Trixylyl phosphate

Neurotoxicity

Description of key information

Assessment of neurotoxicity is discussed below.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: short-term oral

Remarks:

other: acute and subacute.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 12 2004 to November 28th 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, carried out according to recognised guideline.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6100 (Acute and 28-Day Delayed Neurotoxicity of Organophosphorus Substances)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

hen

Strain:

other: White Leghorn

Sex:

female

Details on test animals or test system and environmental conditions:

Test Animals
Adult White Leghorn hens (approximately 15 months old at arrival) obtained from Charles River Laboratories, Roanoke, IL were used in this study. The animals were certified by the Supplier to be free of Marek's and Newcastle Diseases. A random sample of hens weighed approximately 1.2 to 2.5 kg on the day of arrival. The hens were held in quarantine for 5 weeks, during which time they were observed daily for survival and frequency of laying eggs. At the end of the quarantine period, they were examined carefully to ensure their health and suitability as test subjects. Hens selected for the study were identified by a uniquely numbered leg band that was wrapped around the right leg, and by a cage card bearing the corresponding dose group and animal and study numbers.

Food and Water
Spafas Layer, Cargill Nutrina Feed for Chicken was provided ad libitum. City of Chicago water was supplied ad libitum by means of plastic mason watering jars. No known contaminants were present in the food or water provided to the animals that would have adversely interfered with the outcome of this study. Reports for the food and water analyses are maintained with the facility records.

Housing and Environment
Hens were single-housed in suspended stainless steel cages. Absorbent paper was placed below the stainless steel mesh cage floor to absorb liquids. During the treatment phase of the study, the animal room temperature and relative humidity ranged from 68 to 76°F and 51 to 73%, respectively. Minor excursions in relative humidity outside the protocol-specified range (i.e., 50-70 %) occurred, but did not affect the outcome of the study. Fluorescent lighting in the animal room was on a 12-hour light/dark cycle. The housing and environmental conditions in the animal room were in general agreement with the Guide for the Care and Use of Laboratory Animals (1996).

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

All hens were fasted the night before treatment and their food was returned about 3 hours after dosing.
A single bolus dose of the test material, corn oil or the positive control (TOCP) formulation was administered to hens by oral gavage using a syringe equipped with a stainless steel ball-tipped intubation needle; the dose was based on the most recent body weight. The test material was administered neat at 1.76 ml/kg (equivalent to 2000 mg/kg) body weight, while the TOCP was administered in a corn oil vehicle at a volume of 1.67 ml/kg body weight. The negative control group received a dosing volume of corn oil equivalent to the volume of TOCP (i.e. 1.67 ml/kg body weight).

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable.

Duration of treatment / exposure:

Single dose; 4 days exposure (acute); 21-22 days exposure (sub-acute) - see "Further details on study design", below.

Frequency of treatment:

Single dose

Remarks:

Doses / Concentrations:
2000 mg/kg body weight
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Experimental Design

The study consisted of 10 hens treated with the test material (T# 127); their response was compared to concurrent positive (TOCP) and negative (corn oil) control groups of equal size. The control groups were shared with another concurrent study. A single bolus dose of the test material, com oil or TOCP formulation was administered to hens by oral gavage; the dose was based on the most recent body weight. The test material was administered neat at 1.76 ml/kg (equivalent to 2000 mg/kg) body weight, while the TOCP was administered in a corn oil vehicle at a volume of 1.67 ml/kg body weight. The negative control group received a dosing volume of corn oil equivalent to the volume of TOCP (i.e., 1.67 ml/kg body weight). Approximately 48 hours after administration, 4 hens per group were sacrificed and their brains harvested for evaluation of neurotoxic esterase (NTE) and acetyl cholinesterase (AChE) activity. In addition, NTE activity was evaluated in a section of the spinal cord. The remaining 6 hens per group were held for a 21-22 day observation period, during which their walking behavior was monitored. The animals were then euthanized, and their tissues fixed in situ via whole body perfusion. Representative nervous tissues were collected, processed, and examined microscopically.

Methods

Selection of Subjects: Animals were randomly assigned to groups based upon body weight, physical examination, egg production and walking ability,and the procedure documented in the study notebooks. The body weight variation of hens used in the study did not exceed ± 20-26% of the mean weight.

Fasting: All hens were fasted the night before treatment, and their food was returned about 3 hours after dosing.

Administration: All hens received a single bolus dose of the test, negative or positive control material by oral gavage using a syringe equipped with a stainless steel ball-tipped intubation needle.

Moribundity/Mortality Observation: During the quarantine period, animals were observed at least once daily for moribundity and mortality. During the study, all animals were observed twice daily on weekdays and at least once on weekends for mortality or evidence of moribundity.

Body Weights: All study animals were weighed during quarantine (pretest), weekly during the study, and at termination.

Food Consumption: Food consumption was not measured.

Observations and clinical examinations performed and frequency:

Clinical Observations: A detailed clinical examination was performed on all animals during the quarantine period (pretest), and their walking behavior was evaluated. On the day of dosing, the hens were observed frequently after dosing (2-4 hours) for signs of toxicity. Hens selected for the NTE assay were sacrificed 48 hours after dosing. The general appearance and walking behavior (walking behavior was graded/scored) were evaluated in all surviving hens twice per week for 21 to 22 days. During the evaluation, hens were removed from their cage and subjected to a period of forced walking behavior, which included walking along an inclined plane using a stainless steel ladder-like device.

Specific biochemical examinations:

NTE, Protein and AChE Assay: Approximately 48 hours after dosing, the entire brain and a section of the spinal cord were removed from 4 hens of each group. The brain was evaluated for NTE and AChE activity, while the spinal cord was evaluated for NTE activity only. Calculations for NTE activity, AChE activity, and Protein concentration were as follows:
Neurotoxic esterase (NTE) activity was determined by the difference in absorbance of samples preincubated with Tube A (paraoxon) and Tube B (paraoxon + mipafox).

Neurobehavioural examinations performed and frequency:

Criteria for Positive Neurotoxic Effect: Several published studies have shown a correlation between percent NTE inhibition and the induction of neurotoxicity. Brain NTE inhibition of 70 percent or greater consistently correlates with neurotoxicity, which is expressed as ataxia and degenerative changes to the spinal cord and peripheral nerves. Above this threshold level of inhibition, a dose-response is seen between percent inhibition and severity of the neurotoxic effects. Therefore, in this study, 70 percent or greater inhibition of NTE activity was considered a positive neurotoxic effect.
Since neurotoxins are able to cause morphologic changes to nerve tissue, such tissues were examined for abnormal neuropathology, such as demyelination and axonal degeneration. The diagnostic pathology performed as part of this study is intended to assess treatment-related effects on the central and peripheral nerves.

Sacrifice and (histo)pathology:

Final Disposition of Animals
All hens (excluding those designated for NTE, AChE, and Protein Concentration determinations) were subjected to a complete necropsy. Hens scheduled for necropsy were euthanized using sodium pentobarbital and whole body perfused with glutaraldehyde. A gross examination of the brain, heart, lungs, spleen, liver, kidneys, gastrointestinal tract, urinary bladder, and gonads was performed. Tissues collected at necropsy consisted of the brain (entire), medulla oblongata, spinal cord (cervical, thoracic, and lumbar), sciatic nerve, tibial nerves and branches, and identification (leg band); these tissues (except leg band) were obtained from all hens at necropsy, were designated for histopathology (i.e., 6/group) and were evaluated microscopically by a board-certified veterinary pathologist. The brain and spinal cord were embedded in paraffin, while the other nerve tissues were embedded in plastic according to Pathology Associates (PA) Standard Operating Procedures (SOPs). These methods are consistent with USEPA guidelines and standard histological procedures.

Other examinations:

Not applicable

Positive control:

Yes, single dose 500 mg/kg TOCP in corn oil vehicle

Statistics:

Statistical analysis was confined to the generation of means and standard deviations.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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

Behaviour (functional findings):

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, treatment-related

Other effects:

not examined

Description (incidence and severity):

Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):Not applicable. (migrated information)

Details on results:

Mortality
None of the hens died during the study.

Clinical Observations
One hen in the T# 127-treated group had an abnormal walking score towards the end of the study (it achieved a maximum score of l=doubtful change). Also, one hen in the negative control group had a score of 1, but was later scored as normal. Five of six hens in the positive control group had abnormal walking scores ranging from 1 (doubtful) to 5 (score of 5 = immobile and unable to stand). Abnormal gaits were detected as early as study day 13, and, for the majority of hens in the positive control group, the condition progressed from weakened gait to inability to stand by the end of the study. No other signs of toxicity were observed.

Body Weight Changes
The majority of hens lost weight during the study.

Neurotoxic Esterase (NTE) and Acetylcholinesterase (AChE) Activity
Brain AChE activity was not affected by treatment with either T# 127 or the positive control material, TOCP. Brain NTE activity was inhibited by 72% in the T# 127-treated hens, while spinal cord NTE activity was inhibited by 61% in the same group. As expected, NTE activity was inhibited by 97% in the brain and by 100% in the spinal cord of hens treated with TOCP, indicating that the test system was appropriately responsive.

Gross Necropsy Observations
Overall, necropsy findings were unremarkable. One T# 127 treated hen (animal number 138) had a liver lesion described as dark/pale with multiple foci. Two negative control hens (animal numbers 105 and 109) had fluid in the peritoneal cavity. No other gross lesions were observed in any of the remaining hens.

Neuropathology
TOCP produced the expected changes including axonopathy in cerebellar white tracts, and increased incidence and severity relative to negative controls of axonal/fiber degeneration in the spinal tract at all levels, and an increased incidence and severity of axonal degeneration in peripheral nerves. Hens treated with T# 127 had a few lesions similar to those induced by TOCP, but of a lesser incidence for most tissues and lesser group mean severity for all tissues. Subtle minimal brain lesions of comparable nature were seen in three of six T# 127-treated hens; however, only one of these hens (animal number 138) displayed the hallmark peripheral nerve lesions associated with organophosphorous induced delayed, neuropathy (OPIDN).

Key result

Dose descriptor:

NOAEL

Basis for effect level:

other: Neurotoxic effects were noted at the single 2000 mg/kg dose level; hence it is not possible to state a definitive NOAEL or NOEL for the substance on the basis of this test alone.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Conclusions:

Oral administration of T# 127 at 2000 mg/kg resulted in 72% reduction in brain NTE activity, 61% reduction in spinal cord NTE activity, positive walking scores in 1 of 6 hens, and histological changes similar in nature to those induced by TOCP, but of a lesser incidence for most tissues and lesser group mean severity for all tissues. Subtle minimal brain lesions of comparable nature were seen in three of six T# 127-treated hens; however, only one of these hens (animal number 138) displayed the hallmark peripheral nerve lesions associated with OPIDN.
Based on the results of this study, T# 127 can produce neurotoxic sequella but with a much lesser incidence and severity than those produced by TOCP.

Executive summary:

T# 127 was administered neat, at a single dose of 2000 mg/kg of body weight, to 10 adult hens. Responses in the T# 127-treated animals were compared to those of concurrent positive and negative control groups of equal size(i.e.,10 hens per group). The positive control group was treated with triorthocresyl phosphate (TOCP) at a single dose of 500 mg/kg in a corn oil vehicle, while the negative control group received corn oil only. Approximately 48 hours after treatment, 4 hens per group were sacrificed and their brains harvested for evaluation of neurotoxic esterase (NTE) and acetylcholinesterase (AChE) activity. NTE activity was also evaluated in the spinal cord of the same animals. The remaining 6 hens per group were held for a 21-22 day observation period, during which their walking behavior was monitored. At the end of the observation period, the remaining animals were euthanized, and their tissues were fixedin situvia whole body perfusion with gluteraldehyde. Representative nervous tissues were collected, processed, and examined microscopically.

There were no incidences of mortality during the study. The majority of surviving hens lost weight during the study.

All hens in the positive control group had an abnormal gait which was detected as early as study day 13, and progressed in several hens from weakened gait to immobility by the end of the study. One hen in the T# 127-treated group had minimal change in walking (score of l=doubtful change) towards the end of the study. One hen in the negative control group also had a score of 1, but was later scored as normal.

Brain AChE activity was not affected by treatment with either T# 127 or TOCP, whereas brain NTE activity was inhibited by 72% in the T# 127-treated hens, and maximally inhibited (i.e., greater than 97% inhibition) in the TOCP-treated group. Spinal cord NTE activity was inhibited by 61% in the T# 127-treated hens, and by 100% in the TOCP-treated group.

TOCP produced the expected changes including axonopathy in cerebellar white tracts, and increased incidence and severity relative to negative controls of axonal/fiber degeneration in the spinal tract at all levels, and an increased incidence and severity of axonal degeneration in peripheral nerves. Hens treated with T# 127 had a few lesions similar to those induced by TOCP, but of a lesser incidence for most tissues and lesser group mean severity for all tissues. Subtle minimal brain lesions of comparable nature were seen in three of six T# 127-treated hens; however, only one of these hens (animal number 138) displayed the hallmark peripheral nerve lesions associated with OPIDN.

Based on the results of this study, T# 127 can produce neurotoxic sequella but with a much lesser incidence and severity than those produced by TOCP.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Additional information

Neurotoxic effects were noted at the single 2000 mg/kg dose level; hence it is not possible to state a definitive NOAEL or NOEL for the substance on the basis of the observed tests alone.

Justification for selection of effect on neurotoxicity via oral route endpoint:

See discussion below.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimish et al system. This ranking was deemed appropriate because the reports do not detail a specific method but are conducted to GLP are are well documented, so are deemed appropriate for use in the support of a formal registration. 

Justification for classification or non classification

The substance is already classified under the CLP Regulation (EC No 1272/2008) as follows:

H373: May cause damage to organs <or state all organs affected, if known> through prolonged or repeated exposure <state route of exposure if it is conclusively proven that no other routes of exposure cause the hazard>. adrenals, testes, epididymides, ovaries, liver (females only).

The classification for neurotoxic effects will not cause an increase in the classification; hence it is appropriate to consider the labelling as complete. DNEL’s are already available for lower dose levels; hence the effects and risk of neurotoxicity are addressed by these other calculations.