed by a 2 min wait, and then the coloured solution was read at 510 nm. NTE activity was expressed as nanomoles of phenyl valerate hydrolyzed per minute per mg of brain protein.

CHOLINESTERASE ACTIVITY: Yes
The brain AChE activity was determined by measuring the initial rate of acetylthiocholine (ATCH) hydrolysis at 37°C. A 0.02 mL sample of the 10% brain homogenate in the sodium phosphate buffer was added to a reaction mixture containing 1.0 mM ATCH, 100 mM NaCl, 20 mM MgCl2, 20 mM sodium phosphate buffer (pH adjusted to 8.2) and 1.0 mM 5,5-dithiobis-2-nitrobenzoic acid (DTNB) in a final volume of 4 mL. Parallel blank incubations were carried out in the same buffer without acetylthiocholine. The initial hydrolysis rate of ATCH was measured at an absorbance of 412 nm. Brain AChE activity was expressed as micromoles ATCH hydrolyzed per minute per milligram of brain protein.

OTHER:
PLASMA BUTYRYLCHOLINESTERASE
Plasma BuChe activity was assayed by adding 0.02 mL of plasma to a reaction mixture consisting of 0.2 mM butyrylthiocholine (BuTCh), 40 mM MgCl2, 4 mM Tris buffer (pH adjusted to 7.4), and 0.1 mM DTNB in a final volume of 4.0 mL. Initial rate of hydrolysis of BuTCh was measured spectrophotometrically at 412 nm. Plasma BuChE activity was expressed as micromoles of BuTCh hydrolyzed per minute per milligram of plasma protein.

PROTEIN DETERMINATION
Brain and plasma protein levels were determined by the Lowry protein assay, using bovine serum albumin (BSA) as the standard. All spectrophotometric measurements were carried out in a Shimadzo Model 3000 UV-Vis spectrophotometer.

Sacrifice and (histo)pathology:

24 hours after treatments, treated and control hens was anesthetised with carbon dioxide and killed by heart exsanguination followed by decapitation and dissection of the brain.

Positive control:

A group of 5 hens was similarly treated with a single oral dose of 750 mg/kg/bw of TOCP and served as a positive control for delayed neurotoxicity.

Statistics:

The differences between enzyme activity of control and treated hens were assessed by the Student's t-test. A p value of 0.05 or less was considered significant.

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Neither brain NTE nor AChE was significantly inhibited by XP-2563. Only plasma BuChE was significantly inhibited and had activity of 53.77% (46.23% inhibition; P <0.001, P value of 0.05 or less was considered significant) of the controls.

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

The acute effects of a single oral dose of 5000 mg/kg/bw of XP-2563 or a single dose of 750 mg/kg/bw of TOCP on brain NTE and AChE and plasma BuChE were determined 24 hours after administration.

Neither brain NTE nor AChE was significantly inhibited by XP-2563. Only plasma BuChE was significantly inhibited and had activity of 53.77% (P <0.001, P value of 0.05 or less was considered significant) of the controls. In contrast, the OPIDN-producing TOCP inhibited hen brain NTE with activity of 10.12% of control (P<0.001) and brain AChE which had activity of 90.83% (P<0.05) of control. TOCP also inhibited plasma BuChE with activity of 11.22% (P<0.001) of the control

The result that the 5000 mg/kg/bw of XP-2563 was not fatal to the treated hens in consistent with the finding that this dose did not significantly inhibit brain AChE, the target for cholinergic effect of organophosphorus esters, nor did this dose inhibit hen brain NTE. These results suggest that XP-2563 does not have the potential to produce delayed neurotoxity (OPIDN) in hens. An inhibition by approx. 75% or greater of hen brain NTE one day after dosing is used as an indicator for the ability of an organophosphorus compound to produce OPIDN

The dose of 750 mg/kg/bw, of TOCP, the positive control for delayed neurotoxicity, resulted in 89.88% inhibition of hen brain NTE. NTE, an enzyme that has been proposed as the putative target for OPIDN, is inhibited by delayed neurotoxic agents but not by nondelayed neurotoxic organophosphorus compounds. Both XP-2563 and TOCP resulted in severe inhibition of plasma BuChE, an enzyme with unknown biochemical or physiologic functions. Neither brain AChE, nor plasma BuChE seem to play a direct role in the development of OPIDN.

Key result

Dose descriptor:

dose level: 5000 mg/Kg bw

Effect level:

5 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

neuropathology

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

5 000 mg/kg bw (total dose)

System:

central nervous system

Organ:

brain

Effect of a single oral dose of 5000 mg/kg XP-2563 (alkyl diphenyl phosphate ester/dialkyl phenyl phosphate ester mixture) or 750 mg/kg TOCP^aon the activity of hen brain neurotoxic esterase (NTE) and acetylcholinesterase (AChE) and plasma butyryl cholinesterase (BuChE)^b

Enzyme^b	Control		XP-2563		TOCP
	Enzymatic activity	%	Enzymatic activity	%	Enzymatic activity	%
Brain NTE	22.33 ± 0.98	100.00 ± 4.3	21.60 ± 0.50	96.73 ± 2.23	2.26 ± 0.36^c	10.12 ± 1.71^c
Brain AChE	24.0 ± 1.2	100.00 ± 5.0	23.8 ± 0.5	99.17 ± 2.08	21.8 ± 0.7	90.83 ± 2.92^d
Plasma BuChE	0.6928 ± 0.1263	100.00 ± 18.23	0.3725 ± 0.0792^c	53.77 ± 11.43^c	0.0777 ± 0.0243^c	11.22 ± 3.51^c

 

^a A group of 6 hens was given a single oral dose of 5000 mg/kg bw of XP-2563 and a group of 5 hens was given 750 mg/kg bw of TOCP. Treated and untreated control hens were killed 24 hours after dosing

^b Enzymatic activity is expressed as: brain NTE, nmoles phenyl valerate hydrolysed/mg brain protein/min; brain AChE, nmoles ATCH hydrolysed/mg brain protein/min; plasma BuChE, nmoles BuTCh hydrolysed/mg plasma protein/min

^c P<0.001

^d P<0.05

Conclusions:

This study demonstrates that a single oral dose of 5000 mg/kg/bw of XP-5263 did not significantly inhibit hen brain neurotoxic esterase 24 hours after administration. the results suggest that XP-2563 does not produce delayed neurotoxicity in hens.

Executive summary:

The effect of a single oral dose of XP-2563 on white leghorn hen brain neurotoxic esterase (NTE) and acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BuChE) and plasma butylcholinesterase (BuChE) was determined 24 hours after dosing. A 5000 mg/kg bw dose was used since it exceeded the LD₅₀dose in unprotected hens, against the acute cholinergic effects of XP-2563. The dose caused statistically significantly inhibition of plasma BuChE activity (46.23% inhibition), yet neither brain NTE nor AChE was significantly inhibited by this treatment. The inhibition of 3.27% of brain NTE in the present study suggests that XP-2563 does not produce delayed neurotoxicity in hens since an inhibition of approx. 75% of brain NTE 24 hours after dosing is required to have the potential to produce delayed neurotoxicity. This conclusion is supported by the findings that a delayed neurotoxic dose of tri-o-cresyl phosphate (TOCP) significantly inhibited brain NTE (89.88% inhibition), plasma BuChE (88.78% inhibition) and brain AChE (9.17% inhibition), neither of which is directly involved in the development of delated neurotoxicity.

This study demonstrates that a single oral dose of 5000 mg/kg/bw of XP-5263 did not significantly inhibit hen brain neurotoxic esterase 24 hours after administration. the results suggest that XP-2563 does not produce delayed neurotoxicity in hens.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

5 000 mg/kg bw/day

Species:

hen

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The effect of a single oral dose of XP-2563 on white leghorn hen brain neurotoxic esterase (NTE) and acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BuChE) and plasma butylcholinesterase (BuChE) was determined 24 hours after dosing (Monsanto Company, 1990d). A 5000 mg/kg bw dose was used since it exceeded the LD₅₀ dose in unprotected hens, against the acute cholinergic effects of XP-2563. The dose caused statistically significantly inhibition of plasma BuChE activity (46.23% inhibition), yet neither brain NTE nor AChE was significantly inhibited by this treatment. The inhibition of 3.27% of brain NTE in the present study suggests that XP-2563 does not produce delayed neurotoxicity in hens since an inhibition of approx. 75% of brain NTE 24 hours after dosing is required to have the potential to produce delayed neurotoxicity. This conclusion is supported by the findings that a delayed neurotoxic dose of tri-o-cresyl phosphate (TOCP) significantly inhibited brain NTE (89.88% inhibition), plasma BuChE (88.78% inhibition) and brain AChE (9.17% inhibition), neither of which is directly involved in the development of delated neurotoxicity.

This study demonstrates that a single oral dose of 5000 mg/kg/bw of XP-5263 did not significantly inhibit hen brain neurotoxic esterase 24 hours after administration. The results suggest that XP-2563 does not produce delayed neurotoxicity in hens.