Reaction products of phosphoryl trichloride and 2-methyloxirane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: other: DNA Damage and/or repair; Organ: liver.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed to Guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: recommendations of IWGTP workshop

Principles of method if other than guideline:

The Comet assay is a technique for investigating DNA breakage and damage in individual mammalian cells by using electrophoresis of DNA which has been unwound under alkaline conditions (> pH 13). Electrophoresis results in the charged DNA being drawn away from the nucleus, with relaxed and broken DNA fragments migrating further than undamaged DNA complexes. The use of alkaline conditions enables single stranded and alkaline labile sites as well as double stranded DNA breaks to be expressed during electrophoresis.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

1-chloropropan-2-yl bis(2-chloropropyl) phosphate; bis(1-chloropropan-2-yl) 2-chloropropyl phosphate; tris(1-chloropropan-2-yl) phosphate; tris(2-chloropropyl) phosphate

EC Number:

807-935-0

Cas Number:

1244733-77-4

Molecular formula:

C9H18Cl3O4P

IUPAC Name:

1-chloropropan-2-yl bis(2-chloropropyl) phosphate; bis(1-chloropropan-2-yl) 2-chloropropyl phosphate; tris(1-chloropropan-2-yl) phosphate; tris(2-chloropropyl) phosphate

Test material form:

liquid

Details on test material:

Tris (2-chloro-1-methylethyl) phosphate (TCPP), batch number TCPP-TNO, was a colourless liquid. It was received on 25 August 2006 and stored at room temperature. Purity was stated as 100% and expiry date as 1 September 2007.
The sample was prepared by TNO Quality of Life, Zeist, The Netherlands, on 1 February 2006 by mixing equal volumes of:
Fyrol PCF, batch 06 103 A-03-xx, supplied by Supresta Netherlands B.V.
Lupragen TCPP, batch 00000504N0, supplied by Elastogran GmbH
Levagard PP, batch CH2/06016, supplied by LANXESS Deutschland GmbH
Antiblaze TMCP, batch EJ 04AHT 051, supplied by Albemarle Corporation
The certificate of analysis dated 6 March 2006 and supplied by the Sponsor is given in Appendix 5. The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

A sufficient number of out-bred 7-8 week old adult male Sprague Dawley Crl:CD® rats were obtained from Charles River UK Ltd, Margate, UK. They were housed in cages that conformed to the Code of Practice for the housing and care of animals used in scientific procedures (Home Office, London, 1989). They were housed in groups of six. Aspen wood chips (Datesand Ltd, Manchester) were used for bedding. Bottled water (public supply) and diet, (Special Diets Services Ltd, RM1.(E).SQC.) were provided ad libitum. Additionally, in order to enrich the environment and enhance the welfare of the animals, they were provided with wooden Aspen chew blocks and/or nesting material. All of the above were routinely monitored and are not known to contain any biological or chemical entity which might have interfered with the test system.
Routinely, the temperature and relative humidity was 19 to 25°C and 40 to 70%, respectively. No deviation from these ranges occurred for longer than 24 hours throughout this study. The holding rooms were illuminated by fluorescent light for 12 hours out of each 24 hour cycle and are designed to receive at least 15 fresh air changes per hour.
Animals were acclimatised for at least 5 days prior to dosing and were uniquely identified by numbered ear-tag. Rats were individually identified and randomised to groups of six animals using a system of random numbers. Cages were suitably labelled (using a colour-coded procedure) to clearly identify the study number, study type, start date, number and sex of animals, together with a description of the dose level and proposed time of necropsy. Checks were made on the first day of treatment to ensure group weights differed from the overall mean by no more than 5%.
Number of animals used in study
42Males
Weight range on first day of assay (g)
224-268
Approximate age on first day of assay (weeks)
7-8
Acclimatisation period (days)
8

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

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.
Ethylmethanesulfonate was used as the positive control, dissolved at 25mg/ml in purified water.

Duration of treatment / exposure:

The animals were sacrificed in group order, 3or 24 hours after administration. Positiver control animals were killed 3 hours after administration.

Frequency of treatment:

Single dose

Post exposure period:

3 hours or 24 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
750 mg/kg
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

6 males

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:

The liver was chosen for comet analysis as TCPP caused an increased mutation frequency in the mouse lymphoma assay in the presence of S9 and induced liver enlargement in repeated dose toxicity studies.

Details of tissue and slide preparation:

The TCPP or the vehicle control treated rats were killed 3 or 24 hours after dosing. At necropsy, TCPP animals were examined internally for signs of cytotoxicity. For each animal, a section of the liver was removed, cut into small pieces and pushed through bolting cloth of pore size 150 µm 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. At the end of the electrophoresis period slides were neutralised, dried and stained with ethidium bromide for comet analysis. The fourth slide was neutralised and used to determine the degree of highly damaged cells in the cell suspensions (diffusion analysis). Scoring of slides was made using fluorescence microscopy at x 200 magnification and Comet scoring was performed using Perceptive Instruments ¿Comet Assay III¿ image analysis system. Measurements of tail moment and tail intensity (% DNA in tail) were obtained from 100 cells per animal.

Evaluation criteria:

The tail moment is defined as [tail profile centre of gravity ¿ head profile centre of gravity] x tail % DNA, and therefore gives a measure incorporating both tail length and tail content. Each slide was also examined for possible indications of cytotoxicity, with cells with ¿clouds¿, which is a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis, were not included in Comet scoring. Diffusion slides were scored by assessing 100 cells per slide.

Statistics:

Treatment of data
The experimental unit of exposure for in vivo studies is the animal, and all analysis was based on individual animal response. The following was calculated for each animal.
tail moment
tail intensity (i.e. % DNA in the tail)
Values obtained from each parameter were treated as follows:
1. Values were added together from replicate slides
2. The median was calculated
3. The mean of the medians and standard error of the mean was calculated for each group.
No further statistical analysis was performed.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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.Table 4.45below summarises the group mean data, including tail moment and tail intensity values.

Table 4.45  Summary of group mean data forin vivoComet 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	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.

Executive summary:

The potential of TCPP to induce DNA strand breaks or alkali sites by the degree of DNA damage in the liver of treated rats was investigated in the Comet Assay.

The choice of dose levels was based on toxicity studies, which identified 1500 mg/kg as the maximum tolerated dose. In this study, groups of six male rats were administered TCPP at 750 and 1500 mg/kg. The test article was formulated in corn oiland administered as a single dose via oral gavage at a dose volume of 10 mL/kg. The liver of the treated rats was analysed for DNA damage 3 or 24 hours after dose administration. Lethargy was observed in one of the animals receiving the highest dose of TCPP.

The negative (vehicle) control in the study, corn oil, was also administered once via oral gavage to groups of six rats that were killed and sampled 3 or 24 hours after dose administration. Ethyl methanesulfonate (EMS), the positive control, was dissolved in purified water and administred via oral gavage as a single dose at 250 mg/kg (dose volume of 10 mL/kg) to a group of six rats which were killed 3 hours after dose administration.

It is concluded that , unted the conditions of this Comet Assay, TCPP did not induce DNA damage in the liver of rats treated up to 1500 mg/kg/day, when analysed 2 hours or 24 hours after dose administration.