Diquat dibromide

Neurotoxicity

Description of key information

No acute neurotoxic effects at 280 mg test substance/kg bw, rats, single oral administration, EPA 81-8

No subchronic neurotoxic effects at 400 ppm (60.5 mg pure test substance/kg bw/day in males and 71.9 mg pure test substance/kg bw/day in females), rats, 13-week dietary feeding study, EPA OPPTS 870.6200

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:

key study

Study period:

7 January to April 1992 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alpk:ApfSD

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Animal Breeding Unit at ICI Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK
Age: about 28 days at delivery
Acclimatisation: 18 to 20 days
Weight: 173-236 g (males), 126-184 g (females)
Housing: 4 per cage (sexes separated) in stainless steel mesh cages measuring 26.5 x 50 x 20.7 cm, suspended over collecting trays
Feed: CT1 diet supplied by Special Diet Services Limited, Stepfield, Witham, Essex, UK
Water: filter sterilised mains water ad libitum
Temperature: 18.7 to 23 °C
Humidity: 33 to 70%
Air changes: 25 to 30 changes per hour
Photoperiod: 12 hours light to 12 hours darkness cycle

Route of administration:

oral: feed

Vehicle:

other: CT1 diet

Details on exposure:

All diets were based on CT1 diet. The experimental diets were prepared in 30 kg batches from premixes prepared by triturating the appropriate amounts of test substance solutions together with 25 mL of deionised water to 500 g laboratory diet. The compound bottle was washed with 2 x 25 mL deionised water and the washings added to the mixture. The mixture was then sieved and any lumps triturated. After mixing, a further 500 g laboratory diet or CT1 Diet was added to give a 1 kg dry premix. The premixes were added to 29 kg of CT1 Diet and mixed thoroughly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from all dietary levels (including controls) were taken at intervals throughout the study and analysed quantitatively for the substance. The homogeneity of the substance in the diet was demonstrated by analysing samples from the 20 and 400 ppm dose levels. The chemical stability of the substance in the diet was confirmed for the diets over a period of at least four weeks. Concentrations in the diet were analysed by high performance liquid chromatography using extraction by mechanical shaking with 10% trichoracetic acid and a pKb100 column.

Duration of treatment / exposure:

Continuously via feed

Frequency of treatment:

7 days/week for 13 weeks

Dose / conc.:

20 ppm (nominal)

Remarks:

Mean main dose received: 1.6 mg cation/kg bw/day for males and 1.9 mg cation/kg bw/day for females

Dose / conc.:

100 ppm (nominal)

Remarks:

Mean main dose received: 7.9 mg cation/kg bw/day for males and 9.5 mg cation/kg bw/day for females

Dose / conc.:

400 ppm (nominal)

Remarks:

Mean main dose received: 32.4 mg cation/kg bw/day for males and 38.5 mg cation/kg bw/day for females

No. of animals per sex per dose:

Twelve per sex per dose

Control animals:

yes, plain diet

Details on study design:

The study consisted of one control and three treatment groups with 12 males and 12 females per group. The groups were arranged on two racks in six single-sex replicates (randomised blocks). The sequence of the groups within each replicate was determined using computer-generated sequences of the numbers 1-4. The dose levels selected for this study were based on the results of previous studies in the Alpk:APfSD rat carried out in this Laboratory.

Observations and clinical examinations performed and frequency:

Mortality and clinical observations: Cage-side observations which included recording any changes in clinical condition or behaviour were made daily. Detailed clinical observations, including the finding of ‘no abnormalities detected’, were recorded weekly.
Body weight: The body weight of each rat was recorded immediately before feeding the experimental diet commenced and then on the same day of each subsequent week until termination. In addition, body weights were recorded at termination.
Food consumption and utilisation: Food consumption for each cage of rats was recorded continuously throughout the study and calculated on a weekly basis.
Ophthalmoscopy: Ophthalmoscopy was performed prior to randomisation of the study and during week 13 for all rats.

Specific biochemical examinations:

Cholinesterase determination: Cholinesterase activity was not determined in this study.

Neurobehavioural examinations performed and frequency:

Functional Observational Battery (FOB): Detailed observations (during which each rat was removed from its cage and physically examined for changes in general health status) and quantitative assessments of landing foot splay, muscle weakness (fore- and hind-limb grip strength) and sensory perception (tail-flick test) were made in week -1, and in weeks 2, 5, 9 and 14. The clinical observations included, but were not limited to, the following list of measures: assessment of autonomic function (e.g. lachrymation, salivation, piloerection, exophthalmus, urination, defecation, pupillary function, ptosis); description, incidence and severity of any convulsions, tremors, abnormal motor function, abnormal behaviour etc.; reactivity to stimuli; changes in level of arousal; sensorimotor responses; alterations in respiration. The observations were made by one observer who was ‘blind’ with respect to the animals’ treatment, and recorded on a computer system by personnel not directly involved in the clinical observations. The observations were carried out in a room separate from that in which the animals were housed and animals were presented to the observer with no indication of the treatment group. The observations were coded and the degree of condition noted (slight, moderate or extreme) where appropriate. This included the recording of no abnormalities detected.
Motor activity: Locomotor activity was monitored by an automated activity recording apparatus. All animals were tested at weeks -1, 5, 9 and 14 of the exposure period. Each observation period was divided into ten scans of five minute duration. Treatment groups were counter balanced across test times and across devices and, when the trials were repeated, each animal was returned to the same activity monitor at approximately the same time of day. Motor activity was assessed in a separate room to minimise disturbances.

Sacrifice and (histo)pathology:

Termination and pathology: Any rat requiring euthanasia during the study, and also up to six rats/sex/group terminated at the end of the study, were exsanguinated under terminal anaesthesia with halothane Ph Eur vapour and subjected to a full post mortem examination. Also, at the scheduled termination six designated animals/sex/group were deeply anaesthetised with intraperitoneal sodium pentobarbitone and killed by perfusion fixation with modified Karnovsky's fixative. The tissues as listed below were removed:
Tissue submission: The following tissues were taken from all rats killed by perfusion fixation and preserved in an appropriate fixative:
- Brain
- Gasserian ganglia
- Vertebral column including the spinal cord
- Dorsal root ganglia including spinal roots
- Gastrocnemius muscle
- Sciatic nerve
- Sural nerve
- Tibial nerve
- Eyes (and Harderian gland)
- Oral cavity
- Nasal passages

Brain measurements: The brain from all animals was weighed and the length and width measured.
Tissue processing: Only tissues from the perfuse-fixed animals were processed for histology. The eyes (and Harderian Gland), oral cavity and nasal passages were stored and not processed. The brain and gastrocnemius muscle were embedded in paraffin wax, 5 µm thick sections were cut and stained with haematoxylin and eosin. In addition, transverse sections of the vertebral column containing samples from the lumbar and cervical regions of the spinal cord, with dorsal root ganglia and spinal roots attached, were decalcified, embedded in paraffin wax, and 5 µm thick sections were cut and then stained with haematoxylin and eosin. The remaining tissues were embedded in Araldite and semi-thin sections (1-2 µm) cut and stained with toluidine blue. Samples of the spinal cord and peripheral nerves were also embedded in araldite and semi-thin sections cut and stained with toluidine blue. Initially, the brain of 1 male and 1 female rat from the 400 ppm diquat group was examined in the transverse plane at 12 levels. On the basis of this initial examination the brain of the remaining 5 rats/sex from the 400 ppm diquat group and 6 rats/sex from the control group were examined in the transverse plane at the following 6 levels: 2, 5, 6, 7, 8, 9. Spinal cord from the cervical region (C3-C6) and from the lumbar region (L1-L4) was examined in the transverse plane. Spinal roots and dorsal root ganglia were examined from the C3-C6 and L1-L4 levels and the gasserian ganglia from the trigeminal nerve. Transverse and longitudinal sections of the sciatic nerve and transverse sections of the sural and tibial nerves were examined. Samples of gastrocnemius muscle were examined in the transverse plane.
Tissue examination: All submitted tissues from the top dose and control animals, except those indicated for storage, were examined by light microscopy.

Positive control:

Not applicable

Statistics:

All data were evaluated using analysis of variance and/or analysis of covariance for each specified parameter using the GLM procedure in SAS (1985). Least square means for each group were calculated using the LSMEAN option in SAS PROC GLM. Unbiased estimates of differences from control were provided by the difference between each treatment group least square mean and the control group least square mean. Differences from control were tested statistically by comparing each treatment group least square mean with the control group least square mean using a two-sided Student's t-test, based on the error mean square in the analysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Decreased visual placement response was noted for 3/12 males and 1/12 females in the 400 ppm group, at the week 14 assessment. Reduced splay reflex was noted for 2 males and 1 female in the 400 ppm group, but also for 1 female in the 20 ppm group and 1 female in the control. The control animal showed the highest number of observations of this sign. Therefore, this response is considered not to be related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male at 100 ppm was killed for humane reasons during week 12, due to a twisted snout, with associated malocclusion. This death is considered not to be treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A marked reduction in body weight was noted for males and females in the 400 ppm test group in comparison with the controls, causing the final body weight to be lower than the controls by 12% and 10% for males and females respectively.
The body weights for females in the 20 or 100 ppm groups were less than that of the controls, the difference occasionally attained statistical significance during the study. However, the overall body weight gain for females in the 20 ppm group was equivalent to that of the controls, and the absolute weights for females in the 20 or 100 ppm groups were comparable. Therefore, it is considered that there is no treatment-related effect on body weight gain for females in the 20 or 100 ppm groups. The body weights for males in the 20 or 100 ppm groups were comparable with those of the controls throughout the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced during week 1 for males in the 400 ppm group in comparison with controls. Subsequent food consumption for this group was marginally lower than the controls, but, with the exception of week 2, this difference did not achieve statistical significance. For females in the 400 ppm group, food consumption over week 1 was slightly lower than that of controls, but this difference did not achieve statistical significance. Over the remainder of the study, food consumption for females in the 400 ppm group was generally comparable with controls.
There were no treatment-related effects on food consumption for males and females in the 20 or 100 ppm groups.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The efficiency of food utilisation was statistically significantly reduced for males and females in the 400 ppm group over weeks 1-4, with the reduction continuing for males into the period weeks 5-8. Overall, the efficiency of food utilisation was below that of controls for males in the 400 ppm group only. Efficiency of food utilisation for males and females in the 20 or 100 ppm groups was comparable to that of the controls throughout the study.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

At 400 ppm all animals showed treatment-related ophthalmic findings at week 13. The major findings for both males and females were total cataracts and posterior opacities in the lens, with both eyes being affected, although the males also had low incidences of additional minor ophthalmic changes which included prominent suture lines, congested and/or partially fixed iris and increased transparency of the lens.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Landing foot splay measurements: No treatment-related effects.
Time to tail-flick: No treatment-related effects.
Hind-limb grip strength measurements: No treatment-related effects. The hindlimb grip strength at week 14 for females in the 400 ppm group was statistically significantly below that of controls. In the absence of other indicators of neurological dysfunction, including neurohistology, or effects in males this isolated finding was considered spurious.
Fore-limb grip strength measurements: No treatment-related effects.
Motor activity measurements: Total motor activity was statistically significantly increased at week 5 for males in the 20 or 400 ppm groups, and for females in the 20 or 100 ppm groups. There was no evidence of any treatment-related effects on motor activity at other time periods. In the absence of a coherent dose-response and no evidence for a continuous change, these changes are considered not to be treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Brain measurements/weight: No treatment-related effects.
Macroscopic findings: Eye opacities were seen in 8/12 males and 8/12 females in the 400 ppm group. There were no other treatment-related findings.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

400 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

neuropathology

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

ophthalmological examination

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

400 ppm (nominal)

System:

eye

Organ:

iris

lens

Results of analysis of the diets: The mean concentrations for the dosing preparations analysed were within 7% of the nominal concentration. The homogeneity of diquat in the diet was determined at 20 and 400 ppm and was shown to be satisfactory, giving a deviation from the overall mean of within ±6%. The chemical stability of diquat in diet at room temperature was determined, and was shown to be satisfactory over a 38 day period at 20 ppm and over 29 days at 400 ppm, which covered the period of use of the diets.

Conclusions:

A NOAEL of 400 ppm (corresponding to 32.4 and 38.5 mg cation/kg bw/day) for neurotoxicity of the substance to rats was observed in a 13-weeks continuous feeding study. The overall NOAEL was 100 ppm (corresponding to 7.9 and 9.5 mg cation/kg bw/day) based on adverse effects in the eyes (including opacity of the lenses) occurring in animals treated with 400 ppm in the diet.

Executive summary:

In a subchronic neurotoxicity study performed under GLP to EPA OPPTS guideline 870.6200, groups of twelve male and twelve female Alpk:APfSD rats were fed diets containing 0, 20, 100 or 400 ppm substance cation for 13 weeks.

The achieved average doses were 1.6 and 1.9, 7.9 and 9.5 and 32.4 and 38.5 mg cation/kg bw/day in males and females, respectively, for the 20, 100 and 400 ppm groups. A detailed functional observation battery, including quantitative assessments of landing foot splay, sensory perception and muscle weakness, was performed at intervals. Locomotor activity was also monitored at intervals. At the end of the study, the rats were killed and subjected to a full post-mortem examination. Selected nervous system tissues from perfusion-fixed animals were removed, processed and examined microscopically.

Dietary administration of the substance cation at 400 ppm in the diet resulted in reduced growth throughout the study in both males and females, with an associated reduction in food consumption over week 1. Food utilisation efficiency was also low for males for the majority of the study and for females during the initial part of the study. Ophthalmic changes present as total cataract and/or posterior opacity of the lens was seen in both males and females at 400 ppm. No treatment-related effects were noted at 20 or 100 ppm. The neurotoxicity screening battery revealed no evidence of neurotoxic adverse effects of the substance.

The NOAEL for overall toxicity was 100 ppm (7.9 mg substance cation/kg/day in males and 9.5 mg substance cation/kg/day in females). The NOAEL for neurotoxicity was 400 ppm (32.4 mg substance cation/kg/day in males and 38.5 mg substance cation/kg/day in females).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Valid and reliable GLP study to relevant US EPA test guideline

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 substance has been tested for neurotoxicity in acute and repeat dose studies in rats. In neither study was there any indication of overt neurotoxicity / neuropathy.

The NOAEL in the acute study for neuropathy was 150 mg test substance cation/kg bw, equivalent to 280 mg pure test substance/kg bw/day, the highest dose tested. The NOAEL for general toxicity was 75 mg test substance cation/kg bw, equivalent to 140 mg pure test substance/kg bw/day based on a range of findings including clinical signs at 150 mg test substance cation/kg bw.

In a 90 day neurotoxicity study, the NOAEL for overall toxicity was 100 ppm (7.9 mg substance cation/kg bw/day corresponding to 14.8 mg pure test substance/kg b/day in males and 9.5 mg substance cation/kg bw/day corresponding to 17.7 mg pure test substance/kg bw/day in females) based on evidence of eye lesions and reductions in body weight gain and food consumption at 400 ppm. The NOAEL for neurotoxicity was 400 ppm (32.4 mg substance cation/kg bw/day equivalent to 60.5 mg pure test substance/kg bw/day in males and 38.5 mg substance cation/kg bw/day equivalent to 71.9 mg pure test substance/kg bw/day in females), the highest dose tested. There was no evidence that the substance was neurotoxic.

Justification for classification or non-classification

There is no classification category for neurotoxicity under UN GHS or EU CLP. Reliable studies on acute and sub-chronic neurotoxicity provide no evidence for specific target organ toxicity related to neurotoxicity.