Dimethyl octadecylphosphonate

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:

06 November 2012 to 19 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study without deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl octadecylphosphonate

EC Number:

246-904-0

EC Name:

Dimethyl octadecylphosphonate

Cas Number:

25371-54-4

Molecular formula:

C20H43O3P

IUPAC Name:

dimethyl octadecylphosphonate

Constituent 2

Reference substance name:

M-5925

IUPAC Name:

M-5925

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Dimethyl octadecylphosphonate
- Physical state: white powder
- Lot/batch No.: B12-5990
- Expiry date: April 2014
- Purity: >97%
(Refer to report amendment)

Method

Target gene:

The thymidine kinase heterozygote system, where tk+tk- is mutated to tk-tk-, was described by Clive et al (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). In this assay, cells deficient in thymidine kinase (TK) due to the mutation tk+tk- to tk-tk- are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). Thymidine kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The cells used were from the tk+tk- -3.7.2C mouse lymphoma L5178Y cell line obtained from Dr D Clive, Burroughs Wellcome & Company, Research Triangle Park, North Carolina, USA

Additional strain / cell type characteristics:

other: deficient in thymidine kinase

Metabolic activation:

with and without

Metabolic activation system:

exogenous enzyme supplement (liver cell S9 mix)

Test concentrations with justification for top dose:

Test substance concentrations were selected based on a preliminary toxicity test in which concentrations in the range of 0.05 to 1000 uL/mL were investigated.
Assay 1 (in the absence of S9 mix): 3, 6, 9, 12, 16, 20, 25 and 30 μg/mL *
Assay 2 (in the presence of S9 mix): 3, 6, 9, 12, 16, 20, 25 and 30 μg/mL *
Assay 3 (in the absence of S9 mix): 2, 5, 8, 11, 14, 17, 20 and 23 μg/mL **
Assay 4 (in the presence of S9 mix): 6, 10, 14, 18, 22, 26, 30 and 34 μg/mL *
Concentrations of dimethyl octadecylphosphonate were not corrected for percent active ingredient
* experiment using a 4 hour exposure period
** experiment using a 24 hour exposure period

Vehicle / solvent:

Ethanol 1% v/v

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% v/v was used for the 4 hour exposures and 0.5% v/v was used for the 24 h exposures

Positive controls:

yes

Remarks:

All mutation assays included duplicate positive control cultures.

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

CELLS: The cells used were from the tk+tk- -3.7.2C mouse lymphoma L5178Y cell line obtained from Dr D Clive, Burroughs Wellcome & Company, Research Triangle Park, North Carolina, USA, in December 1982. The cells grow in suspension culture, have a generation time of about 11 h, have a stable, near-diploid chromosome number and have a high cloning efficiency in serum-enriched cloning medium.

METHOD OF APPLICATION: soft agar cloning method by Clive and with cloning performed accoring to the microwell method of Cole et al (1983).

COLONY SIZING: Genetic damage involving the TK locus results in 2 phenotypes. Some mutant cells divide at the normal rate, producing large colonies, while other cells divide at a distinctly slower rate, producing small colonies. A high proportion of large type colonies are associated with small chromosomal deletions or point mutations, while a large proportion of the small type colonies are associated with large chromosomal deletions. Assessment of the relative numbers of both colony types can provide information to support results obtained in bacterial mutation and chromosome aberration tests.

DURATION
- Exposure duration: 4 hours but a 24 hour exposure period was also included in assay 3 (absence of S9 mix)
- Expression time (cells in growth medium): cells grown in suspension cuclture have a generation time of about 11 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): After completion of the 4 or 24 h exposure period cells were allowed to express their genetic lesions for 2 days.

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

CULTURE MEDIUM
The basic culture medium (R0P) was RPMI 1640 medium, supplemented with penicillin G (100 units/mL), streptomycin (100 μg/mL), sodium bicarbonate (1.125 g/L) and pluronic acid (0.05%, w/v). For cell growth, heat-inactivated horse serum (10%, v/v) was added to R0P to give R10P.
The medium used during treatment for 4 h was R0P supplemented with 5% (v/v) heat-inactivated horse serum (R5P). The medium used during treatment for 24 h was R10P.
For colony formation, cloning medium was used, consisting of R0P supplemented with heat-inactivated horse serum (20%, v/v), sodium pyruvate (1.9 mM), and amphotericin B (fungizone) (2.5 μg/mL).
For selection of tk-tk- cells, cloning medium was supplemented with trifluorothymidine (TFT) at 3 μg/mL.

NUMBER OF CELLS EVALUATED: for the cloning efficiency assay each culture was diluted into the cloning medium to give an estimated 8 cells/mL. For the mutation selection assay there were 1 x10^4 cells/mL

Evaluation criteria:

The plates were scored using a dissecting microscope fitted to a light box with dark field illumination. The number of empty wells in each plate in the cloning efficiency assay was counted. When scoring the mutant selection assay, separate counts were made of the numbers of wells containing large type and small type colonies. Large colonies are defined as covering greater than ¼ of the floor of the well, while small colonies cover less than ¼ of the well (Moore et al (2000)). In addition, there are morphological differences. Large colonies tend to be similar to those found on the cloning efficiency plates, being generally flat. Small colonies tend to look dense in comparison. Any wells containing both colony types were scored as a large type. (The total number of empty wells is required for the calculation of mutant fraction, so each well can only be scored once).

Statistics:

The results for each experiment were subjected to a test for linear trend in mutant fraction with concentration of Dimethyl Octadecylphosphonate by the recommended UKEMS method.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The tested concentrations were limited by the cytotoxicity of the test item. Relative cell growth at 20 ug/ml without S9 activation was 16% and at 26 ug/ml with S9 activation was 22% for a four hour exposure.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity test - In the preliminary toxicity test, in the absence of the S9 mix, a concentration of 10 ug/mL of the test substance after a 4 hour exposure reduced cell suspension growth to 66% of the vehicle control group. Lethality was seen at 33.3 ug/mL. In the presence of S9 mix, no appreciable toxicity was seen at 10 ug/mL but lethality was still present at 33.3 ug/mL. 24 hour exposure in the absence of S9 showed toxicity (reduced suspension growth 19.4% ) at 16.67 ug/mL and lethality at 50 ug/mL.

Vehicle control groups - solvent control mean mutant fractions met the acceptance criteria.
Positive control groups - The IMF values and colony size ratios for MMS and 3-MC met the acceptance criteria.

Assay 1; absence of S9; 4 hour exposure (Table 1)
All mean IMF values were well below the minimum 126 mutants per million required to indicate a biologically relevant increase, and therefore the test item was considered not to be demonstrating mutagenic activity. The test for linear trend in mutant fraction with concentration of test item was not significant (P=0.25). The RTG at the highest assessed concentration of 20 μg/mL was 16%: a definitive level of toxicity. A call of not mutagenic was made for the assay. As a consequence, the second experiment in the absence of S9 mix was conducted with the extended, 24 h exposure period.
Assay 2; presence of S9; 4 hour exposure (Table 2)
All mean IMF values were well below the minimum 126 mutants per million required to indicate a biologically relevant increase, and therefore the test item was considered not to be demonstrating mutagenic activity. A call of not mutagenic was made for the assay. It was noted that the higher concentrations gave mutant fraction values marginally higher than the vehicle control group and this led to the test for linear trend showing a low level of sigificance (p=0.013).
Assay 3; absence of S9; 24 hour exposure (Tables 3 & 4)
An apparent handling error in the cloning efficiency section of this assessment resulted in a complete absence of cells in the cloning efficiency plates at the 14 and 17 μg/mL treatments. As there were complete results for one lower treatment and for 2 higher treatments, including the definitively toxic highest concentration of 23 μg/mL, these 2 treatments were considered not to be critical, and consideration was given to alternative ways to show that the results were in keeping with the 2 higher and one lower treatments (Table 4). These higher and lower treatments gave no evidence of mutagenic activity. The IMF values were all well below the threshold level of 126 mutants per million, and the test for linear trend with concentration was not significant (P=0.21). The RTG value at the highest concentration of 23 μg/mL was 16%: a definitive level of toxicity.
Assay 4; presence of S9; 4 hour exposure (Table 5)
All mean IMF values were below the minimum 126 mutants per million, required to indicate a biologically relevant increase, and therefore the test item was considered not to be demonstrating mutagenic activity. As in Assay 2, the mutant fraction values for the higher concentrations of test item were marginally higher than the vehicle control group. This resulted in the test for linear trend in mutant fraction with concentration of test item showing significance (P<0.001). The RTG at the highest assessed concentration of 26 μg/mL was 22%. This value is very close to the definitive range of 10-20% (definitive toxicity having already been reached in Assay 2). A call of not mutagenic was made for the assay.

Remarks on result:

other: other: thymidine kinase locus tk+tk- to tk-tk-

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

  Table 1 Mutation assay in the Absence of S9 Mix (4 hour Exposure), Summary of means of data (Assay 1)

 

Chemical	Concentration (µg/mL)	Relative Total Growth %	Mutant Fraction (x 10^-6)	IMF (Induced Mutant Fraction x 10^-6)	Ratio of Small to Large Colonies
Ethanol	(100 µL added)	100	96	N/A	0.62
EMS	250	72	867	771	0.37
MMS	10	34	1021	925	2.28
Dimethyl Octadecylphosphonate	3	NPS	NPS	NPS	NPS
	6	95	96	-	0.99
	9	80	102	6	1.41
	12	58	07	10	1.63
	16	31	103	7	1.57
	20	16	137	41	1.58
	25	NPT	NPT	NPT	NPT
	30	NPT	NPT	NPT	NPT

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

Test for linear trend of mutant fraction with concentration of Dimethyl Octadecylphosphonate = not significant (P=0.25)

- = IMF value ≤1

NPS = Not Plated - Surplus

NPT = Not Plated – Toxic

 

Table 2 Mutation assay in the Presence of S9 Mix (4 hour Exposure), Summary of means of data (Assay 2)

Chemical	Concentration (µg/mL)	Relative Total Growth %	Mutant Fraction (x 10^-6)	IMF (Induced Mutant Fraction x 10^-6)	Ratio of Small to Large Colonies
Ethanol	(100 µL added)	100	87	N/A	2.07
3-MC	2.5	62	924	837	0.90
	10	40	1211	1123	1.22
Dimethyl Octadecylphosphonate	3	NPS	NPS	NPS	NPS
	6	NPS	NPS	NPS	NPS
	9	110	94	6	0.60
	12	100	82	-	0.85
	16	84	134	46	1.21
	20	47	149	62	1.25
	25	16	138	51	1.94
	30	NPT	NPT	NPT	NPT

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

Test for linear trend of mutant fraction with concentration of Dimethyl Octadecylphosphonate = significant (P=0.013)

- = IMF value ≤1

NPS = Not Plated - Surplus

NPT = Not Plated - Toxic

 

Table 3 Mutation assay in the absence of S9 Mix (24 hour Exposure), Summary of means of data (Assay 3)

 

Chemical	Concentration (µg/mL)	Relative Total Growth %	Mutant Fraction (x 10^-6)	IMF (Induced Mutant Fraction x 10^-6)	Ratio of Small to Large Colonies
Ethanol	(100 µL added)	100	129	N/A	1.13
EMS	100	74	1434	1305	0.35
MMS	5	40	2071	1942	1.14
Dimethyl Octadecylphosphonate	2	NPS	NPS	NPS	NPS
	5	NPS	NPS	NPS	NPS
	8	NPS	NPS	NPS	NPS
	11	82	150	20	1.20
	14	$	$	$	1.02
	17	$	$	$	1.26
	20	34	151	22	1.67
	23	16	161	32	0.99

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

NPS = Not Plated - Surplus

Test for linear trend of mutant fraction with concentration of Dimethyl Octadecylphosphonate = not significant (P=0.21)

$ = Values not calculable due to plating error in cloning efficiency assessment. See table on following page for

approximations of values and alternative figures for toxicity and mutant assessment

 

Table 4 Mutation assay in the absence of S9 Mix (24 hour Exposure), Summary of means of data with additional calculations (Assay 3)

 

Chemical	Concentration (µg/mL)	Relative cell suspension growth %	Relative Total Growth %	Mutants per million cells plated	Mutant Fraction (x 10^-6)	IMF (Induced Mutant Fraction x 10^-6)	Ratio of Small to Large Colonies
Ethanol	(100 µL added)	100	100	106	129	-	1.13
EMS	100	72	74	1233	1434	1305	0.35
MMS	5	54	40	1261	2071	1942	1.14
Dimethyl Octadecylphosphonate	2	101	NPS	NPS	NPS	NPS	NPS
	5	89	NPS	NPS	NPS	NPS	NPS
	8	86	NPS	NPS	NPS	NPS	NPS
	11	90	82	114	150	20	1.20
	14	66	(63)$	114	(143)$	(14)$	1.02
	17	55	(52)$	115	(145)$	(16)$	1.26
	20	34	34	125	151	22	1.67
	23	17	16	125	161	32	0.99

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

NPS = Not Plated - Surplus

Test for linear trend of mutant fraction with concentration of Dimethyl Octadecylphosphonate = not significant (P=0.21)

$ = Values are approximations based on the mean cloning efficiency of the 11 and 20 μg/mL treatments combined. Relative Cell Suspension Growth figures are included as an alternative assessment of toxicity

Mutants per Million Cells Plated figures are included as an alternative assessment of the mutant fractions

 

Table 5Mutation assay in the presence of S9 Mix (4 hour Exposure), Summary of means of data (Assay 4)

 

Chemical	Concentration (µg/mL)	Relative Total Growth %	Mutant Fraction (x 10^-6)	IMF (Induced Mutant Fraction x 10^-6)	Ratio of Small to Large Colonies
Ethanol	(100 µL added)	100	155	N/A	0.96
3-MC	2.5	77	1108	953	0.94
	10	51	1714	1559	0.82
Dimethyl Octadecylphosphonate	6	NPS	NPS	NPS	NPS
	10	136	132	-	0.59
	14	100	134	-	0.79
	18	118	170	15	0.70
	22	48	220	65	1.09
	26	22	276	121	1.64
	30	NPT	NPT	NPT	NPT
	34	NPT	NPT	NPT	NPT

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

NPS = Not Plated - Surplus

Test for linear trend of mutant fraction with concentration of Dimethyl Octadecylphosphonate = significant (P<0.001)

- = IMF value ≤1

NPS = Not Plated - Surplus

NPT = Not Plated – Toxic

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In conclusion, Dimethyl Octadecylphosphonate was not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix when tested in ethanol at concentrations extending into the toxic range.

Executive summary:

The test item, Dimethyl Octadecylphosphonate, was assayed for mutagenic potential in the mouse lymphoma L5178Y cell line, clone -3.7.2C, scoring for forward mutations at the thymidine kinase locus: tk+tk- to tk-tk-. Tests were conducted both in the absence and in the presence of a post-mitochondrial supernatant fraction obtained from Aroclor 1254-induced livers of adult male rats and the co-factors required for mixed-function oxidase activity (S9 mix). The study was designed to be consistent with ICH Guidelines, OECD Guideline No. 476 and EC Directive 2000/32/EC B.17.

Preliminary cytotoxicity tests showed that Dimethyl Octadecylphosphonate was of a moderate to high order of toxicity, reducing cell growth over the range 3.3 to 33.3 μg/mL in the absence of S9 mix (4 h exposure period), 10 to 33.3 μg/mL in the presence of S9 mix (4 h exposure period) and over the range 1.67 to 50 μg/mL in the absence of S9 mix (24 h exposure period).

Four independent mutation assays were conducted, as follows:

 

Assay No.	Presence or absence of S9	Treatment time(h)	Concentrations(µg/mL)
1	Absence	4	6,9,12,16,20
2	Presence	4	9,12,16,20,25
3	Absence	24	11,14,17,20,23
4	Presence	4	10,14,18,22,26

 

Positive control cultures were included, and the resultant mutant fractions from these provided the expected increase and proof of adequate recovery of ‘small’ type colonies. Duplicate cultures were carried through the experiments for each treatment point. Vehicle control cultures were also included and were tested in quadruplicate.

Biological relevance was given to any increase in mutant fraction greater than 126 mutants per million above the concurrent control value. In addition, all experiments were tested for dose-related trends in mutant fraction.

No biologically relevant increase in mutant fraction was obtained in either the absence or the presence of S9 mix. (It was noted that small increases considered to be of no biological relevance were obtained in the Dimethyl Octadecylphosphonate treatment groups in the presence of S9 mix.)

Results were obtained at a definitive level of toxicity in both the absence and the presence of S9 mix (Assays 1, 2 and 3).

In conclusion, Dimethyl Octadecylphosphonate was not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix when tested in ethanol at concentrations extending into the toxic range.