Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

Neurotoxicity

Description of key information

Neurotoxicity screening (similar OECD 418): no inhibition of NTE and CHE activity

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:

supporting study

Study period:

01 - 17 Dec 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions. No details on recent historical positive control data provided within the report. Reduced number of animals was tested.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

no details on recent historical positive control data provided within the report; reduced number of animals was tested

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 81-7 (Delayed Neurotoxicity of Organophosphorus Substances Following Acute and 28-Day Exposure)

Deviations:

yes

Remarks:

no details on recent historical positive control data provided within the report; reduced number of animals was tested

GLP compliance:

yes

Species:

hen

Strain:

other: Lohmann Selected Leghorn

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Brinkschulte, Senden, Germany
- Age at study initiation: 13 months and 6 days
- Weight at study initiation: 1.4-1.95 kg
- Housing: hens were kept in floor pen (cages with area of 2.9 m²) in a standard henhouse during acclimation period and study duration. Twenty-four hours after application of the test substance and occasionally during study duration, hens were housed in battery cages (cages with area of 0.15 m²). Cages were provided with low-dust wood granules (Ssniff Spezialdiäten GmbH, Soest, Germany). Cleaning and disinfection of cages was performed on a weekly basis.
- Diet: pelleted whole grain leg horn feed ssniff-LVK (Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: approx. 6 months

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1.5
- Humidity (%): ca. 50
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Cremophor EL in destilled water (2% v/v)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: before preparation of dose formulation, the test substance was warmed to 70 °C using a water bath. Then, the warmed test substance was formulated with Cremophor EL (2% v/v) in destilled water and allowed to cool down to ca. 40 °C before application.

VEHICLE
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The analytical verification of the nominal dose in the formulation regarding stability and homogeneity was not performed, since dose formulations were prepared directly before application. Therefore, no significant alteration of the test substance content in formulation was expected to occur.

Duration of treatment / exposure:

Post-application observation period: 24 h (all 3 control animals and 2/6 test animals) and 16 days (3/6 test animals)

Frequency of treatment:

single administration

Remarks:

Doses / Concentrations:
315 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

3 (control group), 6 (test group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: in preliminary tests, the protective effect of antidotes was investigated in order to prevent death due to acute cholinergic effects of the test substance and thus have the possibility to increase the dose for application. Since the tested antidotes atropine and pralidoxim did not show a positive effect, no protective agent was administered and thus no higher dose than 315 mg/kg bw, which corresponds to the LD50 value from acute oral toxicity testing, was selected.

Observations and clinical examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: appearance (feathering, skin, edema, eyes, tear flow, nose flow and salivation, etc.), behaviour (feather care, utering sounds, agitation, aggressiveness, cannibalism, etc.), nervous system (apathy, motility, reflexes, gait, spasmotic state, tremors, etc.), circulation (heart frequency, paleness, etc.), respiration (respiration frequency and depth), posture (abdominal/lateral position etc.), gastrointestinal function (consistency of faeces etc.), and mortality

CLINICAL OBSERVATIONS FOR NEUROTOXICITY:
- Time schedule: twice per week
- Parameters checked: examination of movement coordination (determination of ataxia, paresis and paralysis) in surviving test animal of the main assay and 4 surviving animals of the preliminary studies (doses: 100 + 315, 250, 315 and 355 mg/kg bw/day, respectively).

BODY WEIGHT: Yes
- Time schedule for examinations: at study begin and then once weekly during the study

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: Yes
- Time schedule for examinations: 24 h post-application
- How many animals: all 3 control animals and 2/6 test animals, 3/6 dead animals (unscheduled)
- Method of sample collection and processing: after decapitation, brain and spinal cord were dissected and the brain was cut longitudinally. Both organs were weighed and frozen on dry ice for NTE activity determination.
- Tissues used: brain and spinal cord
- Animals fasted: No
- Description of methodology for NTE determination: determination of NTE activity was performed according to Johnson (1977) and a modified assay of Johnson and Richardson (1983). The substrate for enzymatic reaction was phenyl valerate, which is enzymatically cleaved into phenol and valeric acid in the presence of esterases. Phenol is photometrically determined after chemical reaction with 4-aminoantipyrine and potassium hexacyanoferrate. Since nerve tissue also contains other esterases than NTE, a differential analysis is applied, in which each one sample is either preincubated with paraoxon or with paraoxon and mipafox. The difference of residual activity in both samples refers to the actual activity of NTE.

CHOLINESTERASE ACTIVITY: Yes
- Time schedule for examinations: 0 and 24 h post-application (blood), 24 h post-application (brain)
- How many animals: all 3 control animals (brain, blood), 2/6 test animals (brain), 6/6 test animals (0 h, blood), 1/6 test animal (24 h, blood), 3 dead animals (brain)
- Method of sample collection and processing: after decapitation, brain was dissected, cut longitudinally and frozen on dry ice for CHE activity determination. Blood (1.3 mL) was obtained from the wing vein of the animals after decapitation.
- Tissues/organs used: brain and blood
- Animals fasted: No
- Description of methodology for CHE determination: CHE activity was determined according to the method described by Ellmann et al. (1961). The substrate for enzymatic reaction was acetylthiocholine iodide, which is enzymatically cleaved into thiocholine and acetate in the presence of CHE. After addition of dithiobisnitrobenzoate, thiocholin reacts to 2-nitro-5-mercaptobenzoate, which is determined by photometric analysis.

OTHER: References:
Johnson, M.K. (1977). Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Arch Toxicol. 37(2):113-5
Johnson, M.K. and Richardson, R.J. (1983). Biochemical endpoints: neurotoxic esterase assay. Neurotoxicology 4(2):311-20
Ellmann, G. et al. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7:88-95

Sacrifice and (histo)pathology:

- Time point of sacrifice: after 24 h and 16 days post-application
- Number of animals sacrificed: 3 control animals, 3 test animals, 3 unscheduled deaths
- Brain weight: yes
- Procedures for perfusion: at the end of the post-exposure observation period, the surviving animal of the test group and all surviving animals of the preliminary experiments were narcotised using Nembutal solution and perfused with buffer solution and 4% aqueous formaldehyde solution for exsanguination and fixation, respectively. Afterwards, nervi ischiadici with nervi tibialis and nervi peronaeus communis as well as brain and spinal cord were dissected and fixed in 4% aqueous formaldehyde solution for optional histological examination.

Positive control:

The sensitivity of the assay was verified in independent experiments using tri-o-cresylphosphate (TOCP) as positive control substance , which resulted in NTE activity inhibition of > 90% in all tissues.

Statistics:

Mean values and standard deviations were determined for body weights and NTE and CHE activities.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Up to 3 days post-application, all test animals showed apathy, staggering gait, temporary tremor, rapid respiration, green excreta, increased salivation, spasmotic state, uttering sounds, bristled feathering

Mortality:

mortality observed, treatment-related

Description (incidence):

mortality: 3/6 test animals (50 min, 1h and 3 h post-application)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

test group: Only one test animal showed transient reduction in body weight during the 16-day post-exposure observation period

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

NTE activity (24 h post-application): only minimal, biologically not significant effects (1 and 6% inhibition in brain and spinal cord, respectively) were observed.
CHE activity (24 h post-application): 22% inhibition in blood was determined, but not the brain of 3/6 treated animals

Behaviour (functional findings):

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Mortalities were observed in 3/6 test animals 50 min, 1 h and 3 h post-application, respectively. Clinical signs were observed in all animals of the test group up to 3 days post-application and involved apathy, staggering gait, temporary tremor, rapid respiration, green excreta, increased salivation, spasmodic state, uttering sounds and bristled feathering (for details see Tables 1 under “Any other information on results incl. tables”).

CLINICAL OBSERVATIONS FOR NEUROTOXICITY
Clinical observation for neurotoxicity did not result in any substance-related findings in the surviving animal of the main test as well as the surviving animals of the preliminary experiments during the respective post-exposure observation periods (16, 17, 20, 21 and 22 days). Thus, there were no indications for delayed neurotoxic effects in the test animals.

BODY WEIGHT AND WEIGHT GAIN
Determination of body weight evolution was only feasible in 1 (surviving) animal during the 16-day post-exposure observation period. This animal showed only a transient and slight reduction in body weight on Day 15 of the study.

BIOCHEMISTRY (see Table 2, 3 and 4 under “Any other information on results incl. tables”)
A minimal inhibition of NTE activity in brain (1%) and spinal cord (6%) in 5/6 animals (2 scheduled and 3 unscheduled deaths) was observed 24 h post-application, which is considered to be within the range of normal variation for this parameter. Since no biologically relevant inhibition of NTE activity was observed in brain and spinal cord, the test substance was not considered to have a potential for delayed neurotoxicity.
In unscheduled deaths, 36% inhibition of CHE activity was observed compared to control. However, this value was considered to be doubtful since the time point of sampling of controls (24 h post-application) was not in agreement with the time points of sampling of the unscheduled deaths (50 min, 1 h and 3 h post-application). Furthermore, no corresponding blood CHE activity could be measured in these animals, which might have substantiated the observations in brain. Twenty-four hours post-application, no inhibition of brain CHE was observed in test animals of scheduled sacrifice, although blood activity was reduced by -22% compared to controls in these animals.
In conclusion, only slight inhibition of NTE and CHE activity was observed, which was either in the range of normal variation or of doubtful origin, and thus not considered be biologically significant to indicate delayed neurotoxicity.

GROSS PATHOLOGY
No substance-related findings were observed at gross pathology of test animals.

Dose descriptor:

dose level:

Effect level:

315 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: NTE activity (24 h post-application): only minimal, biologically not significant effects (1 and 6% inhibition in brain and spinal cord, respectively); CHE activity (24 h post-application): 22% inhibition in blood, but not brain of 3/6 treated animals

Remarks on result:

other:

Table 1. Clinical findings in treated animals

Clinical findings at 315 mg/kg bw	Number of animals affected	Duration	Intensity*
Mortality (unscheduled)	3/6	50 min – 3h	n.a.
Apathy	6/6	10 min – 3 days	3
Uttering sounds	1/6	3h – 3 h	1
Staggering gate	6/6	20 min – 3 days	3
Spasmodic state	1/6	45 min – 50 min	3
Temporary tremor	4/6	30 min – 3 h	3
Dyspnoea	1/6	3h – 3 h	n.a.
Increased salivation	2/6	40 min – 1 h	3
Increased salivation	1/6	3 h – 3h	2
Temporarily rapid respiration	1/6	5 min – 3 h	2
Green excreta	3/6	2 days – 2 days	n.a.
Rapid respiration	3/6	30 min – 3 h	2
Bristled feathering	6/6	10 min – 3 days	3
*1 = weak, 2 = moderate; 3 = strong; n.a. = not applicable

 

Table 2. Mean values of activity and inhibition (% of controls) of NTE in homogenates of brain and spinal cord

Dose (mg/kg bw)	Time p.a.	No. animals	Brain		Spinal cord
			Activity (nmol phenyl valerate/min/g tissue)	Inhibition (% of controls)	Activity (nmol phenyl valerate/min/g tissue)	Inhibition (% of controls)
0 (control)	24 h	3	3286	-	820	-
315 (test group)	50 min – 3 h* 24 h	3 2	3252 3333	1 0	767 785	7 4
Total (mean)		5	3285	1	774	6

p.a. = post application; * unscheduled deaths

Table 3. Mean values of activity and inhibition (% of controls) of CHE in brain

Dose (mg/kg bw)	Time p.a.	No. animals	Brain
			Activity (U/g tissue)	Inhibition (% of controls)
0 (control)	24 h	3	27.86	-
315 (test group)	50 min – 3 h* 24 h	3 2	17.96 30.03	36 0

p.a. = post application; * unscheduled deaths

Table 4. Mean values of activity and inhibition (% of controls) of CHE in blood

Dose (mg/kg bw)	Time p.a.	No. animals	Brain
			Activity (U/g tissue)	Inhibition (% of controls)
0 (control)	0 h 24 h	3 3	0.93* 0.97	-
315 (test group)	0 h 24 h	6 3#	0.90* 0.76	22

p.a. = post application; * activity of CHE before application of the test substance; #surviving animals at scheduled death

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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

Neurotoxicity screening

To assess the potential to induce delayed neurotoxicity following acute exposure to Amines, C12-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 96690-34-5), activities of neuropathy target esterase (NTE) and cholinesterase (CHE) were investigated in a GLP-conform screening assay similar to OECD guideline 418 (Bayer AG, 1993). Six Lohmann Selected Leghorns received a single dose of the test material at 315 mg/kg bw in Cremophor EL (2% v/v in distilled water) via oral gavage. In preliminary tests, the protective effect of antidotes was investigated in order to prevent death due to acute cholinergic effects of the test substance and thus have the possibility to increase the dose for application. Since the tested antidotes atropine and pralidoxim did not show a positive effect, no protective agent was administered and thus no higher dose than 315 mg/kg bw, which corresponds to the LD50 value from acute oral toxicity testing, was selected. In addition to the test group, 3 hens received the vehicle only and served as controls. After treatment with the test substance, mortalities were observed in 3/6 test animals 50 min, 1 h and 3 h post-application, respectively. Clinical signs were observed in all animals of the test group up to 3 days post-application and involved apathy, staggering gait, temporary tremor, rapid respiration, green excreta, increased salivation, spasmodic state, uttering sounds and bristled feathering. After 24 h, 2 further animals were sacrificed, and brains and spinal cords of in total 5 animals (including the unscheduled deaths) were dissected and homogenised for the biochemical assessment of NTE and CHE activity. The mean NTE activity in brain, expressed as nmol phenyl valerate/min/g tissue, was 3285 and 3286 for treated and control groups, respectively. In spinal cords, the NTE activity for control and treated group was 774 and 820 nmol phenyl valerate/min/g tissue, respectively. The results showed only a minimal inhibition of NTE activity in brain (1%) and spinal cord (6%) in treated compared to control animals 24 h post-application, which is considered to be within the range of normal variation for this parameter, and thus of no biological relevance. Additionally, CHE activity was assessed in brain and blood of the treated animals and compared to those of controls. In unscheduled deaths, 36% inhibition of CHE activity was observed compared to control. However, this value was considered to be doubtful since the time point of sampling of controls (24 h post-application) was not in agreement with the time points of sampling of the unscheduled deaths (50 min, 1 h and 3 h post-application). Furthermore, no corresponding blood CHE activity could be measured in these animals, which might have substantiated the observations in brain. Twenty-four hours post-application, no inhibition of CHE was observed in test animals of scheduled sacrifice, although blood activity was reduced by -22% compared to controls in these animals. In conclusion, only slight inhibition of NTE and CHE activity was observed, which was either in the range of normal variation or of doubtful origin, and thus not considered be biologically significant. Together with the clinical observations for neurotoxicity showing no substance-related impairment of movement coordination in the surviving treated animal of the main test as well as the surviving treated animals of preliminary experiments during the respective post-exposure observation periods (16, 17, 20, 21 and 22 days), the test substance was not considered to have a potential to induce delayed neurotoxic effects following acute exposure.