Alkyl phosphites

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was carried out to GLP standard, in accordance with OECD guideline 473. Some details on the test material are missing.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A Medium containing 10% fetal bovine serum and 2mM L-glutamine at 37 ± 2 degrees centigrade.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: Not stated
- Periodically "cleansed" against high spontaneous background: Not stated

Cells supplied by Merck Research Laboratories, West Point Pennsylvania, 19486.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Liver of Aroclor-Pretreated Sprague Dawley Rats (S9 mix)

Test concentrations with justification for top dose:

Solubility Test

A vehicle solubility test was performed with the test substance, it was found to be soluble in both acetone and tetrahyrofuran. Acetone was chosen as the vehicle.

Culture Medium Solubility: A solubility test was carried out at the concentrations listed below.

10, 20, 39, 78, 156, 313, 625, 1250 and 2500 µg/mL (rounded to the nearesr whole number).

Toxicity Pretest

2.5, 5.0, 10, 20, 40, 80, 160, 320, 640 and 1280 µg/mL

Chromosomal Abberation Assay

2, 4, 8, 10, 12, 16, 20, 24 µg/mL without metabolic activation (-S9)

10, 20, 40, 60, 80, 100 and 120 µg/mL with metabolic activation (+S9).

Vehicle Control: 50 µl/10 mL

Positive Control: 7, 12-Dimethylbenz[a]anthracene (DMBA) 10 µg/mL, N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG) 0.6 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Considered stable for the duration of the assay.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

other: N-Methyl-N-Nitro-N-Nitrosoguanidine and 7, 12-Dimethylbenz[a]anthracene

Details on test system and experimental conditions:

see below

Evaluation criteria:

Criteria for a valid assay: For an assay to be considered valid the following criteria must be established:
1. The percentage of cells with one or more aberrartion (% aberrant cells) must be between 0-5% for the vehicle control.

2. The positive control susbtances must induce frequencies of chromosomal aberration outside the normal range of the vehicle control (0-5% aberrany cells per treatment group) and be at least twice the value of the vehicle control.

Criteria for Evaluation of Data

For a test to be considered as inducing a positive result when compared to the vehicle control one or of the following condtions must be met.
1. A statistically siginificant dose related increase in the mean percentage of aberrant cells and in at least one of the treatment groups the mean percentage of aberrant cells exceeds 5%

or

2. A reproducible and statistically significant response for at least one of the treatment groups observed. In addition the mean percentage of aberrant cells exceeds 5%.

A positive result indicates that under the test conditions the test substance induces chromosome aberrations in cultured mammalian somatic cells. If neither of the above conditions are met the test substance is considerd negative for inducing chromosomal aberrations in this system.

Statistics:

The following analyses were performed for each of the activated and nonactivated series of experiments

1. The number of cells with at least one aberrant chromosome and the number of cells examined in each replicate were used for statistical analysis. The number of aberrant individual chromosomes per were not staitically analysed (Richardson et al 1989)

2. The positive control group results were compared to the vehicle control group by fisher Exact Test (Bradley, J. V, 1968: Metha et al 1983) to assure that the assay perfomred in an appropriate manner.

3. To test for homogenity of the replicates in the control and treated groups each pair of replicates were compared by Fisher Exact Test, then 2 times the sum of the log of the individual two sided significant levels were compared by chi squared distribution with 2K dgrees of freedom (k is the number of replicate pairs). If the test failed further investigation would be pursued and the remaining analysis not performed.

4. To test for differences among the control and treated group a 2x4 Fisher Exact Test was performed. If differences were shown to exsist at least at the 0.05 level, individual 2x2 Fisher Tests would be performed to determine which of the treated groups differed from the control group.

5. A permutation Test (Hoeffding, W, 1950) was performed to test for dose related trends.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 80 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 10 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 80 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 16 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Preliminary Test

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Culture Media Soubility Test

Precipitation was observed microscopically at all thest concetrations =10 µg/mL (with metabolic activation) and in all concetrations

= 80 µg/mL (without metabolic activation). Test concentrations selected by the study director for the toxicity pretest were 2.5, 5.0, 10, 20, 40, 80, 160, 320, 640 and 1280 µg/mL.

Toxicity Pretest

A noteable decrease in the mitotic index was observed at concetrations = 10 ug/mL(without metabolic activation) and 80 ug/mL (with metabolic activation). In the nonactivated series cell confluency at 20 ug/mL was below 5% and complete cell death (0% confluency) occured at higher test concentrations. In the activated series cell confluency was reduced to 20% at 80 ug/mL (as compared to 50% confluency for the vehicle control) and complete cell death occured at higher concentrations. The percent cells surviving was reduced to 22% at 20 ug/mL without activation and to 61% at 80 ug/mL with activation. Therefore the doses selected for chromosomal aberration assay were 2, 4, 8, 12, 16, 20 and 24 ug/mL (without metabolic activation) and 10, 20, 40 ,60, 80, 100 and 120 ug/mL (with metabolic activation).

Aberration Assay Results

Initial Assay

In the initial assay a 50% or greater reduction in cell confluency was observed at 80 ug/mL with activation and 20 ug/mL without activation (as compared with the vehicle controls). Thus test concentrations selected for the evaluation of chromosomal aberration for the 16 hour harvest were 40, 80 and 100 ug/mL for activated series and 8, 16 and 24 ug/mL for the nonactivated series.

There was no dose related trend or statistically significant difference in the percentage of aberrant celss in the initial assay for the activated series. The percentage of aberrant cells in the vehicle control group with metabolic activation was 0.5%. The percentage of aberrant cells in the treatment groups for the series with metabolic activation ranged from 0.5 to 2.5%.

In the non activated group trend analysis showed a statistical difference (p <0.05) however the number of aberrant cells in each test group was within the expected value of the vehicle control. Therefore the trend was not considered biologically significant.There was no statistically significant difference in the number of aberrant cells. The number of aberrant cells in the treatment groups for the series without metabolic activation ranged from 0.5 to 2.5%. The percentage of aberrant cells for the vehicle control group without activation was 0.5%. In contrast the positive control group for both the metabolically activated and non activated series (DMBA and MNNG respectively) showed aberration percentages which were well above the vehicle control frequencies and statistically significant at p<0.01.

Repeat Assay

Based on the results of the inital assay the same test concetrations were selected for analysis of chromosomal aberration in the activated series for the 16 and 40 hour harvests in the repeat assay. Test concetrations selected for analysis of the nonactivated series were also identical to those evaluated in the initial assay for the 16 hour harvest group. However a narrower range of concetrations were selected to evaluate the 40 hour harvest, since the exposure to 20 and 24 ug/ml resulted in complerte toxicity. Therefore 8, 12 and 16 ug/mL dose levels were selected for evaluation of the 40 hour nonactivated harvest.

In the repeat assay trend analysis by permutation test showed a statsitical difference (p<0.01) in the 16 hour harvest of the nonactivated series. In addition the percentage of aberrant cells at the 16 ug/mL level was statistically increased at the p<0.01 level as compared to the respective vehicle control group. However neither the 16 hour harvest interval of the activated series nor the 40 hour activated and nonactivated harvest showed statistically significant goup trend analysis differences.

The vehicle control groups of the 16 and 40 hour harvests (with and without metabolic activation) had aberrant cell percentages of 2.0% or less. The highest percentage of aberrant cells observed for the test susbtance treated groups in the 16 hour harvest was 4% at 80 ug/mL for the activated series and 6.0% at 16 ug/mL for the nonactivated series. The highest percentage of aberrant cells observed in the 40 hour harvest was 3.5% at 40ug/mL for the activated series and 2.5% or less for all treated groups in the nonactivated series.As observed in the initial assay the percentage of aberrant cells noted in the positive control groups was well above that of the vehicle control and was statistically siginificant at the p<0,02 level. No positive control was required for the 40 hour harvest.

In the repeat assay an increased number of pulverized cells was observed at the 16 hour harvest (16 and 24 yg/mL) and 40 hour harvest (16 ug/mL) of the nonactivated series. The increases indicated severe cytotoxicity.

Increased numbers of endoreduplicated metaphases were observed at the 16 ug/mL level of the 40 hour harvest nonactivated series. Endoreduplication does not represent chromosome breakage. It is the result of two successive round on DNA replication without an intervening cell division (i.e. mitotic disruption); when the cells subsequently reach mitosis the chromosome appear as pairs or diplochromosomes. Possible mechanisms of endoreduolication involve inihbition of normal DNA synthesis and or temporary cell cycle block in G2 phase.

Statistical Analyses

The test for homogeneity of the replicate pairs indicated homogenity beyond the 0.05 level of significance for all treatment groups, vehicle controls and positive controls in both the metabolically activated and nonactivated series in both the initial assay and repeat assay.

Analytical Chemistry Satisfactory uniformity of the mixture was observed. Concentration verification analyses indicated that the test substance concentration was within 7% of the nominal value. Since the mixtures were does immediately after preperation and the concentraion verification was within acceptable range, the mixtures were considered stable.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

There was no statistically siginifcant difference or dose related trend in the percentage of aberrant cells in the intial assay for the activated series. In the non activated series trend analysis showed a statistical difference however the number of aberrant cells in each thest group was within the expected values of the vehicle control. Therefore the trend was not considered biologically significant. In the repeat assay trend analysis showed a statistical difference in the 16 hour harvest of the nonactivated series and the percentage of aberrant cells was significantly higher than the control group at the mid dose (16 µg/mL) level. The statistical increase at this level (16 µg/mL) was not considered biologically significant since 1) a similar increase was not observed in the high dose levels (24 µg/mL) of the repeat assay, 2) no significant differences were observed in the percentage of aberrant cells in the initial assay for either the activated or nonactivated series and 3) no differences were observed in the percentage of aberrant cells at the 40 hour harvest of the repat assay. In addition in the activated series the percentage of aberrant cells remianed unaffected in both the initial and repeat assays.

Increases in endoreduplicated cells were not observed at the 40 hour harvest without metabolic activation. The production of these cells does not represent chromosome breakage but does indicate that the test substance is capable of mitotic disruption.

The positive control materials DMBA and MNNG produced statistically significant increases in percentage of aberrant cells when tested with activation (DMBA) or without aactivation (MNNG). Thus the test system responded in an appropriate manner to these known clastogens. The vehicle control used in this study (acetone) also performed in a manner consistent with the criteria established for a valid assay. In conclusion under the conditions of this study the test substance did not induce chromosomal aberrations in CHO cells.

No circumstances occured which would have affected the quality of the data.

Executive summary:

The study was performed in accordance with OECD guideline 473 to evaluate the potential of the test susbtance to induce structural chormosome aberrations in cultured chinese hamster ovary cells. The study consisted of two phases, an initial 16 hour chromosomal aberration assay and a repeat assay with both 16 and 40 hour cell ahrvest times. In each phase the test susbtance was dissolved in acetone and the dissolved test substance was added to the media to achieve final test concentrations of 2, 4, 8, 12, 16, 20 and 24 µg/mL without metabolic activation (-S9) and 10, 20, 40, 60, 80, 100 and 120 µg/mL with metabolic activation (+S9). The procedures were performed in general agreement with those published by Galloway et al 1985.

The CHO cells were exposed to test substance, two sets of duplicate cultures were prepared, one with metabolic activation of test substance and one without. Positive controls (N-methyl-N-Nitro-N-Nitroguanidine or 7, 12 -Dimethylbenz[a]anthracene) and vehicle controls (acetone) were also tested. Flasks with and without metabolic activation were treated for 3 and 16 hours respectively. The cultures were incubated to the respective harvest times (16 and 40 hours). A spindle inhibitor was added to the flasks approximately 2 hours prior to harvest to arrst the cells in c-metaphase. The cells were then harvested and slides prepared for evaluate chromosomes.

Selection of test concetrations for the evaluation of chromosomal aberrations was based on percent cell confluency, such as the highest concentrations selected for the evaluation induced at least 50% reduction in confluency when compared to the vehicle control. Thus concentrations of 40, 80 and 100 µg/mL were selected for evaluation in the 16 and 40 hour cell harvests with activation. Concentrations of 8, 16 and 24 µg/mL were selected for evaluation in the 16 hour cell harvests without activation and concentrations of 8,12 and 16 µg/mL were selected for evaluation in the 40 hour cell harvest without activation.

There was no statistical difference or dose related trend in the percentage of abberant cells in the initial assay for the activated series. In the non activated series trend analysis showed a statistical difference however the number of aberrant cells in each test groups was within the expected values of the vehicle control. Therefore the trend was not considered biologically significant. In the repeat assay, trend analysis showed a statistical difference in the 16 hour harvest of the non activated series and the percentage of aberrant cells was significantly higher than the control group at the mid dose (16 µg/mL) level. The statistical increase at the (16 µg/mL) was not considiered biologically significant since 1) a similar increase was not observed in the high dose levels (24 µg/mL) of the repeat assay, 2) no significant differences were observed in the percentage of aberrant cells in the initial assay for either the activated or nonactivated series and 3) no differences were observed in the percentage of aberrant cells at the 40 hour harvest of the repat assay. In addition in the activated series the percentage of aberrant cells remianed unaffected in both the initial and repeat assays.

Increases in endoreduplicated cells were not observed at the 40 hour harvest without metabolic activation. The production of these cells does not represent chromosome breakage but does indicate that the test substance is capable of mitotic disruption.

In conclusion under the conditions of this study the test substance did not induce chromosomal aberrations in CHO cells. The vehicle and positve control used in this study performed in an appropriate manner, indicating that the test system could detect both activation dependant and direct acting clastogens.