O,O,O-triphenyl phosphorothioate

Neurotoxicity

Description of key information

No indication of organophophorus induced delayed neurotoxicity was seen in hens treated with 30 mg/kg bw/day of the test substance.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 419 (Delayed Neurotoxicity of Organophosphorus Substances: 28-Day Repeated Dose Study)

Deviations:

yes

Remarks:

(90 days of exposure as opposed to 28 days, dosing frequency only 5 days/week, only one concentration tested)

GLP compliance:

not specified

Limit test:

yes

Specific details on test material used for the study:

- Name as cited in the publication: Triphenylphosphorothionate (TPPT)
- Purity: 100%

Species:

hen

Strain:

other: Leghorn

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Seaboard, Inc (Roanoke, VA)
- Age at study initiation: at least 1 yr
- Weight at study initiation: 1.1-1.8 kg
- Housing: 4 per cage (2 x 2x 2 ft)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS: no data

Route of administration:

oral: gavage

Vehicle:

other: synthetic polyol based engine oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 wks

Frequency of treatment:

once daily, 5 times per week

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

polyol based lubricating oil containing the 3% of TPPT was administered at a dose of 1 g/kg bw (1.02 mL/kg bw) = 30 mg TPPT/kg bw

No. of animals per sex per dose:

17-20

Control animals:

other: yes (saline)

Details on study design:

- Dose selection rationale: based on the EPA guidelines for delayed neurotoxicity of organophosphorus substances

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: signs of behavioural changes or physical changes. e.g mobility scores (according to Cavenagh 1954) were assigned based on the abilities of the hens to move around the cage. 0 = normal; 8 = paralysed

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice weekly
- Parameter checked: ataxia (the ability to climb an inclined walkway (3 ft, 20°) was used as measure). Scoring using an 8 point scale was used (0 = normal; 8 = paralysed).

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY AND CHOLINESTERASE ACTIVITY: Yes
- Time schedule for examinations: after 6 weeks and 13 weeks of dosing
- How many animals: 4
- Method of sample collection and processing: no data
- Tissues used: brain and spinal cord
- Animals fasted: no data
- Description of methodology for NTE determination: as described in Sprague et al 1981 (J. Toxicol. Environ. Health 8, 507-518)
- Description of methodology for AChE activity determination: as described in Ellman et al 1961 (Biochem. Pharmacol. 1, 88-95)

Sacrifice and (histo)pathology:

- Time point of sacrifice: at termination, with heparin (1000 U/ml) and sodium pentobarbital
- Number of animals sacrificed: all animals
- Parameters measured: no data
- Brain weight: no data
- Length and width of brain: no data
- Procedures for perfusion: with room temperature 5% glutaldehyde in 0.1 M phosphate buffer, pH 7.4
- Number of animals perfused: all animals at termination
- Tissues evaluated: brain (forebrain, cerebellum, pons), spinal cord (cervical, thoracic, lumbar), sciatic nerve segment and tibial nerve branch
- Methodology of preparation of sections: no data
- Embedding media: Epoxy and paraffin embedding
- Type of staining: toluidine blue - safranin (epoxy embedded tissue), hematoxylin and eosin (paraffin embedded tissues)
- Thickness: 1 µm (epoxy embedded tissues), 8 µm (paraffin embedded tissues)
- Number of sections: no data
- Number of animals evaluated from each sex and treatment group: no data

OTHER
- Scoring of sections was semi-quantitative and based upon the intensity of degenerating myelinated fibers. Degeneration changes were graded according to a severity scale ranging from 1 - 4
- Scoring was performed blinded

Positive control:

- Name: TOCP (tri- ortho-tolyl phosphate), administered by gavage in corn oil
- Dose 1: 7.5 mg/kg bw given once a day, 5 days/week for 13 weeks
- Dose 2: 500 mg/kg bw given once, 12 days before the end of the study

Statistics:

Analysis of variance was used to compare differences among groups, followed, if indicated by Duncan's test with p < 0.05 considered statistically significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Symptoms indicative of delayed neurotoxicity were not observed in the hens treated with the test susbstance. Mean scores obtained from cage side observations and the inclined walkway did not indicate the presence of ataxia or difficulties in standing and or walking in the treated birds. In contrast the positive control substance induced the expected responses; with a statistically significant (p < 0.05) increase in the mean cage side scores 39 days into the study. A statistically significant (p < 0.05) difference in the ability to climb the inclined walkway was also noted on day 88 in the positive control group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Necropsy and neuropathology was performed on all animals that died. Mortality was judged as not directly related with the administration of the jet oil. A variety of avian diseases were identified as being causal for death.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights were not adversely affected over the 13 week dosing period. There was no statistical significant difference between the negative control (saline), positive control and the test formulation. Although the mean body weight of the positive control was less at week 13 than in week 1, this decrease was not statistically significant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- NTE activity: While NTE activity was not influenced after 6 weeks, the test substance caused a statistically significant (p< 0.05) inhibition of NTE activities after 13 weeks compared to the saline controls (22.6 % in the brain and 17.5 % in the spinal cord). Although for subchronic studies, the threshold generally considered necessary for the induction of organophosphate induced delayed neurotoxicity (OPIDN) has not been delineated, the level of brain NTE inhibition is well below the threshold of 70 % inhibition necessary for the induction of OPIDN in hens following acute dosing (Johnson 1982, Rev. Biochem. Toxicol. 4, 141-212; Johnson 1990, Toxicol. Appl. Pharmacol. 102, 385 -399). For subchronic studies, this threshold has been suggested by Johnson (1990) to lie in the range of 45 - 65%). The findings of the NTE assay are in line with the lack of neuropathological lesions in hens treated with the test substance. The positive control TOCP induced the expected response. NTE activity in TOCP treated hens was significantly (p < 0.05) reduced both at the 6 week as well as the 12 week sacrifice compared to controls. The inhibition was stronger at 13 weeks both in the brain (76 %) and spinal cord (50 %) than at 6 weeks (50 and 43 %, respectively).
- AChE activity: No test substance dependent effect was detected. The positive control substance also did not inhibit AChE activity either. Neither after 6 weeks nor after 13 weeks. This is an appropriate response because it permits the study of OPIDN without confounding effects of cholinergic poisoning.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Microscopic examination of the central and peripheral nervous system of hens treated with the test substance did not reveal lesions characteristic of OPIDN. The positive control substance (TOCP) in contrast caused pathological changes consistent with OPIDN in all birds. Bilateral degeneration of the myelinated nerve fibres was the most conspicuous finding. This effect was observed in both the peripheral and the central nervous systems and was associated in the CNS with signs of gliosis. CNS lesions were seen in the lumbar levels of the medial pontine spinal and rostral (cervical) levels of the spinocerebellar tracts and fasciculus gracilis. These lesions consisted of dense-staining collapsed myelinated axons with associated myelin-rich masses. In addition, some myelinated fibers had swollen, edematous and/or debris-laden axons. A striking feature was the association of gliosis with these neuropathic events. The peripheral nerve lesions (tibial nerve branch) consisted of various stages of Wallerian like degeneration. The peripheral nerve lesions were present in all positive controls but none in any the hens treated with the test substance.

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Only dose tested

Remarks on result:

other:

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subchronic

Species:

hen

Quality of whole database:

A published study comparable to OECD TG 419 with acceptable restrictions is available (Klimisch score: 2).
In addition, a published in vitro study is considered (Klimisch score: 2).

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

In an in vivo study comparable to OECD guideline 419, 17 – 20 hens were treated with synthetic polyol based engine oil containing 3% of the test substance. This corresponds to a dose of 30 mg/kg bw/day. The hens were treated once daily, 5 days per week for the duration of 90 days via gavage. Control hens received saline solution. Tri-ortho-tolyl phosphate, administered by gavage in corn oil was included as the positive control.

Besides cage side and detailed clinical observations, body weights of the treated animals were also monitored. In addition, to appraise organophosphate induced delayed neurotoxicity (OPIDN), the activities of two enzymes were determined, i.e. neuropathy target esterase (NTE) and cholinesterase (AChE) isolated from brain (forebrain, cerebellum, pons) and spinal cord (cervical, thoracic, lumbar); measurements conducted after 6 and 13 weeks of dosing. Histopathological observations were performed in the following tissues: brain (forebrain, cerebellum, pons), spinal cord (cervical, thoracic, lumbar), sciatic nerve segment and tibial nerve branch.

Mortality and body weights were not influenced. Clinical symptoms indicative of delayed neurotoxicity were not observed in the hens treated with the test substance. Mean scores obtained from cage side observations and the inclined walkway did not indicate the presence of ataxia or difficulties in standing and or walking in the treated birds. In contrast the positive control substance induced the expected responses; with a statistically significant (p < 0.05) increase in the mean cage side scores 39 days through the study. A statistically significant (p < 0.05) difference in the ability to climb the inclined walkway was also noted on day 88 in the positive control group.

While NTE activity was not influenced after 6 weeks, the test substance caused a statistically significant (p< 0.05) inhibition of NTE activities after 13 weeks compared to the saline controls (22.6 % in the brain and 17.5 % in the spinal cord). Although for sub-chronic studies, the threshold generally considered necessary for the induction of organophosphate induced delayed neurotoxicity (OPIDN) has not been delineated, the level of brain NTE inhibition is well below the threshold of 70 % inhibition necessary for the induction of OPIDN in hens following acute dosing (Johnson 1982, Rev. Biochem. Toxicol. 4: 141-212; Johnson 1990, Toxicol. Appl. Pharmacol. 102: 385 -399). For sub-chronic studies, this threshold has been suggested by Johnson (1990) to lie in the range of 45 - 65%. The findings of the NTE assay are in line with the lack of histopatholocigal findings in the respective tissues in hens treated with the test substance. Microscopic examination of the central and peripheral nervous system of hens treated with the test substance did not reveal lesions characteristic of OPIDN. Hence the NOAEL for neurotoxicity in hen is 30 mg/kg bw/day.  

The positive control TOCP induced the expected response. NTE activity in TOCP treated hens was significantly (p < 0.05) reduced both at the 6 week as well as the 13 week sacrifice compared to controls. The inhibition was stronger at 13 weeks both in the brain (76 %) and spinal cord (50 %) than at 6 weeks (50 and 43 %, respectively). The positive control substance (TOCP) caused pathological changes consistent with OPIDN in all birds as detected via microscopic examination.

 

No test substance dependent effect on AChE activity was detected in treated hens (Daughtrey W et al. 1996). In support of this finding are data from an in vitro cholinesterase assay which indicated no inhibitory effect of the test substance on AChE activity after treatment in vitro with concentrations ranging from 39.06 to 5,000 µg/mL.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification. Based on the criteria laid down in Regulation (EC) No.1272/2008, classification for neurotoxicity is not warranted.