[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro study was performed to assess the detection of structural chromosomal aberrations in cultured mammalian cells (Human lymphocytes).

Link to relevant study records

Reference

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:

The experimental phases of the study were performed between 23 October 2013 and 19 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 10 July 2012. Date of Signature: 30 November 2012

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitabilityThe volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Cell Growth Inhibition Test:
4(20)-hour without S9: 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL.
4(20)-hour with S9: 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL.
20-hour without S9: 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL.

Experiment 1
4(20)-hour without S9: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL
4(20)-hour with S9: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL

Experiment 2
24-hour without S9: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL
4(20)-hour with S9: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
The test item was insoluble in Eagle's minimal essential medium with HEPES buffer (MEM) at 50 mg/mL but was soluble in dimethyl sulphoxide at 500 mg/mL in solubility checks performed in house. Therefore, dimethyl sulphoxide was used as the solvent vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9

Migrated to IUCLID6: (CP)EXAMPLE:

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9

Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHODS OF APPLICATION:
In medium

DURATION
- Pre-incubation period:
48 hours
- Exposure duration:
Experiment 1 – 4 hours with and without S9. Experiment 2 – 24 hours without S9, 4 hours with S9.
- Expression time (cells in growth medium):
20 hours for 4 hours exposure
- Selection time (in incubation with a selective agent):
Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):
24 hours

SELECTION AGENT (MUTATION ASSAYS):
No selection agent selected

SPINDLE INHIBITOR (Cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When slides were dry they were stained in 5% giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS:
Duplicate cultures

NUMBER OF CELLS EVALUATED:
100/culture

DETERMINATION OF CYTOTOXICITY
-Method:
Mitotic index – A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells

OTHER:
none

Evaluation criteria:

The following criteria were used to determine a valid assay:

The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range. All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

Criteria for determining the Study Conclusion
A test item can be classified as non-genotoxic if:

1: The number of induced chromosome aberrations in all evaluated dose groups is within the range of the laboratory historical control data.
2: No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.

A test item can be classified as genotoxic if:

1: The number of induced structural chromosome aberrations is not in the range of laboratory historical control data.
And
2: Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.

Biological relevance of the results will be considered first. Statistical methods will be used to analyze the increases in aberration data as recommended in the OECD 473 guidelines. However, statistical significance will not be the only determining factor for a positive response.

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable
- Precipitation: See below

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Cell Growth Inhibition Test

The dose range for the Cell Growth Inhibition Test was 19.53 to 5000 µg/mL where the maximum dose was the maximum recommended dose level. 

 

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in the presence of S9 only, at and above 1250 µg/mL.  No haemolysis was observed following exposure to the test item in any exposure group.

 

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 µg/mL in all of the exposure groups. However, there was evidence that there was some modest toxicity at the upper dose levels of the test item in the 4(20)-hour exposure group, with S9 and the continuous exposure group. The results of the cell counts and the mitotic index of the Cell Growth Inhibition Test are presented in sections 5(2) and 6(2) of Appendix1.

 

The selection of the maximum dose level was based on the maximum recommended dose level in both Experiments 1 and 2.

 

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 FAT65088/A TE (µg/mL)
4(20)-hour without S9	0*, 156.25, 312.5, 625, 1250*, 2500*, 5000*, MMC 0.4*
4(20)-hour with S9 (2%)	0*, 156.25, 312.5*, 625, 1250*, 2500, 5000*, CP 5*

*: dose levels selected for metaphase analysis

MMC: Mitomycin C

CP: Cyclophosphamide

 

The qualitative assessment of the slides determined that the toxicity was somewhat different to that observed in the Cell Growth Inhibition Test and that there were metaphases suitable for scoring present up to 5000 µg/mL in both exposure groups. The precipitate was similar to that observed in the Cell Growth Inhibition Test. 

 

Precipitate observations were made at the end of exposure and precipitate was noted at and above 625 µg/mL in the exposure group in the presence of S9 only. No precipitate was observed in the absence of S9. Haemolysis was observed following exposure to the test item at and above 1250 µg/mL and 2500 µg/mL in the absence and presence of S9, respectively. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. These data are presented in Appendix1section 5(3).

 

The mitotic index data confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed, and that 22% mitotic inhibition was achieved at 5000 µg/mL in the absence of S9. In the presence of S9, no dose-related inhibition of mitotic index was observed. The results of the cell counts and the mitotic indices (MI) from the cultures after their respective exposures are presented as cell growth indices in Form 1 and Form 2, Appendix 2.

 

The maximum dose level selected for metaphase analysis was the maximum recommended dose level (5000 µg/mL).

 

The chromosome aberration data are given in Form 1and Form 2, Appendix 2. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. 

 

The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

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.

 

The polyploid cell frequency data are givenin Figure 2 and Figure 3, Appendix 2. 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. There was no indication of endoreduplication noted.

 

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 FAT65088/A TE (µg/mL)
24-hour without S9	0*, 156.25, 312.5*, 625, 1250*, 2500*, 5000, MMC 0.2*
4(20)-hour with S9	0*, 156.25*, 312.5*, 625*, 1250, 2500, 5000, CP 5*

*: dose levels selected for metaphase analysis

MMC: Mitomycin C

CP: Cyclophosphamide

 

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 5000 µg/mL in both the absence and presence of S9. However, in the absence of S9, the maximum test item dose level with metaphases suitable for scoring was 2500 µg/mL whereas in the presence of S9, it was 625 µg/mL. 

 

A precipitate of the test item was observed at the end of exposure in the presence of S9 only at and above 625 µg/mL. No precipitate was observed in the 24‑hour continuous exposure group. Haemolysis was observed following exposure to the test item at 5000 µg/mL in the presence of S9 only.

 

These data are presented in Appendix1sections 5(3) and 6(3). They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 26% and 60% mitotic inhibition were achieved at 1250 and 2500 µg/mL, respectively, in the absence of S9. In the presence of S9 a dose-related inhibition of mitotic index was observed at 625 µg/mL (42%).

 

The maximum dose level selected for metaphase analysis was based on toxicity, and was 2500 µg/mL in the absence of S9 and 625 µg/mL in the presence of S9.

 

The chromosome aberration data are presented in Form 3 and Form 4, Appendix 2. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. 

 

The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test item was modestly toxic but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that generally included a dose level that induced mitotic inhibition.

 

The polyploid cell frequency data are given in Figure 4 and Figure 5, Appendix 2. 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. There was no indication of endoreduplication noted. 

 

Conclusions:

Interpretation of results (migrated information):
negative

The test item, FAT 65088/A TE was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

SUMMARY

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1991). 

 

Methods…….

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4‑hour exposure with addition of S9 was repeated, whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

 

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

 

Group	Final concentration of test item (µg/mL)
4(20)-hour without S9	156.25, 312.5, 625, 1250, 2500, 5000
4(20)-hour with S9 (2%)
24-hour without S9
4(20)-hour with S9 (2%)

 

 

Results…….

All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

 

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test item 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 metabolizing system, in either of two separate experiments.

 

 

Conclusion

The test item, FAT65088/A TEwas considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

1) An in vitro test was performed to assess the detection of structural chromosomal aberrations in cultured mammalian cells (Human Lymphocytes). 

 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4‑hour exposure with addition of S9 was repeated, whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

 

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

 

Group	Final concentration of test item (µg/mL)
4(20)-hour without S9	156.25, 312.5, 625, 1250, 2500, 5000
4(20)-hour with S9 (2%)
24-hour without S9
4(20)-hour with S9 (2%)

 

 Results…….

All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item 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 metabolizing system, in either of two separate experiments.

Conclusion

The test item, FAT65088/A TEwas considered to be non-clastogenic to human lymphocytesin vitro.

2) A second experiment, a reverse mutation test, was performed according to the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test"

Salmonella typhimuriumstrains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test item using the Ames plate pre-incubation method (Prival and Mitchell modification) at five dose levels, in triplicate, both with and without the addition of a hamster liver homogenate metabolizing system (30% liver S9 in modified co‑factors). The dose range for the range-finding test (Experiment 1) was determined in a Preliminary Toxicity Test and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was the same as the range-finding test (50 to 5000 µg/plate).  

Results…….

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver S9-mix and the uninduced hamster liver S9-mix were validated.

 

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 mg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the range-finding test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the main test. 

  

Conclusion

FAT 65088/A TEwas considered to be non-mutagenic under the conditions of this test.

Justification for selection of genetic toxicity endpoint
Two in vitro studies were perfomed, (Ames test using Salmonella typhimurium) and chromosome aberration in Human Lymphocytes cells.
In both experiment the results are negative.
As the results are negative, the experiment perfomed using Human lymphocytes cells was selected as key value for the Chemical safety assessment.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).