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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication/study report which meets basic scientific principles.

Data source

Reference
Reference Type:
review article or handbook
Title:
European Union Risk Assessment Report TRIS(2-CHLORO-1-METHYLETHYL) PHOSPHATE (TCPP)
Author:
Ireland (lead) and United Kingdom
Year:
2008
Bibliographic source:
Covance Laboratories Ltd.

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The Single Cell Gel Electrophoresis assay (also known as comet assay) is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It was first described by Singh et al. in 1988. It has since gained in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by an imaging software.
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
TCPP
There are differences in the isomer content from each supplier,but these are not important given that the properties of the isomers are expected to be very similar.
Purity
A typical purity(total of the four isomers)is>97.9%.All testing described in this report is for the commercial product.
Impurities
The impurity profile of the commercial product TCPP is specific to individual manufacturers. It is not likely that the impurities will have had particular influence on any of the results obtained.
Additives
No additives are used.

Test animals

Species:
rat
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
In this study,groups of six male rats were administered TCPP in corn oil as a single dose via oral gavage at either 750 or 1500 mg/kg.The choice of dose levels was based on previous toxicity studies on TCPP,which identified 1500 mg/kg as the maximum tolerated dose.In the
absence of any gender differences in the acute toxicity studies with rats,only male animals were tested.The negative(vehicle)control group received corn oil only.
Duration of treatment / exposure:
3 or 24 after dosing.
Frequency of treatment:
single dose
Post exposure period:
not applicable.
Doses / concentrations
Remarks:
Doses / Concentrations:
750, 1500 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
six
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control group received a single gavage dose of ethyl methansulfonate(EMS)at 250 mg/kg three hours prior to necropsy.

Examinations

Tissues and cell types examined:
liver cell
Details of tissue and slide preparation:
For each animal, a section of the liver was removed,cut into small pieces and pushed through bolting cloth of pore size 150μm to produce single cell suspensions.An aliquot of the cell suspension was then added to agarose,plated onto four slides and allowed to gel.Three slides were placed in
lysis buffer for 1 hour,then transferred to electrophoresis buffer(pH>13)to allow DNA to unwind for 30 minutes,after which the slides were electrophoresed at 0.7 V/cm for 40 minutes.
Evaluation criteria:
no data
Statistics:
no data

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
Cloud assessment and analysis of diffusion slides of TCPP and vehicle control treated animals demonstrated low levels of cells (less than 15%)with significantly fragmented DNA,indicating little cytotoxicity,necrosis or apoptosis in the cell preparations.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
Nothing to add in this field.

Any other information on results incl. tables

Lethargy was observed in one animal at 1500 mg/kg, with no other clinical signs noted.At necropsy, the livers of animals dosed at 1500 mg/kg were noted to be darker in appearance than those of the 750 mg/kg or vehicle control groups. Cloud assessment and analysis of diffusion slides of TCPP and vehicle control treated animals demonstrated low levels of cells (less than 15%) with significantly fragmented DNA, indicating little cytotoxicity,necrosis or apoptosis in the cell preparations. Comet analysis of livers treated with TCPP, sampled at either 3 or 24 hours post dosing,had slightly elevated group mean tail moments and intensities compared with the concurrent control.However,there was no dose response, the increases were within the degree of variation frequently seen with this assay and also fell within the historical control range.The positive control induced a clear increase in tail

moment and tail intensity.

Summary of group mean data for in vivo Comet assay with TCPP

 Treatment group (mg/kg/day)   Sample time (hrs after dosing)   Group mean % clouds   Group mean % diffused cells   Tail Moment ± SEM   Tail Intensity ± SEM 
 Vehicle (0)   3   2.17   6.33   0.29 ± 0.04   2.20 ± 0.20 
 TCPP (750)   3   3.08   4.83   0.48 ± 0.04   2.94 ± 0.12 
 TCPP (1500)   3   2.50   8.83   0.51 ± 0.05   3.46 ± 0.25 
 Positive control EMS   3   2.17   11.33   1.40 ± 0.05   6.77 ± 0.31 
 Vehicle (0)   24   2.17   5.50   0.41± 0.04   2.91 ± 0.20 
 TCPP (750)   24   1.42   6.67   0.41 ± 0.02   2.90 ± 0.14 
 TCPP (1500)   24   1.33   7.50   0.49 ± 0.05   3.29 ± 0.32 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It was concluded that TCPP did not induce DNA damage in the liver or rats treated up to 1500 mg/kg.