Triphenyl phosphite

Neurotoxicity

Description of key information

Oral: NOAEL ≤ 250 mg/kg bw; hen, non-guidline, non-GLP, K2

Dermal: NOAEL = 50 mg/kg bw; hen, non-guidline, non-GLP, K2

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: oral

Remarks:

other: screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was designed to determine the neurotoxic effects following oral dosing of the test substance to adult hens.

GLP compliance:

yes

Limit test:

no

Species:

other: chicken

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mr. Odlin, Graygable Poultry Service, Bury St. Edmunds, Suffolk
- Age at study initiation: over 14 months
- Weight at study initiation: 2030 - 2915 g
- Housing: floor pens
- Diet: standard HRC laying bird ration in pellet form
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (mean; range 17-30)
- Humidity (%): 73 (mean)
- Photoperiod (7 hrs dark /17 hrs light):

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

single administration, followed by a 21 day observation period

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Group 2

Dose / conc.:

750 mg/kg bw (total dose)

Remarks:

Group 3

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

Group 4

Dose / conc.:

250 mg/kg bw (total dose)

Remarks:

Group 5

No. of animals per sex per dose:

Group 1 (control) : 5 hens
Group 2 (1 g/kg): 10 hens
Group 3 (0.75 g/kg): 10 hens
Group 4 (0.5 g/kg: 10 hens
Group 5 (0.25 g/kg): 10 hens

Control animals:

other: treated with water at the maximum dose volume of this study

Details on study design:

- Dose selection rationale: based on a range finder

Observations and clinical examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days -14, -7, 0* (immediately prior to dosing), 4, 7, 11, 14 and 21.

FOOD CONSUMPTION
group msan food consumption was recorded over weekly intervals throughout the study.

EGG PRODUCTION
all eggs laid during the study were recorded

ATAXIA ASSESSMENT
daily

Neurobehavioural examinations performed and frequency:

The movements of each bird were examined daily to assess muscle coordination. The degree of ataxia was scored on a point award system similar to that described by Cavanagh et al (1961). The scale ranges from 0 to 8, increasing scale number indicating increasing degree of ataxia.

Sacrifice and (histo)pathology:

All birds were subjected to gross post mortem examination. Tissues for histological examination were taken only from birds showing Grade 3 ataxia and upwards at termination of the study. The brain, spinal cord and sciatic nerve from each bird were fixed in 10% neutral buffered formalin, and sections of these tissues ware stained with haematoxylin and eosin.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Shortly after dosing, a number of birds in Groups 2 (1g/kg) and 3 (0.75 g/kg) became unsteady or unable to stand, and within 4 hours following dosing, further birds In all groups were similarly affected. Most surviving birds appeared to have recovered by the end of Day 1, although five birds subsequently developed ataxia, and further mortalities were recorded. On Day 6 one bird In Group 5 (0.25 g/kg) was sacrificed as it had been bullied by other birds. On Day 15 one bird In Group 3 also showed signs of being bullied, but Its condition improved before the study was terminated.

Mortality:

mortality observed, treatment-related

Description (incidence):

5 hens of group 2 (1g/kg), 4 hens of group 3 (0.75g/kg), 3 hens of group 4 (0.5g/kg) and 1 hen of group 5 (0.25 g/kg) died. Most mortalities occurred within 24 hours after dosing. No mortalities occurred in the control group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Over Days - 7 to 0 , all groups showed mean bodyweight losses. Over Days 0 to 7 following dosing, most birds Increased In bodyweight, although mean weight increases in Groups 2 and 3 were smaller than in the other groups. During the remainder of the study, bodyweight changes were variable, but were considered to be within normal limits.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption figures were variable, partly because of spillage which was impossible to measure. However, there was a general tendency for food consumption to increase as the study progressed. No clearly defined differences were observed between the results for the test and control groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A small number of birds showed abnormalities, which included a red appearance to the intestine In two of the birds that died following dosing. However, no abnormalities were detected in the majority of birds.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Two birds from Group 5 that showed Grade 4 and Grade 7 ataxia were examined, and both showed morphological evidence of neurotoxicity. The neuropathological changes observed ware reasonably consistent with the clinical ataxia gradings, and indicated a treatment-related effect.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

ATAXIA ASSESSMENT
Three birds from Group 4 and two birds from Group 5 developed ataxia, although no neurotoxic signs were observed In any surviving birds from Groups 2 and 3, which were dosed at higher levels. The first positive signs of ataxia (Grade 2 and upwards) appeared on Day 10, and the grades reached at death or termination ranged from 2 to 7.

EGG PRODUCTION
In Group 5, egg production was variable. In Group 3 and the control group, production was low during the settling-in period and the first week following dosing, but increased thereafter. Overall production by Group 5 birds was slightly lower than that of the control birds, and this was in turn slightly lower than that of Group 3. The mean egg weights of the control group and Group 5 were higher than that of Group 3.

Dose descriptor:

LOAEL

Effect level:

<= 250 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Ataxia/Neuronal signs

Dose descriptor:

NOAEL

Effect level:

< 250 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw (total dose)

System:

nervous system

Organ:

other: nervous system

Conclusions:

Under the conditions of this study, oral administration of the test substance produced clinical signs of neurotoxicity in a number of adult hens dosed at levels of 0.25 and 0.50 g/kg. Among the five birds affected, the two dosed at 0.25 g/kg developed more severe ataxia, and histopathological examination of these birds showed evidence of neurotoxicity. However, surviving birds dosed at the higher levels of 0.75 and 1.0 g/kg showed no signs of neurotoxicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

250 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

Link to relevant study records

Reference

Endpoint:

neurotoxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May and June 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was designed to determine the neurotoxic effects following dermal dosing of the test substance to adult hens.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot number of test material: Supplied by sponsor, 01B46920A
- Appearance: Clear, colorless liquid

Species:

hen

Strain:

other: Gallus pallus domesticus

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mr. M. D. Odlin, Graygable Poultry Service, Bury St, Edmunds, Suffolk
- Age at study initiation: 14 months (adult)
- Weight at study initiation: 2000 - 2440 g
- Housing: In treatment groups in flour pens
- Diet: ad libitum, standard HRC laying bird ration (pellet form, Josph Odom, EYE, Peters)
- Water: ad libitum (automatic drinker)
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: mean 20 - 24°C (range 15 - 28 °C)
- Humidity: 65 %
- Ventilation as required
- Photoperiod: 7 hrs dark / 17 hrs light

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

in all cases the test material was spread over the surface of the comb using a disposable syringe.
Volumes of individual doses were calculated as follows: (Dose level in g/kg / bodyweight in kg)/specific gravity (SG); with SG of test material = 1.184, water = 1, TOCP = 1.17

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Single administration.
Exemption: Because of limits to the maximum dose volume that could be administered in single application, multiple dosing was carried out as shown under "Frequency of treatment".

Frequency of treatment:

Group 3: 1 dose on day 1 (1 x 50 mg/kg bw)
Group 4: 3 doses on day 1 (1 x 100 mg/kg) + (2x 200 mg/kg)
Group 5: 5 doses on days 1- 5 (25 x 200 mg/kg)

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Control (Water)

Dose / conc.:

50 mg/kg bw (total dose)

Remarks:

Group 3

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

Group 4

Dose / conc.:

5 000 mg/kg bw (total dose)

Remarks:

Group 5

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Rationale for animal assignment: Based on body weight (achieving similar initial group mean body weights)

Observations and clinical examinations performed and frequency:

Observation period: 21 days following first treatment

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: Bird health and mortality

BODY WEIGHT: Yes
- Time schedule for examinations: Days -14, -7, 0 (= immideatelyprior to first dosis), 7, 14 and 21

FOOD CONSUMPTION: Yes (group mean)
- Time schedule: weekly (days -7 to 0, 0 to 7, 8 to 14, 15 to 21)

ATAXIA ASSESSMENT
- Time schedule: daily
- The degree of ataxia was scored on a point system, ranging from 0 to 8, with increasing scale number indicating increasing degree of ataxia.

Specific biochemical examinations:

Six days after start of the treatment, and 24h after the occurrence of ataxia in all birds of group 5, blood samples for cholinesterase analysis were taken from eight birds in group 5, and from two negative control birds for comparative purposes. This was followed by intramuscular injection with atropine sulphate (10 mg/kg) in group 5 birds in an attempt to counteract possible cholinesterase inhibition by the test compound.

Sacrifice and (histo)pathology:

- Number of animals sacrificed: All birds
- Number of animals gross examined: All birds
- Number of animals with histopathological examination of tissues: 3 per treatment group (except for group 4 (500 mg/kg) where all 10 animals were examined)
- Tissues evaluated: brain, spinal cord, sciatic nerve
- Embedding media: Fixed in 10% buffered formalin
- Type of staining: Hematoxylin and eosin

Positive control:

Tri-ortho-cresyl-phosphate (TOCP, 250 mg/kg), treated with one dose/day at days 1-2 (2 x 125 mg/kg)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

By the end of day 6, the eyes of all birds in Groups 4 and 5 had become partially or fully closed and did not appear to return to normal in surviving birds until Day 11.

All birds in the negative control group and in Group 3 (50 mg/kg) remained in apparent good health throughout the study.

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

By the end of day 4 following the first treatment, blistering of the comb had developed in all birds in Groups 4 & 5 (500 mg/kg and 5000 mg/kg, respectively).

Mortality:

mortality observed, treatment-related

Description (incidence):

Due to severe neurotoxic signs, all birds of group 5 were sacrificed over days 7 to 9.
Four birds of group 4 had to be sacrificed during day 14 to 18 with high grades of ataxia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Over Days -7 to 0, bodyweight changes were variable. During days 0 to 7, all groups showed mean bodyweight losses which are more marked in Groups 4 and 5 than in the other groups. Over days 7 to 21, group mean bodyweights increased in the negative control groups and in Group 3. However, in the positive control group and Group 4 in which most birds developed ataxia, mean weight decreases occurred, particularly towards the end of the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Over Days -7 to 0, food consumption figures were similar in all groups. Following the start of treatment, food consumption declined rapidly in Group 5, and was noticeably reduced in Group 4 during the remainder of the study. In the positive control group, food consumption decreased as the study progressed. In the negative control group and group 3, all food consumption figures were considered to be within normal limits.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cholinesterase analysis
Plasma cholinesterase concentrations were consistently lower in the blood samples from Group 5 birds than these from the negative controls. Although administration of atropine sulphate produced no apparent change in the condition of the birds, the results indicated that cholinesterase inhibition was occurring.

Endocrine 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:

effects observed, non-treatment-related

Description (incidence and severity):

A small number of birds Groups 1 – 4 showed minor clinical abnormalities, none of which could be attributed to treatment.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

In general, the neuropathological findings had a responsible degree of correlation with the clinical ataxia gradings. Neuropathological changes were observed in all birds in the positive control group and in birds dosed with the test substance at 500 mg/kg. Early changes were observed in one bird dosed at 5000 mg/kg, and changes were also observed in one bird dosed at 50 mg/kg. However, because of the small number of birds examined in the latter group, it is difficult to determine whether the effect was clearly treatment-related.

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Except for brain, spinal cord, sciatic nerve no histopathology was performed. Histopathological findings of these three tissues are reported under "neuropathological findings".

Other effects:

effects observed, treatment-related

Description (incidence and severity):

ATAXIA ASSESSMENT
In group 5, ataxia developed only five days after the first treatment, and was relatively severe in all birds, progressing rapidly to grades 7 and 8. No improvement was observed in the condition of the birds (ataxia) after injection of atropine sulphate. Thus, all birds were sacrificed by day 9.
In group 4, the first signs of ataxia appeared on day 7 and were less severe than in group 5. There was considerable variation in the timing and rate of development of neurotoxic signs in the other birds. Only one bird did not show any signs of ataxia.
In the positive control group, only two birds in the latter group showed no signs of ataxia. However, one of them was sacrificed on day 11 after being bullied by other birds, and the other was found dead on day 18. In general, ataxia tended to develop slightly earlier in group 4 than in the positive control groups (day 11).

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

other:

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw (total dose)

System:

nervous system

Organ:

other: nervous system

Treatment related:

yes

Conclusions:

Under the conditions of this test, dermal administration of the test material produced clinical signs of neurotoxicity in adult hens dosed at levels of 500 mg/kg and 5000 mg/kg. Histopathological examination confirmed the clinical observations on birds dosed at 500 mg/kg.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Species:

hen

Additional information

Neurotoxicty (oral)
In two oral neurotoxicity studies in hens (K2, non-GLP; Huntington 1981/1982) the test substance was administered by gavage as single doses at concentrations of 250 – 2000 mg/kg bw with 10 animals/sex/group. The hens were observed for 21 days for, among others, signs of ataxia and general clinical signs. Brain, spinal cord and sciatic nerve were examined histologically for all animals that showed clear ataxia.
The test substance produced clinical signs of neurotoxicity in a number of adult hens dosed all dose groups, and histopathological examination of affected birds showed evidence of neurotoxicity. The NOAEL was not determinable due to presence of neurotoxic effects at the lowest dose.

 

Neurotoxicicty (dermal)
A similar study was conducted using the dermal route (K2, non-GLP; Huntingdon 1982) with application of 50, 500 and 5000 mg/kg bw (in total) test substance on the comb. Because of limits to the maximum dose volume that could be administered in single application, that is 200 mg/kg bw, multiple dosing was carried for the two high dose groups. In the 21-day observation period, identical assessments were performed as in the oral toxicity studies. 
Next to blistering of the comb, dermal administration of the test material produced clinical signs of neurotoxicity in adult-hens dosed at levels of 500 mg/kg and 5000 mg/kg. Histopathological examination confirmed the clinical observations on birds dosed at 500 mg/kg.

 

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable as additional information for classification purposes under Regulation 1272/2008. There were significant neurotoxic effects at the lowest tested dose of 250 mg/kg bw upon acute oral exposure in hens, including signs of ataxia accompanied with morphological evidence of neurotoxicity found during neuropathological examination. Similar neurotoxic effects were also observed after dermal application at 500 mg/kg bw. These findings of test substance related toxicity on the nervous system serve as supporting data for the classification as STOT RE 2 (nervous system) under Regulation (EC) No. 1272/2008, as amended for the fifteenth time in Regulation (EC) No. 2020/1182.