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Toxicological information

Additional toxicological data

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Administrative data

Endpoint:
additional toxicological information
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Experimental dates: Start: 08 July 1997, End: 14 July 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Type of study / information:
In vitro study on the toxicity to primary cultures of hepatocytes of rats and mice.
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Stock solutions of thiamethoxam in DMSO were prepared and added to the culture medium to give the desired final concentrations of 10, 30, 60, 100, 300, 600, 1000, 2000 and 5000 μM. (the final concentration of the vehicle was 0.1% or 0.5% v/v (concentrations ≥600 μM). A positive control material, dibucaine was also tested at concentrations of 50, 100, 200 and 300 μM.
The morphology of hepatocyte monolayers was evaluated by light microscopy after 1, 4 and 24 hours exposure. Results of the evaluations, no morphological effect or irregular cell surface, formation of blebs, cell spreading, intracellular granulation or vacuolation, and cell disaggregation were recorded and the severity graded (1-3). Treatments resulting in 100% cell death and/or detachment of the monolayer were also recorded.
GLP compliance:
yes
Remarks:
The study was performed in accordance with GLP but since the purpose of the study was investigative in nature, no procedural quality assurance inspections or report audits were performed.

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-ylidene-N-nitroamine
Cas Number:
153719-23-4
Molecular formula:
C8H10ClN5O3S
IUPAC Name:
3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-ylidene-N-nitroamine
Test material form:
solid: particulate/powder

Results and discussion

Any other information on results incl. tables

Cytotoxic potential in rat and mouse hepatocytes: Thiamethoxam-exposed rat and mouse cultures were unaffected morphologically at all concentrations up to the maximum of 5000 µM.

The positive control material, dibucaine, induced morphological changes in both rat and mouse cultures at concentrations between 50 and 300 µM. The severity of cytotoxicity was both time and concentration dependent. Morphological effects observed in rat hepatocytes were slight to severe granulation, vacuolation and hypertrophy of cells and 100% cell death with detachment of the monolayer. In the mouse, slight granulation, vacuolation and hypertrophy of cells and 100% cell death with detachment of the monolayer occurred.

Release of enzymes: Thiamethoxam-exposed cultures did not release intracellular LDH into the medium at any concentration up to the maximum of 5000 µM.

LDH release in both species in response to dibucaine paralleled the morphological changes. LDH release increased dose- and time-dependently from approximately 15% of total intracellular activity at 50 µM to approximately 95% at 300 µM.

Table 1: Morphology of primary cultured rat and mouse hepatocytes after 1, 4 and 24 hours treatment.

Test substance

Incubation

(h)

Concentration (µM)

10

30

50

60

100

200

300

600

1000

2000

5000

Rat

CGA293343

1

0

0

-

0

0

-

0

0

0

0

0

4

0

0

-

0

0

-

0

0

0

0

0

24

0

0

-

0

0

-

0

0

0

0

0

Dibucaine

1

-

-

0

-

0

0

0

-

-

-

-

4

-

-

0

-

1HV

1HV

3GHV

-

-

-

-

24

-

-

1GV

-

2GHV

K

K

-

-

-

-

Mouse

CGA293343

1

0

0

-

0

0

-

0

0

0

0

0

4

0

0

-

0

0

-

0

0

0

0

0

24

0

0

-

0

0

-

0

0

0

0

0

Dibucaine

1

-

-

0

-

0

0

0

-

-

-

-

4

-

-

0

-

1HV

1H

K

-

-

-

-

24

-

-

1G

-

K

K

K

-

-

-

-

0 = no, 1 = slight, 2 = moderate and 3 = strong effect. K = dead cells (detached monolayer). D = detached and dead cells, G = granulation, H = hypertrophy and/or vacuolisation of cells. - = not determined.

 

Applicant's summary and conclusion

Conclusions:
Thiamethoxam is not cytotoxic to rat and mouse primary hepatocytes in short-term culture at concentrations up to 5000 µM.
Executive summary:

Primary cultures of hepatocytes were prepared from young adult male Tif:RAIf (SPF) rats and Tif:MAGf mice. Hepatocytes were isolated by in situ perfusion of the liver with collagenase solution. Hepatocyte viability was confirmed to be >90% by the trypan blue exclusion method. Isolated hepatocytes were suspended in Williams Medium E with supplements and seeded at confluency on collagen-coated 24-well plates at an initial cell density of 2 x 105cells/well. The experiment was started 24 hours after establishment and attachment of the monolayers.

Solutions of thiamethoxam in DMSO solvent (0.1 or 0.5%) at concentrations of 10, 30, 60, 100, 300, 600, 1000, 2000 and 5000 µM were added to the culture medium. A positive control material, dibucaine was also tested at concentrations of 50, 100, 200 and 300 µM. The morphology of hepatocyte monolayers was evaluated by light microscopy after 1, 4 and 24 hours exposure. Results of the evaluations, no morphological effect, or irregular cell surface, formation of blebs, cell spreading, intracellular granulation or vacuolation, and cell disaggregation were recorded and the severity graded. Treatments resulting in 100% cell death and/or detachment of the monolayer (K) were also recorded. Lactate dehydrogenase (LDH) activity in the culture medium was assayed, in triplicate, spectrophotometrically after 4 and 24 hours exposure. Total intracellular LDH activity was determined in 3 additional cultures before exposure to thiamethoxam.

Thiamethoxam-exposed cultures were unaffected morphologically and did not release intracellular LDH into the medium at any concentration up to the maximum of 5000 µM.

The positive control material, dibucaine, induced morphological changes in both rat and mouse cultures at concentrations between 50 and 300 µM. The severity of cytotoxicity was both time and concentration dependent. Morphological effects observed in rat hepatocytes were slight to severe granulation, vacuolation and hypertrophy of cells and 100% cell death with detachment of the monolayer. In the mouse, slight granulation, vacuolation and hypertrophy of cells and 100% cell death with detachment of the monolayer occurred. LDH release in both species in response to dibucaine paralleled the morphological changes. LDH release increased dose- and time-dependently from approximately 15% of total intracellular activity at 50 µM to approximately 95% at 300 µM.

Thiamethoxam is not cytotoxic to rat and mouse primary hepatocytes in short-term culture at concentrations up to 5000 µM.