4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Neurotoxicity

Description of key information

NOAEL (rat): 1200 ppm (corresponding to 77 and 92.9 mg/kg bw/day in males and females, respectively); no effects indicative for neurotoxicity observed.

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:

21 July 2003 - 05 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Health Canada PMRA DACO No. 4.5.11

Qualifier:

according to guideline

Guideline:

other: Notification No. 12-Nousan-8147 (Japan)

Version / remarks:

24 November 2000

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hannover Crl:WI[Glx/BRL/Han]IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: 8 weeks
- Weight at study initiation: 254.5 (males), 165.1 (females)
- Housing: individually housed in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least six days

ENVIRONMENTAL CONDITIONS
- Temperature: 18°C - 26°C
- Humidity: 30% - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the ration was measured by liquid chromatographic analysis.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

400 ppm

Remarks:

24.3 mg/kg bw/day (males) and 31.1 mg/kg bw/day (females)

Dose / conc.:

1 200 ppm

Remarks:

77.0 mg/kg bw/day (males) and 92.9 mg/kg bw/day (females)

Dose / conc.:

3 600 ppm

Remarks:

229.2 mg/kg bw/day (males) and 273.8 mg/kg bw/day (females)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent no treatment

Details on study design:

analytically-determined concentrations of the test substance were 377, 1143 and 3387 ppm

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (on 9/10/03, the morning mortality check was inadvertently not performed)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations: pre-terminal (week 12)
- Dose groups that were examined: all

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY:
- Description of procedures: Score intensity of the observation as follows: 1) Slight (barely perceptible or infrequent); 2) Moderate to Severe. Record colors as follows: C = Clear; W = White; R = Red; Y = Yellow; B = Brown; G = Green O = Other (list color) Home cage observations: Posture, piloerection, involuntary motor movements (clonic, tonic), gait abnormalities, vocalizations, other.
Observations during handling: Ease of removing the rat from its cage, reaction to being handled, muscle tone, palpebral closure, lacrimation, salivation, nasal discharge, stains, other.
Open field observations (The rat is placed in the center of a flat surface with a perimeter barrier, such as a cart, for 2 minutes. During this time, the number of rears is counted and other observations are made): Piloerection, respiratory abnormalities, posture, involuntary motor movements (clonic, tonic), stereotypy, bizarre behaviour, gait abnormalities, vocalizations, arousal, rearing, other, excretion (defecation, urination)
Refelx/Physiologic observations and measurements: Approach response (approach the rat head-on with the end of a blunt object, such as a pencil, then hold the object approximately 3 cm from the face for approximately 4 seconds), touch response (coming in from the side, touch rump gently with a blunt object), auditory response (position clicker approximately 5 cm above the back of the rat and make a sudden sound), tail pinch response (metal forceps are used to squeeze the tail approximately 2-3 cm from the tip), pupil size (assess both eyes under standard lighting conditions), pupil response (assess both eyes after adaptation in a darkened enclosure. Assess the reaction of both pupils to dark adaptation and the reaction of both pupils to the beam of a penlight as it is brought in from the side of the rat's head.), righting reflex (the rat is held supine, then dropped from approximately 30 cm. If the rat is severely affected, this test may not be performed), grip strength, body weight, body temperature, landing foot splay
- Minimization of bias: Studies have been conducted with acrylamide, carbaryl and untreated rats to establish the sensitivity, reliability, and validity of these test procedures, the adequacy of training of technical personnel and to serve as a historical control.
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes
- Site of testing: No data
- Time schedule for examinations: 5 times; once during the week prior to initiating the exposure and again during weeks 2, 4, and 13
- Environmental conditions: No data
- Noise level: Broad-spectrum background noise (approximately 74dB(A)

LOCOMOTOR ACTIVITY: Yes
- Length of session, number and length of subsessions: Motor and locomotor activity were examined for the 60-minute session and during each ten-minute interval

Sacrifice and (histo)pathology:

- Time point of sacrifice: at termination of the study
- Number of animals sacrificed: all
- Parameters measured: The necropsy involved an examination of all organs, body cavities, cut surfaces, external orifices and surfaces; the entire brain and spinal cord, both eyes (with optic nerves) and selected (bilateral) peripheral nerves (sciatic, tibial and sural), the gasserian ganglion, gastrocnemius muscle, both forelimbs, gross lesions in neural tissues or skeletal muscle and physical identifier were dissected from each animal.
- Brain weight: Yes
- Length and width of brain: No data
- Procedures for perfusion: animals were deeply anesthetized using an intraperitoneal dose (50 mg/kg) of pentobarbital and then perfused via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by Universal fixative (1% (w/v) glutaraldehyde and 4% (w/v) EM-grade formaldehyde) in phosphate buffer.
- Number of animals perfused: 6 per sex/dose level

- Type of staining: hematoxylin and eosin (H&E); Lee's stain
- Embedding media: glycol methacrylate (GMA)
- Number of sections: eight

Statistics:

Statistical evaluations were performed on INSTEM Computer Systems or SAS. With the exception of Bartlett's test (p<0.001) the statistical significance level was p<0.05.
For the FOB, continuous data were first analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA. For weeks on which there was a significant treatment effect, Dunnett's test was applied to determine which groups were significantly different from the control group. Categorical data collected in the FOB were analyzed in a similar manner, using General Linear Modeling (GLM) and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using Dunnett's test and an Analysis of Contrasts, respectively.
Motor and locomotor activity were analyzed using ANOVA procedures. Session activity data were first analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA. For weeks on which there was a significant treatment effect, Dunnett's test was performed. Interval data were subjected to a twoway Repeated-Measures ANOVA, using both test interval and test occasion as the repeated measures, followed by a Repeated Measures ANOVA. For those weeks, the data for each interval were subjected to analysis using a oneway ANOVA and/or Dunnett's test.
For pathology, continuous data were evaluated initially using Bartlett's Test to analyze for homogeneity of variances among groups. Homogeneous data were further analyzed using an ANOVA followed by Dunnett's Test for pair-wise comparisons. In the event of non-homogeneous data, statistical analysis was performed using the non-parametric Kruskal-Wallis Test followed by a Mann-Whitney U Test for pair-wise comparisons. Micropathology frequency data were analyzed using a Chi-Square Test followed by a one-tailed Fisher's Exact Test. A probability value of p<0.05 was accepted as significant for all statistical tests with the exception of Bartlett's Test in which a probability value of p<0.001 was used.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Red lacrimal stain and red nasal stain (one male) and areas of alopecia (forelimbs of one female) were observed in individual controll animals. The only clinical observation in treated animals consisted of a lesion, described as a scab, on the neck of one high-dose female (day 4 only).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For males, body weight was significantly reduced relative to controls at the highest dietary level but was not different from control at lower dose levels. For high-dose males, differences from control ranged from 6-9% and were statistically significant beginning on day 35 and continuing until study termination. For females, body weight was not statistically different from controls at any dietary level. However, the trend for high-dose females was very similar to the males, with body weight consistently less than controls (5-6%) beginning on day 49 and continuing until study termination.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For males, food consumption was not different from controls at any dietary level. For females, food consumption was significantly less than controls at the 1200 ppm dose for days 35 (-13%), 84 (-9%) and 91 (-12%) and in high-dose animals for days 35 - 42 (-14% and -15%, respectively) and days 84 - 91 (-14% to -16%, respectively). Food consumption for low-dose females was not different from controls on any occasion.
Daily food consumption, averaged over the duration of the study on a per kg body weight basis, was not affected by treatment at any dietary level.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were statistically significant decreases in terminal body weight and increased relative fixed brain weight in males at the highest dietary level. This same trend was evident in high-dose females without reaching statistical significance. The increase in relative fixed brain weight is attributed to decreased body weight rather than to treatment. Relative liver weight was also significantly increased in high-dose males, compared to controls. This increase in relative and not absolute liver weight is also attributed to significantly lower body weight and not to treatment. In females, relative liver weight was significantly increased, compared to controls, at the mid- and high-dose, while absolute liver weight was only significantly increased, compared to controls, at the highest dietary level. The increased absolute liver weight in high-dose females is attributed to treatment. The increased liver weight in high-dose females, in the absence of microscopic changes, is not considered an adverse effect.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no compound-related microscopic lesions in the high-dose males or females. Therefore, tissues from animals that received a lower dose of the test substance were not examined.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were observed at this dose

Remarks on result:

other: corresponding to 77.0 and 92.9 mg/kg bw/day in males and females, respectively

Key result

Dose descriptor:

LOAEL

Effect level:

3 600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

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

Several studies have been conducted to evaluate the neurotoxic properties of the test substance. In detail, acute and long-term neurotoxicity studies have been performed in rats and hens which were supplemented by in vitro and ex vivo mechanistical studies. The following specific investigations on neurotoxicity are available.

 

Rodent in vivo studies

Acute neurotoxicity

An acute oral neurotoxicity screening study in Wistar rats was performed according to GLP and US-EPA OPPTS 870.6200 (2002b).

Technical grade test substance was administrated orally by gavage (single administration) to groups of 12 fasted rats (12 animals per sex/dose level) using nominal doses of 200, 500 and 2000 mg/kg bw/day. Based on analytical results, the actual doses were 223, 576 and 2009 mg/kg bw/day. Concurrent control animals were included in the study.

The test substance was suspended in 0.5% methylcellulose/0.4% Tween 80 in deionized water and administered at a dosing volume of 10 mL/kg. The following observations and measurements were included in the study: clinical observations, mortality checks, body weight, automated measurements of activity (figure-eight maze), a functional observational battery, brain weight, and a gross necropsy. Skeletal muscle, peripheral nerves, eyes (with optic nerves), and tissues from the central nervous system were examined microscopically for lesions.

No deaths occurred at any dose level prior to scheduled terminal sacrifice on day 15 after treatment. Compound-related clinical signs were not evident at any dose level in either sex. Body weight was not affected by treatment in males or females at any dose level. For the functional observational battery (FOB), compound-related effects were not evident at the time when peak neurobehavioral effects should occur on day 0 in either sex at any dose level. There were no compound-related effects on motor or locomotor activity in the figure-eight maze at any dose level in either sex. Habituation was not affected by treatment at any dose level.

Further, no compound-related gross lesions in males or females at terminal sacrifice have been observed. Brain weight was not affected by treatment in males or females at any dose level. Compound-related microscopic lesions were not evident in high-dose males or females.

In conclusion, acute exposure to the test substance produced no evidence of toxicity in males or females at a limit dose of 2000 mg/kg bw/day. Based on these results, the acute NO(A)EL for test substance is 2000 mg/kg bw/day for males and females.

 

Subchronic neurotoxicity

In addition to the acute neurotoxicity study, a GLP-conform subchronic neurotoxicity screening study (2004a) was performed according to US-EPA OPPTS Guideline No. 870.6200, OECD Guideline 424, Health Canada PMRA DACO No 4.5.11 and the Japanese Testing Guideline Notification No 12-Nousan-8147.

Test substance of technical grade was administered in the diet for 13 weeks to young-adult Wistar rats (12/sex/dietary level), using nominal concentrations of 400, 1200 and 3600 ppm for males and females (corresponding to 24.3, 77.0 and 229.2 mg/kg bw/day for males and 31.1, 92.9, 273.8 mg/kg bw/day for females). Respective control animals were included. All 12 rats/sex/dietary levels were used for neurobehavioral evaluation, with six/sex/dose used for micro-pathology. The following observations and measurements were included in the study: clinical observations, mortality, body weight, food consumption, automated measurements of activity (figure-eight maze), functional observational battery, ophthalmic exams, brain and liver weight, and a gross necropsy. Skeletal muscle, peripheral nerves, eyes (with optic nerves) and tissues from the central nervous system were also examined microscopically for lesions.

Based on analytical results, the actual mean concentrations of the test substance in the diet were 377, 1143 and 3387 ppm for males and females. No deaths occurred at any dietary level prior to scheduled terminal sacrifice. Compound-related clinical signs were not evident in males or females at any dietary level. Body weight was reduced at the 3600 ppm dietary level for males and females but not at lower levels. Differences from control at the highest dietary concentration ranged from 6-9% (statistical) for males and 5-6% for females (not statistically significant). Food consumption was not affected by treatment in males at any dietary level. For females, food consumption was significantly less than control at the 1200 ppm and 3600 ppm dietary levels but the difference from control for mid-dose females was not considered as an adverse effect since it was not associated with a difference in body weight. For the functional observational battery (FOB), compound-related effects were not apparent in males or females at any dietary level.

There were no compound-related effects on motor and locomotor activity in the figure-eight maze at any dietary level for either sex. Habituation was not affected by treatment. There were no compound-related ophthalmic findings and there were no compound-related gross lesions at terminal sacrifice in males or females. Terminal body weight was significantly decreased compared to controls in males and moderately decreased (non-statistical) in females at the highest dietary level. There were no differences observed in animals at lower dietary levels. Brain weight was not affected by treatment in males or females at any dietary level. Increased relative brain weight for males (statistical) and females (non-statistical) at the highest dietary level were due to decreased terminal body weight. Liver weight was increased by treatment in high-dose females but was not affected by treatment in females at lower dietary levels nor in males at any dose. The increased liver weight in high-dose females, in the absence of microscopic changes, is not considered as an adverse effect.

Compound-related microscopic lesions were not evident in high-dose males or females.

In conclusion, the present study established an overall NOAEL of 1200 ppm for males and females, based on decreased body weight in both sexes and decreased food consumption in females at 3600 ppm. The increased liver weight in high-dose females, in the absence of microscopic changes, is not considered as an adverse effect. There was no evidence of neurotoxicity at any dietary level.

 

In vivo studies performed in hens

Acute neurotoxicity

A GLP-conform study for delayed neurotoxicity following acute oral administration to hens was performed according to the guidelines ‘Acute Delayed Neurotoxicity Study, Guidance on Toxicological Study Data for Application of Agricultural Chemical Registration, 59 NohSan No., January 28, 1985’, ‘OECD Draft Updated Guideline No. 418: Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure, OECD Guideline for the Testing of Chemicals, (ENV/MC/CHEM(94)16) (TGP/GL418/October 1994)’ and ‘EPA-FIFRA guideline 81-7: Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure, Pesticide Assessment Guidelines - Subdivision F, Hazard Evaluation Human and Domestic Animals, Addendum 10, Neurotoxicity, Series 81, 82 and 83, March 1991’ (1998g).

A single oral dose of 5000 mg test substance/kg bw was orally administered by gavage twice at an interval of 3 weeks. In the 3-week post-treatment observation periods which followed each administration, daily observations and 2 forced running tests per week were performed to detect gait abnormalities. From a dose finding study it was known that the limit dose of 5000 mg/kg bw caused marked clinical signs and lethality. The positive control substance TOCP was administered orally by gavage at a dose of 400 mg/kg bw. The TOCP-treated hens developed typical signs of OPIDP, like slight gait abnormalities and ataxia (from 7 days after treatment) and severe ataxia and complete paralysis (from 10 and 14 days after treatment, respectively), so that the sensitivity of this test model was confirmed. A 2nd treatment was not possible and the animals had to be sacrificed for animal welfare reasons during the observation period. No acute clinical signs were observed in either the negative or the positive control groups. Administration of 5000 mg target substance/kg bw caused acute clinical signs for up to 9 days after the first and up to 13 days after the second test substance administration. The main signs were diarrhea, reduced reactivity and motility which secondarily resulted in transiently uncoordinated gait. 1 animal died 6 days after the first and another one 13 days after the second test substance administration. Slight body weight reductions were observed in the test substance-treated group after each treatment which was compensated by the end of the study.

In the forced running tests during the post-treatment periods no gait abnormalities which are typical for OPIDP were observed in animals treated with 5000 mg test substance/kg bw. This correlates well with the negligible NTE inhibition exerted by the test substance. After the first and second administration, acute symptoms were so marked that scheduled forced running tests were not possible at 1 or 5 occasions. 24and 48 hours and 7 days after the first treatment, determinations were conducted of the neuropathy target esterase (NTE) activity in brain, spinal cord and sciatic nerve, and of the acetylcholinesterase (AChE) activity in brain. The AChE determinations did neither reveal an AChE-inhibiting potential of test substance nor TOCP. The test substance caused an inhibition of the NTE, which was slight, however, and did not reach the threshold of 70-80 % which is considered to be indicative of a delayed neurotoxic potential. The average inhibitions were between 8 and 56 % in different nerve tissues. Highest inhibitions were determined 2 days after treatment, whereas inhibitions were negligible after 7 days. TOCP, however, induced the expected NTE inhibitions of more than 90 % in all tested nerve tissues, thus confirming the sensitivity of the test model.

No treatment-related changes in brains, spinal cords or sciatic nerves of the animals treated with 5000 mg test substance/kg bw were detected at the histopathological examination. This confirms the negative outcome of the clinical and biochemical measurements so that no evidence of a delayed neurotoxic potential of the test substance exists. However, the TOCP-treated animals showed the typical histopathological changes for OPIDP in all nerve tissues examined.

In conclusion, the clinical, biochemical and histopathological results of this study, which used the hen as a sensitive test model for the detection of OPIDP, gave no evidence of a delayed neurotoxic potential of the test compound.

Subacute neurotoxicity

A CLP-conform subacute 4-week oral dosing study with a subsequent 2-week recovery period was performed in adult hens in order to determine the possible potential of the target substance to cause organophosphate-induced delayed neuropathy (OPIDN) (1999a). The study was performed in accordance with internationally accepted guidelines: Guidelines on the Compiling of the Results on Toxicity (Draft), 1998, 28 Day Multiple Dose Delayed Neurotoxicity Test (Japan), Draft Updated Guideline No. 419: Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study, OECD Guideline for the Testing of Chemicals, (ENV/MC/CHEM(94)16) (TGP/GL418/October 1994), Annex IV, Part B, B.38 (Delayed Neurotoxicity of Organophosphorus Substances 28-Day Repeated Dose Study) to Directive 67/548/EEC of the Council of the European Communities of June 27, 1967 (Official Journal of the European Communities L196/1 of August 16, 1967); Health Effects Test Guidelines (OPPTS 870.6100), Acute and 28-Day Delayed Neurotoxicity of Organophosphorus Substances (US Environmental Protection Agency, EPA 712-C-98-190, August 1998).

The hens were administered daily doses of 50, 200 or 750 mg/kg bw/day; the group sizes ranged from 17 in the untreated control to 24 in the high-dose group. The high dose caused marked lethality and was reduced to 500 mg/kg bw/day after 2 weeks of treatment. The high dose of 750 mg/kg bw/day caused the death of 9 of 24 hens and hence had to be reduced to 500 mg/kg bw/day after 2 weeks of treatment. Following 28 days of administration, the hens were observed for an additional period of 2 weeks.

Administration of > 500 mg target substance/kg bw/day caused acute clinical signs starting from day 2 or 3 and lasting to either spontaneous death of the hens or up to the end of treatment. The signs consisted of decreased motility and reactivity, spasmodic state, pale comb and dark-green faeces. In all, 13 of 24 high-dose hens (54%) and 2/18 middle-dose hens (11%) died during the treatment period. No acute clinical signs were observed in the negative control group and the groups treated with up to 200 mg/kg bw/day. Slight body weight reductions were observed in all high-dose hens, which, in surviving hens, were generally compensated by the end of the study.

24 and 48 hours after the last treatment, NTE determinations (high-dose only 24 h) were conducted in brain, spinal cord and sciatic nerve, and of the acetylcholinesterase (AChE) activity in brain.

Repeated administration of the target substance for 28 day resulted in some inhibition of the NTE, which was slightly and did not reach the range of > 60% inhibition which is considered to be a threshold beyond which a delayed neurotoxic potential may be expressed. At 500 mg/kg bw/day, the average inhibitions of NTE activity determined 1 day after the end of treatment were 45% in the brain, 35% in the spinal cord and 30% in the sciatic nerve. TOCP induced NTE inhibition was known to be more than 90% in all tested nerve tissue. The AChE determinations did not reveal a relevant AChE-inhibiting potential of the target substance.

No gait abnormalities typical for OPIDN were observed in animals treated with up to 500 mg /kg bw/day in the forced running tests conducted during the treatment and the post-treatment periods. This correlates well with the negligible NTE inhibition exerted by the target substance.

No test-substance-induced histopathological findings were observed. Any findings were evenly distributed among control and treated animals and comprised well known background lesions expected in healthy adult laying hens.

In conclusion, the clinical, biochemical and histopathological results of this study, which used the hen as a sensitive test model for the detection of OPIDN, gave no evidence of a delayed neurotoxic potential of the target substance when administered in daily doses of up to 500 mg/kg body weight for 28 consecutive days.

 

In vitro studies

Based on the results obtained in a previous chronic study in rats, in which the highest dose group increased the incidences of degenerative myelinopathies, the following in vitro study was performed to clarify if the target substance might inhibit NTE and whether the induction of degenerative myelinopathies in rats chronically treated with the target substance might be an initial sign of OPIDN-like effects (1997t). Accordingly, inhibition of NTE in vitro was studied using rat and hen brain homogenates as enzyme sources.

Following 20 min pre-incubation, 90 µM test substance inhibited rat brain NTE half-maximally. Hen brain NTE was less sensitive; an IC50 value for the maximally employable concentration (300 µM) could not be established under these conditions. When the pre-incubation period was prolonged to 2 h 20 min, increased inhibition was observed, corresponding IC50 values for rat and hen brain NTE were approximately 10 µM and 32 µM, respectively. Further investigations were made using rat brain only. Studies varying pre-incubation time and substrate concentrations suggested that the target substance only initially inhibited NTE in a competitive manner. Inhibition of NTE was persistent suggesting covalent modification of the enzyme, and inhibited NTE could not be reactivated by fluoride. Racemic methamidophos-(pre)inhibited, target substance-treated NTE was largely reactivatable, as was simply racemic methamidophos-inhibited NTE, strongly suggesting that the target substance modifies the catalytic centre of the enzyme.

In conclusion, the target substance seems to share some characteristics of delayed neuropathic OPs which might point to a delayed neuropathic potential. However, compounds are known that inhibited NTE in vivo beyond the threshold, but had no potential to induce OPIDN when compared to delayed neuropathic OPs in the acute hen model known to be predictive for humans. Since the target substance structurally differs from known NTE inhibitors, especially OPs, no conclusion on a delayed neuropathic potential can be drawn solely based on in vitro data. Taking into consideration the available in vivo neurotoxicity data and the available in vitro data, the target substance is not considered to induce a delayed neurotoxic potential with human relevance although the in vitro test indicates a possible NTE inhibition property.

 

Further, mechanistic investigations have been performed in primary rat cortical neurons, rodent cell lines of various other organs as well as rat liver mitochondria (1998f). The cells were treated with the target substance and with various well characterized neurotoxic compounds (2,5-hexanedione, acrylamide, paraquat, KCN, TOCP, mipafox, paraoxon). Additionally, toxicokinetic measurements were performed.

Primary neurons were isolated from embryonal brain of Wistar rats at a developmental stage E 18 - E19. Additionally, mouse N-18 cell line, NRK 52e (kidney; rat), L6 (skeletal muscle; rat), H9C2 (heart muscle; rat), Hepa 1-6 (liver, mouse) and 3T3 (fibroblasts; mouse) were tested at following doses: 0.1, 1, 5, 10 and 20 µg/mL for the target substance and 0.1, 1, 5, 10 and 50 µg/mL for all other compounds. The treatment period was 7 days with dosing by medium change at day 1, 3, and 5. Evaluations were made 7 days after first dosing and after a treatment free (recovery) period of 7 days. In another experiment the effects on mitochondrial respiration were investigated using fresh rat livers (Wistar, Hsd Cpb).

Read-out parameter as index for (neuro)toxic properties were viability, rhodamine 123 incorporation, Glucose (intra- and extracellular), pyruvic acid (intracellular), lactic acid (intracellular), ATP determination, visualisation of neurofilaments, mitochondrial respiration, binding of the target substance to rat plasma proteins and cell culture medium constituents.

The target substance was not very cytotoxic to neuronal cells. The NOEC of 10 µg/mL at day 3 slightly decreases at day 7 (NOEC 5 µg/mL) and more distinctly at day 14 (NOEC 1 µg/mL). The cytoskeleton was initially not specifically compromised and behaved similarly to the cell viability. However, at day 14 the cytoskeleton was altered more strongly than the cell viability.

Interestingly, all endpoints characterizing the cellular energy status reacted drastically on target substance treatment. At day 3 the mitochondrial potential as measured by rhodamine 123 was distinctly more strongly influenced than the cell viability. At days 7 and 14 glucose consumption, mitochondrial potential, and ATP content were strongly depressed with ATP being the most sensitive parameter (NOEC < 0.1 pg/mL, IC50 1µg/mL). Pyruvate abolished or at least strongly diminished the effects of the target substance on cytotoxicity, ATP depletion, and cytoskeleton, indicating that in fact impairment of the cellular energy supply was an initial event in toxicity on neuronal cells.

The effects of the target substance on cortical rat neurons were in principle comparable to those of the mitochondrial toxins with respect to the primary involvement of cellular energy supply. Their sensitivity to pyruvate and their relative slow development as compared to KCN, however, point to differences in the molecular mechanism.

Effects of the target substance similar to those on primary neuronal cells were only observed in the cell line of neuronal origin (neuroblastoma N18). Although less sensitive than in the primary rat neurons the effects in the neuroblastoma cells underlined the role of energy depletion in neuronal cells. All other cell lines did not show any effect after treatment with the target substance indicating a very low unspecific cytotoxic potential. Therefore, it was concluded that the effects observed were specific for neuronal cells.

In primary cortical neurons of rats, specific effects on cellular energy supply have been observed following treatment with 2,5-hexanedione, acrylamide, paraquat, KCN as well as the target substance. In a separate series of experiments it was investigated whether these compounds might exert a direct inhibitory effect on mitochondrial respiration and the subsequent ATP formation. For this purpose the effects of increasing concentrations (3 to 300 µmol/L) of the test compounds on freshly isolated mitochondria from rat liver were investigated with the endpoints malate induced respiration (complex I, III - V) and succinate induced respiration (complex II - V). None of the substances tested showed decoupling activity. As to be expected KCN - a direct inhibitor of cytochrome C oxidase (complex IV) - dose dependently inhibited both types of mitochondrial respiration. The EC50 of 3 µg/mL nicely corresponds to the EC50 observed in neuronal cells. None of the other model neurotoxins revealed any effect on the mitochondrial respiration. In contrast, the target substance revealed to be a weak complex I inhibitor as could be seen by its selective inhibition of the glutamate/malate induced mitochondrial respiration. However, this direct effect on mitochondrial respiration had an EC50 > 100 µg/mL and a NOEC of 4 µg/mL which were by far higher than the corresponding values for mitochondrial membrane potential (rhodamine 123) or ATP production in neuronal cells. This strongly indicates that a direct effect of the target substance on mitochondrial respiration is not the primary reason for the impaired energy supply in the neuronal cells.

Different endpoints characterizing the function of the glucose uptake and utilisation were measured in neuroblastoma N18 cells. Neuroblastoma cells had to be used instead of primary neurons due to the higher cell numbers and, therefore, analyte concentrations available in these cultures which allowed a reliable determination of the different endpoints in question. After treatment with the target substance, neuroblastoma cells showed in principle comparable effects to primary rat neuronal cultures. The treatment conditions were as described before but with determination of glucose uptake, intracellular glucose, pyruvate, and lactate concentrations at day 3 and day 7 of treatment with increasing concentrations of the target substance. The target substance strongly reduced concentration-dependently the glucose consumption as measured by the decrease of the extracellular glucose concentration. However, the intracellular glucose concentration increased up to 3.4 times that of control cells, indicating that sufficient glucose was available intracellularly for metabolism and the glucose uptake into the neuroblastoma cell was not disturbed. The strong increase of intracellular glucose under target substance treatment probably reflects the high basal energy and thus, glucose demand of the neuroblastoma cells which was then dose-dependently reduced by inhibitory effects of the target substance downstream in the glucose utilisation. As already observed for ATP depletion these effects evolved slowly and were only fully expressed at day 7 of the treatment.

The target substance distinctly reduced dose-dependently the intracellular concentrations of the product of the glycolysis, pyruvate as well as of lactate which could be formed from pyruvate under anaerobic conditions. Glucose consumption as well as pyruvate and lactate concentrations were affected at distinctly lower concentrations of the target substance than ATP concentration and cell viability indicating that these endpoints might reflect the primary targets of the target substance. Pyruvate and lactate were reduced simultaneously. This indicates that the formation of pyruvate via aerobic glycolysis might be supressed by the target substance rather than that there was a shift to anaerobic glycolysis which in contrast should increase the lactate concentration. The further metabolism of pyruvate via citrate cycle and respiratory chain seemed to be unaffected since a downstream blockade would elevate both substrates. This complies with the protective effects of exogenous pyruvate which thus seem to bypass a block in the glycolysis and normalises the ATP deficit via a still functional citrate cycle and respiratory chain. It can be concluded that by the slowly evolving deficit of pyruvate the substrate supply to the respiratory chain will be reduced resulting in a drop of the mitochondrial membrane potential and ATP production.

Primary neuronal cells react very sensitively to the target substance. The most sensitive endpoint ATP was already affected at total concentrations as low as 0.1 µg/mL. However, only the target substance is not bound to constituents of the cell culture medium will be able to react with intracellular targets. To account for binding to the proteins in the culture medium, the free (unbound) fraction in the culture medium was determined in vitro by the ultrafiltration method. The target substance was bound in the medium used during treatment of the cells to approximately 50%. Therefore, the free low effect concentration in vitro could be assumed to be approx. 0.05 µg/mL for the most sensitive parameter ATP. In a toxicokinetic subacute rat study plasma concentrations of the target substance of 0.40 µg/mL in female rats dosed with 4,000 ppm and 0.14 µg/mL (calculated for 3,000 ppm) in male rats were determined. Since only the free (unbound) test substance can be assumed to penetrate the blood/brain barrier and to reach the cellular targets these total plasma concentrations have also to be corrected for the protein binding of the target substance. The free (unbound) fraction in rat plasma was determined by the ultrafiltration method to be 22%. Free plasma concentration, therefore, can be assumed to be 0.03 (male) and 0.09 µg/mL (female) at the high doses where degenerative myelinopathy was observed. The similarity of the free concentrations reached in vivo at the effect dose and the low effect concentration for ATP reduction in vitro (0.05 µg/mL) further substantiate the potential role of cellular energy effects for the observed neurodegenerative effects. The very low free concentrations of the test substance reached in vivo, however, might also explain the mildness of the in vivo effects, since all other substantial effects on cell viability and cytoskeleton became observable at least 10 times higher concentrations in the in vitro model.

The high basal energy demand of neuronal cells - necessary for preservation of their complex architecture - and their exclusive dependence on glucose as energy source, make the neuronal cells specifically vulnerable to this mode of action. However, at the low concentrations of the test substance reached in vivo under the conditions of the chronic study, a possible effect on cellular energy production could be regarded to be negligible. For inducing the morphological consequences - the degenerative myelinopathy - aging of the rat as another factor compromising the energy production of the neuronal mitochondria has to be considered as additional prerequisite. Therefore, it is concluded that these effects of the target substance – occurring only after chronic influence of high doses of the compound in old rats - pose no specific risk under the exposure conditions relevant to humans.

 

Overall conclusion

Based on the available data, the target substance is not considered to induce an OPIDN-like effect (organophosphate-induced delayed neuropathy). The slightly increased incidence / degree of the common age-related degenerative myelinopathy which was observed in the chronic rat study might rather be age-related than a specific neurotoxic effect induced by the target substance. The available mechanistic studies indicate that the exposure to the target substance might be related to deficits of the energy metabolism (aerobic glycolysis). Especially the in vivo studies performed in rats and hens after acute and repeated exposures show that an OPIDN (organophosphate-induced delayed neuropathy) -like effect is not induced due to target substance administration. Thus, no evidence of adverse effects on neurophysiological functions have been observed.