benzyl(diethylamino)diphenylphosphonium 4-[1,1,1,3,3,3-hexafluoro-2-(4-hydroxyphenyl)propan-2-yl]phenolate

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

July 29 - November 18, 2013

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - 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

Reference substance name:

XA31

IUPAC Name:

XA31

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: XA31
Batch: 3706OB
Purity: > 99%
Expiry Date: 31/12/2018
Storage Conditions: Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Group 4(20)-hour exposure to the test item without S9 mix: 1.88, 3.75, 7.5, 15, 30 and 60 ug/mL;
Group 4(20)-hour exposure to the test item with S9 mix (2%): 1.88, 3.75, 7.5, 15, 30 and 60 ug/mL;
Group 24-hour exposure to the test item without S9 mix: 3.75, 7.5, 15, 30, 45, 60 and 90 ug/mL;
Group 4(20)-hour exposure to the test item with S9 mix (1%): 7.5, 15, 30, 60, 90 and 120 ug/mL.

Vehicle / solvent:

Dimethyl sulphoxide

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 19.53 to 5000 ug/mL. The test item was considered to be a mixture and therefore the maximum recommended dose was initially set at 5000 ug/mL.

A precipitate of the test item was observed in teh parallel blood-free cultures at the end of the exposure, at and above 312.5 ug/mL, in the exposure groups dosed in the absence of S9. However, in the presence of S9, precipitate was observed in the blood-free cultures at and above 156.25 ug/mL.

Haemolysis was observed following exposure to the test item. In the 4(20)-hour exposure group (in the absence of S9), haemolysis was observed between 156.25 and 2500 ug/mL. In the 4(20)-hour exposure group in the presence of S9, haemolysis was observed at and above 156.25 ug/mL and at and above 78.13 ug/mL in the 24 -hour continuous exposure group. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 39.06 ug/mL in all three exposure groups. Qualitative assessment indicated that the test item had a very steep toxicity curve. In the 4(20)-hour exposure groups, there were substantial decreases in mitotic index at the dose levels analyzed. In the 24 -hour continuous exposure, increases in mitotic index were observed at both dose level analyzed, possibly due to cell cycle delay.

Chromosome Aberration Test - Experiment 1

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of XA31 (ug/mL)
4(20)-hour without S9	0*, 1.88, 3.75, 7.5*, 15*, 30*, 60*, MMC 0.4*
4(20)-hour with S9	0*, 1.88, 3.75, 7.5, 15*, 30*, 60*, CP 5*

The qualitative assessment of the slides determined thata the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the maximum dose level of test item in both the presence and absence of S9 (60 ug/mL). Precipitate observations were made at the end of exposure but no precipitate was noted at any dose level, either in the presence or absence of S9. No haemolysis was observed at the end of exposure in either exposure group. In the absence of S9, 15%, 42% and 54% mitotic inhibition was achieved at 15, 30 and 60 ug/mL, respectively. In the presence of S9, a modest dose-related inhibition of mitotic index was observed at 30 and 60 ug/mL. The maximum dose level selected for metaphase analysis was, therefore, 60 ug/mL for both exposure groups. The positive control item induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation. Although there was a small increase in polyploid cells at 60 ug/mL in the presence of S9, the increase was only just outside the historical maxima and was considered to have no biological relevance. Therefore, the test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups. Chromosome Aberration Test - Experiment 2

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of XA31 (ug/mL)
24-hour without S9	0*, 3.75, 7.5, 15*, 30*, 45*, 60, 90, MMC 0.2*
4(20)-hour with S9	0*, 7.5*, 15*, 30, 60*, 90*, 120, CP 5*

 

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to 60 ug/mL in the both the absence of S9. Precipitate observations were made at the end of exposure but no precipitate was noted at any dose level, either in the presence or absence of S9. However, haemolysis was observed at the end of exposure at 90 ug/mL in the absence of S9, at 120 ug/mL in the presence of S9. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

The qualitative assessment of the slides prior to analysis suggested that toxicity was apparent at and above 90 ug/mL in both the absence and presence of S9. However, in the 24-hour continuous exposure group, no dose-related inhibition of mitotic index was observed, and 42% mitotic inhibition was achieved at 15ug/mL. In fact, the mitotic index increased with concentration.. This phenomenon is indicative of cell cycle delay where the toxicity of the test item delays the cell cycle process causing synchronization of the cells, and confirmed the observed response in the preliminary toxicity test. In the presence of S9, inhibition of the mitotic index was observed but it was not clear cut with 66% and 50% mitotic inhibition occurring at 30 and 60 ug/mL, respectively.

The maximum dose level selected for metaphase analysis was based on toxicity. The maximum dose level selected for metaphase analysis were 45 ug/mL and 60 ug/mL in the absence and presence of S9, respectively.

All the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control item induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

There was no incidence of polyploidy cells at any dose level in either of the exposure groups. This confirms that the response observed in the presence of S9 in Experiment 1 was spurious and had no toxicological significance.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative non-clastogenic to human lymphocytes in vitro

The test item was non toxic and did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme meaboiizing system, in either of two separate experiments, even when the dose range included a dose level which induced approximately 50% mitotic inhibition.
The test item was therefore considered to be non-clastogenic to humna lymphocytes in vitro.