Reaction products of phosphoryl trichloride and 2-methyloxirane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation 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:

The test substance comprised a mixture of four commercial samples of TCPP, mixed in equal measure.
Identifier: Fyrol PCF
Supplier: Supresta Netherlands BV
Batch no: 04 103 F-09-04
Physical Appearance: clear, colourless liquid
Received:22 Dec 2004
Storage: room temperature in the dark
MW: 327.5
CAS no: 13674-84-5
purity: >99%
Expiry date: 22 Mar 2005

Identifier: Antiblaze TMCP
Supplier: Albemarle Chemicals UK ltd
Batch no: DM29HT051
Physical appearance: clear colourless liquid
Received: 20 Jan 2005
Storage: room temperature in the dark
MW: 327.6
CAS no: 13674-84-5
purity: >95%
Expiry date: 20 Apr 2005

Identifier: TCPP
Supplier: Elastogran GmbH
Batch no: 00000204N0
Physical appearance: yellow liquid
Received: 17 Jan 2005
Storage: room temperature in the dark
MW: 327.75
CAS no: 13674-84-5
purity: >98%
expiry date: 17 Apr 2005

Identifier: TCPP-Levagard PP
Supplier: LANXESS Deutschland GmbH
Batch no: CH1/14124
Physical appearance: clear colourless liquid
Received: 21 Dec 2004
Storage: under desiccant at room temperature in the dark
MW: 327.6
CAS no: 13674-84-5
purity: 97%
expiry date: 21 Mar 2005

All four components were prepared at identical concentrations (w/v) in sterile anhydrous DMSO, then mixed in the ratio of 1:1:1:1 (v/v) to give a pooled solution of the test article.

Method

Target gene:

thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not recorded
- Periodically "cleansed" against high spontaneous background: not recorded

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Initially examined both in the absence and presence of Aroclor 1254 induced rat liver S9 fraction at doses of 62.5, 125, 250, 500, 1000 and 2000 µg/ml. Complete toxicity was observed at 500 µg/ml in the absence of S9 and at 250 µg/ml in the presence of S9. Therefore, for the first experiment doses of 150, 200, 250, 300, 400 and 450 µg/ml without S9 and 80, 100, 112.5, 125, 137.5, 150 and 200 µg/ml with S9 were tested for viability and trifluorothymidine (TFT) resistance. For the second experiment the following doses were plated for viability and TFT resistance: 100, 150, 200, 250, 300, 350, 400 and 450 µg/ml without S9 and 25, 50, 75, 100, 125, 150, 175 and 200 µg/ml with S9. The highest doses 450 and 500 µg/ml without S9 and 250 and 300 µg/ml with S9 were excluded from plating due to excessive cytotoxicity.

Vehicle / solvent:

DMSO- the test substance has a solubility of 500mg/ml in DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: with metabolic activation

Controls 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: without metabolic activation

Details on test system and experimental conditions:

A 3-hour treatment incubation period was employed for all treatments in the presence and absence of S9 mix. The highest dose of 500 µg/ml without S9 was considered too toxic and was excluded from viability and (TFT) plating. For the second experiment the following doses were plated for viability and TFT resistance: 100, 150, 200, 250, 300, 350, 400 and 450 µg/ml without S9 and 25, 50, 75, 100, 125, 150, 175 and 200 µg/ml with S9. The highest doses of 475 and 500 µg/ml without S9 and 250 and 300 µg/ml with S9 were excluded from plating due to excessive toxicity.

Evaluation criteria:

The test article was considered to be mutagenic if all the following criteria were met:
the assay was valid according to the Acceptance criteria; the mutation frequency at one or more doses was significantly greater than that of the negative control (p<0.05); there was a significant dose-relationship as indicated by the linear trend analysis (p<0.05)

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was carried out according to UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment dose based on Dunnett's test for multiple comparisons. The data was checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test is one tailed, therefore a negative trend wasnot considered significant. These tests require the calculation of a heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

In the absence of metabolic activation, there was no significant increase in mutation frequency in either the first or second experiment up to toxic doses. In the presence of S9, statistically significant increases in mutation frequency were observed at the highest doses in both the first (137.5, 150 and 200 µg/ml) and second (150, 175 and 200 µg/ml) experiment. The relative total growth (RTG) at these doses were 52, 58 and 41% in the first experiment and 28, 18 and 10% in the second experiment, respectively. Large and small colonies were scored for the doses at which statistically significant increases in mutation frequency were observed. Increases in both small and large colony mutant frequencies were noted as well as a clear increase in the proportion of small colony mutants, indicating potential clastogenic activity.

Applicant's summary and conclusion

Conclusions:

When tested up to toxic doses, tris(2-chloro-1methylethyl) phosphate induced mutation at the tk locus of L5178Y mouse lymphoma cells in two independent experiments in the presence of S9, but did not induce mutation in two independent experiemnts in the absence of S9. It is concluded that under the conditons employed in the study, Tris(2-chloro-1-methylethyl) phosphate was mutagenic in this test system in the presence of S9, but not in the absence of S9.

Executive summary:

When tested up to toxic doses, tris(2-chloro-1methylethyl) phosphate induced mutation at the tk locus of L5178Y mouse lymphoma cells in two independent experiments in the presence of S9, but did not induce mutation in two independent experiemnts in the absence of S9. It is concluded that under the conditons employed in the study, Tris(2-chloro-1-methylethyl) phosphate was mutagenic in this test system in the presence of S9, but not in the absence of S9.