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Genetic toxicity: in vitro

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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:
1995-04-24 - 1995-10-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Scientifically valid, well documented GLP study according to EU method B.10 and the requirements of the Japanese New Chemical Substance Law (MITI) on the registered substance itself.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: requirements of the Japanese New Chemical Substance Law (MITI)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction Products of Diphosphorus Pentaoxide with n-Alcohols, C8-10 (even), salted with Amines, C12-14, Tert-alkyl
EC Number:
943-016-1
Molecular formula:
Too complex
IUPAC Name:
Reaction Products of Diphosphorus Pentaoxide with n-Alcohols, C8-10 (even), salted with Amines, C12-14, Tert-alkyl
Test material form:
liquid: viscous
Details on test material:
- Substance type: pure substance
- Physical state: liquid
- Storage condition of test material: room temperature

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
mammalian cell line, other: CHL cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's Minimal Essential medium with Earle's Salts (MEM), supplemented with 10% foetal bovine serum and antibiotics, at 37'C with 5% CO2 in air.
Metabolic activation:
with and without
Metabolic activation system:
Microsomal Enzyme Fraction: Lot No. Aro. S9/31/5/95 was prepared 'in-house' on 31/5/95 from the livers of male Sprague-Dawley rats weighing ~ 200g which had received a single ip. injection of Aroclor 1254 at 500 mg/kg, up to 5 days before S9 preparation.
Test concentrations with justification for top dose:
0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000 µg/ml (±S9, all treatments, preliminary cytotoxicity test)
0, 5, 10, 20, 40, 60, 80 µg/ml without S9 (Experiment 1, 12h harvest, 4h treatment, 8h recovery)
0, 10, 20, 40, 80, 120, 160 µg/ml with S9 (Experiment 1, 12h harvest, 12h treatment)
0, 10, 20, 40, 60, 70, 80 µg/ml without S9 (Experiment 2, 12h harvest, 4h treatment, 8h recovery)
0, 40, 60, 80, 100, 120, 160 µg/ml with S9 (Experiment 2, 12h harvest, 12h treatment)
0, 10, 20, 40, 60, 80, 120 µg/ml without S9 (Experiment 2, 24h harvest, 6h treatment, 18h recovery)
0, 40, 80, 100, 120, 160 µg/ml with S9 (Experiment 2, 24h harvest, 6h treatment)
0, 2.5, 5, 10, 20, 40 µg/ml (Experiment 2, 24h harvest, continuous treatment)
0, 1.25, 2.5, 5, 10, 20 µg/ml (Experiment 2, 48h harvest, continuous treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controls
Untreated negative controls:
yes
Remarks:
vehicle controls
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Mitomycin C without S9, 0.075 µg/ml (12h cultures) / 0.05 µg/ml (24h & 48h cultures); Cyclophosphamide with S9, 10 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration:
a) 12h / 24h / 48h (-S9, preliminary cytotoxicity test)
b) 4h / 6h (+S9, preliminary cytotoxicity test)
c) 12h (-S9, Experiment 1)
d) 4h (+S9, Experiment 1)
e) 24h (-S9, Experiment 2)
f) 48h (-S9, Experiment 2)
g) 6h (-S9, Experiment 2)
h) 12h (-S9, Experiment 2)
i) 6h (+S9, Experiment 2)
j) 4h (+S9, Experiment 2)

- Expression time (cells in growth medium):
a) none (-S9, preliminary cytotoxicity test)
b) 8h (4h exposure) / 18h (6h exposure) (+S9, preliminary cytotoxicity test)
c) none (-S9, Experiment 1)
d) 8h (+S9, Experiment 1)
e) none (-S9, Experiment 2)
f) none (-S9, Experiment 2)
g) 18h (-S9, Experiment 2)
h) none (-S9, Experiment 2)
i) 18h (+S9, Experiment 2)
j) 8h (+S9, Experiment 2)

- Fixation time (start of exposure up to fixation or harvest of cells):
a) 12h / 24h / 48h (-S9, preliminary cytotoxicity test)
b) 12h (4h exposure) / 24h (6h exposure) (+S9, preliminary cytotoxicity test)
c) 12h (-S9, Experiment 1)
d) 12h (+S9, Experiment 1)
e) 24h (-S9, Experiment 2)
f) 48h (-S9, Experiment 2)
g) 24h (-S9, Experiment 2)
h) 12h (-S9, Experiment 2)
i) 24h (+S9, Experiment 2)
j) 12h (+S9, Experiment 2)

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 µg/ml) 2h prior to harvest
STAIN (for cytogenetic assays): 2% Gurrs Giemsa R66 for 5 min

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 100 consecutive well-spread metaphases per culture for Chromosome damage, 1000 cells per culture for mitotic index

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
For Chromosome Damage, if a cell of the first 100 consecutive well-spread metaphases from each culture had 23 to 27 chromosomes, any gaps, breaks or rearrangements, those were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix III). Cells with 28 to 31 chromosomes were scored as aneuploid cells. Cells with greater than 31 chromosomes were classified as polyploid cells with the % incidence of polyploid cells reported. The percentage of cells showing structural chromosome aberrations (gaps, breaks and exchanges) were calculated and reported as both including and excluding those with gaps.

Historical Aberration Ranges for Vehicle and Untreated Control Cultures
Many experiments with the CHL cell line have established a range of aberration frequencies acceptable for control cultures, these are commonly in the range of 0 to 3% cells with aberrations (Ishidate, 1987), Data Book of Chromosomal Aberration Test In Vitro, (Revised Edition).
A positive response was recorded for a particular treatment if the % cells with aberrations (gaps included) was equal to or exceeded 10%, an equivocal response was recorded for values between 5 and 10% and a negative response for values less than 5%. For polyploid cells, an incidence greater than 10% is generally recorded as positive.
However, consideration is given to a number of factors, such as the frequency of chromosome exchange events which are comparatively rare in control cultures, and the ultimate designation must rely upon experience and sound scientific judgement (UKEMS Guidelines for Mutagenicity Testing, 1983).
Statistics:
Besides the evaluation of criteria are given in Appendix III of the 1983 UKEMS guidelines for mutagenicity testing, the chromosome aberration data were statistically analysed using Fisher's Exact Test.

Results and discussion

Test results
Species / strain:
mammalian cell line, other: CHL cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose-related
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation: observed at the upper dose levels
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the Preliminary Cytotoxicity Test, Microscopic assessment of the slides prepared from the preliminary cytotoxicity test showed metaphases present at up to 78.13 µg/ml in the 12-hour with and without S9-mix and the 6-hour without S9-mix treatment cases, and at up to 156.25 µg/ml with S9-mix treatment case. The maximum dose with metaphases present was in the 24-hour and 48-hour exposure cases was 39.06 µg/ml.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

1 Preliminary Cytotoxicity Test

The results of the preliminary cytotoxicity test are presented in Table 1. It can be seen that the test material showed evidence of a dose-related increase in cell toxicity in every exposure case. The presence of a precipitate was observed at the upper dose levels. Microscopic assessment of the slides prepared from the preliminary cytotoxicity test showed metaphases present at up to 78.13 µg/ml in the 12-hour with and without S9-mix and the 6-hour without S9-mix treatment cases, and at up to 156.25 µg/ml with S9-mix treatment case. The maximum dose with metaphases present was in the 24-hour and 48-hour exposure cases was 39.06 µg/ml.

 

2 Chromosome Aberration Test - Experiment 1

The results of the cell counts from the cultures after their respective treatments are presented in Table 2 and the results of the mitotic index are presented in Table 3. It can be seen that the test material showed a toxicity profile similar to that seen in the preliminary toxicity study. The test material gave a very steep toxicity curve and the point where there was 50% toxicity was within a very narrow dose range.

The dose levels that were considered to be the most appropriate for metaphase analysis were 20, 40 and 60 µg/ml in the without-S9 group and 40, 80 and 120 µg/ml with-S9.

The vehicle control cultures gave values of chromosome aberrations within the expected range.

The MMC positive control gave a significant increase in the frequency of cells with aberrations indicating that the test method was operating as expected. CP gave a small non-significant increase in aberration frequency and it was considered that the typical poor responses observed were due to toxicity-induced cell cycle delay.

The test material was seen to induce no statistically significant increases in the frequency of cells with aberrations in any treatment cases.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in any of the six treatment cases .

 

Chromosome Aberration Test - Experiment 2

The results of the cell counts from the cultures after their respective treatments are presented in Table 4 and the results of the mitotic index are presented in Table 5. It can be seen that the test material showed the expected level of toxicity similar to that seen in the preliminary cytotoxicity study and Experiment 1. In some cases the cell counts and the mitotic index data gave conflicting estimates of toxicity, however, in each case one or both methods of toxicity measurement indicated that the maximum dose level selected for metaphase analysis had resulted in approximately 50% cell growth inhibition. Furthermore, in each exposure case complete mitotic inhibition was achieved in at least one dose level using narrow dose ranges.

The vehicle control cultures gave values of chromosome aberrations within the expected range.

The positive control cultures, except cyclophosphamide without S9 treatment, gave significant increases in the frequency of cells with aberrations indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected. It was considered, as in Experiment 1, that toxicity-induced cell cycle delay was responsible for the absence of a response in both of the 12-hour positive control groups. The cell cycle time for CHL cells is approximately 12 hours. Toxicity-induced cell cycle delay means that the toxic effects of the positive control materials increases the cell cycle time. Therefore, any clastogenic response induced by the positive controls will not be apparent at the 12-hour harvest time. This is a typical response for this time-point and does not invalidate the study.

The test material was seen to induce no statistically significant increases in the frequency of cells with aberrations in any of the treatment cases.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in any of the six treatment cases.

 

 

Due to the limited space in this IUCLID field, the respective tables are presented as attachment. 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The study is well-documented and was performed according to EU Method B.10 and the requirements of the Japanese New Chemical Substance Law (MITI) under GLP conditions. Hence, the information provided in the report can be considered reliable. Both positive and negative controls gave the appropriate response, so the test system and the obtained results can be regarded as valid. In consequence, the results are suitable for the assessment of the capability of the test item to induce chromosome aberrations in mammalian cells. The test material demonstrated no statistically significant dose-related increases in the frequency of cells with aberrations in Experiment 1 or Experiment 2. The test material was shown to be toxic to CHL cells in vitro in all six treatment cases, with a very steep dose response curve. The test material was shown to be non-clastogenic to CHL cells in vitro.
Executive summary:

In a mammalian cell chromosome aberration assay, Chinese hamster lung (CHL) cells cultured in vitro were exposed to the test item at concentrations up to 160 µg/mL in the main tests in the presence and absence of mammalian metabolic activation (S9).

 

Duplicate cultures of CHL cells were treated with the test material at a minimum of four dose levels, in each treatment case, together with vehicle and positive controls in each of two experiments. In Experiment 1 a single cell-harvest time-point at 12 hours, both with and without metabolic activation, was used. In Experiment 2 six treatment regimes were used: 6 hours exposure both with and without the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 18 hours treatment-free incubation); 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 8 hours treatment-free incubation); continuous exposures of 12, 24 and 48 hours.

The dose range for metaphase analysis was selected from a series of at least four dose levels chosen on the basis of the results of a preliminary toxicity test. The test material was evaluated at doubling dose levels between 1.25 and 120 µg/ml depending on the particular treatment regime used, the continuous exposures were more toxic than the pulse exposures.

 

The vehicle (solvent) controls gave frequencies of aberrations within the range expected for the CHL cell line. The positive control treatments gave significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. A typical poor response was seen in the 12-hour treatment case which was probably due to toxicity-induced cell cycle delay, such that any clastogenic responses induced by the positive control materials were not apparent at the 12-hour harvest time.

 

The test material demonstrated no statistically significant dose-related increases in the frequency of cells with aberrations in Experiment 1, or in Experiment 2. The test material was shown to be toxic to CHL cells in vitro in all six treatment cases, with a very steep dose response curve. The test material was shown to be non-clastogenic to CHL cells in vitro.

 

This study is classified as acceptable and study satisfies the requirements for EU Method B.10 and the requirements of the Japanese New Chemical Substance Law (MITI).