3-iodo-2-propynyl butylcarbamate

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-01-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test:
Acetylcholinesterase inhibition in rat and human blood samples applying test item at two different concentrations, positive and negative control

- Short description of test conditions:
A subsample of the blood samples at 0 h was taken and the plasma and red cell cholinesterase levels estimated (0 h measurement).
Negative control, positive control and test material were added to the blood samples, mixed thoroughly and incubated at 37 °C. At 15 min, 30 min and 60 min plasma and red cell cholinesterase were assayed.

- Parameters analysed / observed: Acetylcholinesterase activity in red blood cells and plasma

GLP compliance:

yes

Type of method:

in vitro

Endpoint addressed:

neurotoxicity

Species:

other: rat and human

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified
- Age at study initiation: not specified

Route of administration:

other: exposure of blood samples in vitro

Vehicle:

polyethylene glycol

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Samples were taken at 15, 30, and 60 min after applying test item to blood samples.

Frequency of treatment:

single application

Post exposure period:

not applicable

Dose / conc.:

1 other: mg/mL

Dose / conc.:

2 other: mg/mL

No. of animals per sex per dose:

ten male sprague dawly rats were sacrificed for blood collection

Control animals:

yes, concurrent vehicle

Details on study design:

Ten male Sprague Dawley rats were separated into 2 groups of 5 animals each. These animals were sacrificed for collection of blood samples in two batches. Immediately after death blood samples were taken from the aorta.
The following procedure was performed in duplicates:
Blood samples were placed in an incubator at 37 °C. A 1 mL subsample was taken and the plasma and red cell cholinesterase levels estimated (0 h measurement).
Negative control, positive control and test material were added to the blood samples. Each sample was mixed thoroughly and incubated at 37 °C. At 15 min, 30 min and 60 min a further 1 mL of blood was removed and assayed for plasma and red cell cholinesterase.
Plasma and red cell measurements were performed within 10 minutes of subsampling. The 1 mL aliquot of blood was centrifuged at high speed in a microcentrifuge for 1 minute. The plasma was removed for direct measurement of cholinesterase activity.
A sample of packed red cells was lysed with distilled H2O. The haemolysate was analysed immediately for cholinesterase activity.

Examinations:

Acetylcholinesterase activity in plasma and red blood cells of rat and human blood samples, at different exposure times.

Positive control:

physostigmine salicylate

Details on results:

The negative control, polyethylene glycol, showed no evidence of effects in either plasma or red blood cell acetylcholinesterase activity.

5 x 10^-7 M physostigmine salicylate was found to cause a marked reduction in plasma cholinesterase from 0.25 h onwards. The degree of the effect on the pooled male rat, female rat and female human blood samples was generally similar although it started from varying baselines. The effect of physostigmine salicylate on rat red blood cell acetylcholinesterase was less consistent. Overall results tended to be lower than control but this effect was not pronounced. However, physostigmine salicylate markedly reduced the acetylcholinesterase level in female human red cells from 0.25 h onwards.

50 µL of 1.0 mg/mL and 2.0 mg/mL test item added to 5 mL rat and female human blood samples did not affect either plasma or red blood cell acetylcholinesterase levels. Results were reproducible and correlated closely with those obtained for the negative control group.

Conclusions:

The test item at two incremental concentrations showed no evidence of plasma or red blood cell cholinesterase inhibition in rat blood smaples.

Executive summary:

Two sets of rat blood samples were tested for plasma and red blood cell acetylcholinesterase inhibition in the presence of negative control, positive control and the test material. An additional set of human blood samples was included in the second phase of the study to examine the lack of consistent response for rat red blood cell acetylcholinesterase.

The positive control, physostigmine salicylate, was found to markedly inhibit plasma cholinesterase in each of the 3 sets of samples. Rat red blood cell acetylcholinesterase response was inconsistent. However, human red blood cell acetylcholinesterase was markedly inhibited.

The test item at two incremental concentrations showed no evidence of plasma or red blood cell cholinesterase inhibition. Data for the test groups were similar to control.

The concentrations of the test item used in this in vitro study showed no evidence of inhibition of either plasma or red blood cell cholinesterase. The concentration of test material in the first phase was based on the LD50 value of 20 mg/kg bw in rats. For the second phase the initial concentration of test item was doubled. The validity of the methodology for estimation of carbamate acetylcholinesterase inhibition was confirmed by the use of physostigmine salicylate as a positive control.Physostigmine salicylate markedly inhibited plasma acetylcholinesterase in both rat and human samples. Rat red blood cell acetylcholinesterase data were inconsistent although human red cell acetylcholinesterase data demonstrated marked inhibition.

Reference 2

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988-04-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: US EPA Pesticide Assessment Guideline Subdivision F, 81-3

Deviations:

no

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

neurotoxicity

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: P-2848-8603-P100

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (U. K.) Limited, Kent, U. K.
- Weight at study initiation: First batch: males: 176 - 190 g, females: 163 - 196 g; second batch: males 185 - 219 g, females: 180 - 211 g
- Housing: two per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approx. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 23 °C
- Humidity: 43 %
- Air changes: 15 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Route of administration:

intravenous

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were freshly prepared immediately prior to use. Appropriate weighed amounts of test item were dissolved in PEG 400/water for injection B.P. (70:30 v/v) to give concentrations of 4, 10 or 16 mg/mL. Solvent without test material was used for control purposes.

VEHICLE
- Justification for use and choice of vehicle: polyethylene glycol was shown to be a suitable vehicle in a dose range finder study
- Concentration in vehicle: 4, 10 or 16 mg/mL
- Dose volume: 1 mL/kg bw; except 2 mg/kg bw dose group, they received 2 mL/kg bw

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

single injection

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

2 mg/kg bw/day (actual dose received)

Dose / conc.:

4 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

16 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

main study: 10

Control animals:

yes, concurrent vehicle

Details on study design:

Test item was administered to animals intravenous via lateral tail vein. blood samples were taken at 15, 30, 60 min and 2 and 5 h after dosing for measurement of erythrocyte cholinesterase.

Examinations:

Clinical observations and mortalities were recorded as appropriate during the investigation.
Erythrocyte cholinesterase activity was assayed in blood samples taken.

Positive control:

not applicable

Details on results:

Dose Ranging Study

Clinical Observations:
Marked clinical signs of hyperkinesia, vocalisation, aggressiveness and reduced heart rate were noted for most treated animals. Some of these clinical signs persisted up to 1 - 1.5 h after dosing. Convulsions were noted in some animals at dose levels of 20 mg/kg bw and above. The incidence and severity of the convulsions increased with increasing dose. Prostration was noted for some animals following convulsion, this was generally followed by death. All surviving treated animals showed no abnormalities (NAD) after 2 h post dosing.

Mortalities:
There were no mortalities at 12.5 or 20 mg/kg bw. There was 1/4 mortalities in the 25 mg/kg dose group and all animals dosed at 37.5 mg/kg bw or greater died within a few minutes of dosing.
The estimated LC50 for the test item administered by intravenous injection to the rat was 22 mg/kg bw.


Main Study

Clinical Observations:
Two animals showed severe convulsions after dosing at 16 mg/kg bw. It was decided at this point not to dose animals at the estimated LC50 level, but to administer an extra low dose of 2 mg/kg bw (1/2 the dose volume using 4 mg/mL dosing solution). This decision was made by the Study Director on humane as well as scientific grounds.
All animals dosed at 16 mg/kg bw showed convulsions and/or marked hyperkinesia. All the females and occasional males dosed at 10 or 16 mg/kg bw showed red stained urine ca 15 min after dosing. Animals receiving 0, 2 or 4 mg/kg bw appeared NAD during the study.
A number of female animals showed poor or lack of ability to provide blood samples after the +30 min or 1 h sampling.

Mortalities:
One animal was found dead after the 1 h blood sampling in the 10 mg/lg bw dose group. Within the 16 mg/kd bw dose group five out of 20 animals died.
Erythrocyte Cholinesterase Activity:
The results indicated a large inter-individual variation in RChe activity. There appeared to be no effect on RChe activity with time or concentration as a result of treatment with the test item. It was therefore not possible to calculate nor estimate an IC50 concentration of for RChe enzyme.

Conclusions:

The test item administered as a single intravenous injection of doses up to 16 mg/kg bw did not appear to inhibit RChe activity in rats.

Executive summary:

In this investigation the inhibition of erythrocyte cholinesterase (RChe) was assessed following intravenous administration of a single injection of test item to the rat. Dosages of 0, 2, 4, 10, 16 mg/kg bw were applied. At the highest dose increased mortality following treatment was observed.

The increased sensitivity to the test item toxicity in the main study compared to the dose ranging study, as indicated by mortalities at lower doses, was probably attributable to the additional stress of blood sampling of the animals on the main study.

The RChe data appeared to show no effect with time nor concentration resulting from a single intravenous dose of the test item. The dose levels tested ranged up to the highest feasible dose for the study.

In conclusion, the test item administered as a single intravenous dose of up to 16 mg/kg bw did not appear to inhibit RChe activity in rats.

Description of key information

Data of both in vivo and in vitro studies indicate that the test item showed no evidence of plasma or red blood cell cholinesterase inhibition in rats and human blood.

Additional information

Cholinesterase inhibition in rats

In this investigation the inhibition of erythrocyte cholinesterase (RChe) was assessed following intravenous administration of a single injection of test item to the rat. Dosages of 0, 2, 4, 10, 16 mg/kg bw were applied. At the highest dose increased mortality following treatment was observed.

The increased sensitivity to the test item toxicity in the main study compared to the dose ranging study, as indicated by mortalities at lower doses, was probably attributable to the additional stress of blood sampling of the animals on the main study.

The RChe data appeared to show no effect with time nor concentration resulting from a single intravenous dose of the test item. The dose levels tested ranged up to the highest feasible dose for the study.

In conclusion, the test item administered as a single intravenous dose of up to 16 mg/kg bw did not appear to inhibit RChe activity in rats.

Acetylcholinesterase inhibition in vitro with rat blood

Two sets of rat blood samples were tested for plasma and red blood cell acetylcholinesterase inhibition in the presence of negative control, positive control and the test material. An additional set of human blood samples was included in the second phase of the study to examine the lack of consistent response for rat red blood cell acetylcholinesterase.

The positive control, physostigmine salicylate, was found to markedly inhibit plasma cholinesterase in each of the 3 sets of samples. Rat red blood cell acetylcholinesterase response was inconsistent. However, human red blood cell acetylcholinesterase was markedly inhibited.

The test item at two incremental concentrations showed no evidence of plasma or red blood cell cholinesterase inhibition. Data for the test groups were similar to control.

The concentrations of the test item used in this in vitro study showed no evidence of inhibition of either plasma or red blood cell cholinesterase. The concentration of test material in the first phase was based on the LD50 value of 20 mg/kg bw in rats. For the second phase the initial concentration of test item was doubled. The validity of the methodology for estimation of carbamate acetylcholinesterase inhibition was confirmed by the use of physostigmine salicylate as a positive control. Physostigmine salicylate markedly inhibited plasma acetylcholinesterase in both rat and human samples. Rat red blood cell acetylcholinesterase data were inconsistent although human red cell acetylcholinesterase data demonstrated marked inhibition.