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Genetic toxicity: in vitro

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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 27 January 2012 and 11 May 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 473 using the Genetic Toxicology: Chromosome Aberration Test method in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Sponsor's identification : IFF TM 11-212
Storage conditions : Room temperature in the dark (up to 28 March 2012)
Approximately 4ºC in the dark (28 March 2012 onwards)*
* The change in storage conditions was not considered to impact on the stability/quality of the test item

Method

Target gene:
Not applicable.
Species / strain
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 suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.
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:
Preliminary Toxicity Test
The dose range of test item used was 19.53 to 5000 µg/ml.
The dose levels used in the main experiments were selected using data from the preliminary toxicity test.

Group Final concentration of test item (µg/ml)
4(20)-hour without S9 1, 2, 4, 8, 16, 24, 32, 48
4(20)-hour with S9 (2%) 2, 4, 8, 16, 24, 32, 48, 64
24-hour without S9 2, 4, 8, 16, 24, 32, 48, 64
4(20)-hour with S9 (1%) 2, 4, 8, 16, 24, 32, 48, 64
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent because the test material was readily soluble in it at the required concentrations.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9

Migrated to IUCLID6: (CP)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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:
METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period: 48 hrs

- Exposure duration: Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent): Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs.

SELECTION AGENT (mutation assays): No selection agent.

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine

STAIN (for cytogenetic assays): When the 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 and 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 was approximately 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. 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

Cell Culture
Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37ºC with 5% CO2in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Preparation of Test Item and Control Items
The test item was accurately weighed, dissolved in dimethyl sulphoxide (DMSO) and serial dilutions prepared. The test item was considered to be a complex mixture; therefore the maximum recommended dose level was 5000 µg/ml. The purity of the test item was initially supplied as 96.1% and an allowance for this was made in the formulations. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al 1991). 

The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Vehicle and positive controls were used in parallel with the test item. The positive control items were as follows:
In the absence of S9, mitomycin C (MMC) (Sigma, Batch No. 089K0731) was used at 0.4 and 0.2 µg/ml for cultures in Experiment 1 and 2 respectively. It was dissolved in Minimal Essential Medium.

In the presence of S9, cyclophosphamide (CP) (Acros, Batch No. A0277203) was used at 5 µg/ml in both experiments. It was dissolved in dimethyl sulphoxide.

Microsomal Enzyme Fraction
Lot No. PB/betaNF S9 27/11/11 was prepared in-house from the livers of male Sprague-Dawley rats weighing approximately 250g. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day. 
The S9 homogenate was produced by homogenising the liver in a 0.15 M KCl solution (1g liver to 3ml KCl) followed by centrifugation at 9000g. The protein content of the resultant supernatant was adjusted to 20 mg/ml using 0.15 M KCl and aliquots of the supernatant were frozen and stored in ampoules at approximately -196ºC in a liquid nitrogen freezer.

Each batch of S9 has been routinely tested for its capability to activate known mutagens in the Ames test.
The above procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedures) Act 1986.

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (10 to 20% (v/v)), MgCl2(8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2% in the Preliminary Toxicity Test and Experiment 1 and 1% in Experiment 2.

Culture Conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 ml MEM, 10% (FBS) 0.1 ml Li-heparin 0.1 ml phytohaemagglutinin 0.75 ml heparinised whole blood

With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at 37ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 ml of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1ml of the appropriate solution of vehicle control or test item was added to each culture.  For the positive control, 0.1 ml of the appropriate solution was added to the cultures. 1 ml of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co‑factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.

In Experiment 2, 1 ml of 10% S9-mix (i.e. 1% final concentration of S9 in standard co‑factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 ml.
After 4 hours at 37ºC, 5% CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium.  After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at 37ºC in 5% CO2 in humidified air.

 Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at 37ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 ml of the culture medium was removed and reserved. The cells were then re-suspended in the required volume of fresh MEM (including serum) and dosed with 0.1 ml of the appropriate vehicle control, test item solution or 0.1 ml of positive control solution. The total volume for each culture was a nominal 10 ml.

After 4 hours at 37ºC in 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 ml.  After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 ml of vehicle control, test item dose solution or 0.1 ml of positive control solution. The nominal final volume of each culture was 10 ml. The cultures were then incubated at 37ºC, 5% CO2 in humidified air for 24 hours.
The Preliminary Toxicity Test was performed using the methodology of Experiment 1 and Experiment 2 in the absence of S9 except that positive controls were not included.

Preliminary Toxicity Test(Cell Growth Inhibition Test)
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test item used was 19.53 to 5000 µg/ml. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Experiment 1
i)      4-hour exposure to the test item without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 1 to 48 µg/ml.
ii)     4-hour exposure to the test item with S9-mix (2% final concentration) followed by 20‑hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 2 to 64 µg/ml.
Experiment 2
i)      24-hour continuous exposure to the test item, without S9-mix, prior to cell harvest. The dose range of test item used was 2 to 64 µg/ml.
ii)     4-hour exposure to the test item with S9-mix (1% final concentration) followed by 20‑hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 2 to 64 µg/ml.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl.  After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4ºC to ensure complete fixation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several ml of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerised random number generator and supplementary slides were coded manually.

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 and as a percentage of the vehicle control value.
Evaluation criteria:
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 4). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
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.

Results and discussion

Test results
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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, test material dissolved in DMSO
- Precipitation: Refer to results section below

RESULTS
Preliminary Toxicity Test (Cell Growth Inhibition Test)
The dose range for the Preliminary Toxicity Test was 19.53 to 5000 µg/ml. The maximum dose was the maximum recommended dose level. A
greasy/oily precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 156.25 µg/ml, in the
4(20)-hour exposure group in the absence of S9 and in the 24-hour exposure group in the absence of S9. Cloudy precipitate was also noted in the
4(20)-hour exposure group in the presence of S9 from 312.5 to 2500 µg/ml and in the 24-hour exposure group from 312.5 to 1250 µg/ml. In the
4(20)-hour exposure group in the presence of S9 greasy/oily precipitate was observed in the parallel blood-free cultures at the end of the exposure
period at and above 312.5 µg/ml with cloudy precipitate at 312.5 and 625 µg/ml.

Haemolysis was observed at the end of exposure at and above 19.53 µg/ml in the 4(20) hour exposure groups in the absence and presence of S9
and at and above 39.06 µg/ml in the 24-hour exposure group. It should be noted that haemolysis is caused by a disruption of the erythrocyte
membranes and is not considered to be indicative of toxicity to the lymphocytes.
Microscopic assessment showed that metaphase cells were present up to 19.53 µg/ml in the 4(20)-hour exposure in the absence of metabolic
activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure and the 4(20)-hour exposure in the presence of
metabolic activation (S9) was 39.06 µg/ml. The test item induced marked evidence of toxicity in all three exposure groups.
The selection of the maximum dose level was based on toxicity and was 48 and 64 µg/ml for the 4(20)-hour exposure groups in the absence and
presence of S9 respectively and was 64 µg/ml for the continuous exposure group used in Experiment 2.

Chromosome Aberration Test – Experiment 1
The dose levels of the controls and the test item are given below:
Group Final concentration of IFF TM 11-212 (μg/ml)
4(20)-hour without S9 0*, 1, 2, 4, 8*, 16*, 24*, 32*, 48, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 2, 4, 8, 16, 24*, 32*, 48*, 64*, CP 5*

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to 32 µg/ml in the absence of S9 and
up to at 64 µg/ml in the presence S9. No precipitate of the test item was observed at the end of exposure in either exposure group. Haemolysis was
observed at the end of exposure at and above 32 µg/ml, in the 4(20)-hour exposure group in the absence of S9, and at and above 48 µg/ml, in the
4(20)-hour exposure group in the presence of S9.

The results of the mitotic indices (MI) from the cultures after their respective treatments confirm the qualitative observations in that a dose-related inhibition of mitotic index that 76% mitotic inhibition was achieved at 32 µg/ml in the absence of S9. In the presence of S9 62% mitotic inhibition was achieved at 64 µg/ml. Although these dose levels achieved greater than optimum toxicity they were selected as the maximum dose levels for metaphase analysis for the exposure groups of Experiment 1 as they provided an intermediate dose in a relatively steep toxicity curve. Due to the toxicity at 32 µg/ml in the 4(20)-hour exposure group in the absence of S9 it was not possible to score 100 metaphases from either of the replicate cultures, however since there was no indication of a response this was considered to be acceptable.

The chromosome aberration data were recorded. 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. The ‘B’ replicate of the positive control in the 4(20)-hour exposure group in the absence of S9 demonstrated a weak response initially and therefore extra metaphases were selected for scoring to verify the response. The ‘A’ replicate of the positive control in the 4(20)-hour exposure group in the presence of S9 proved to be excessively toxic with only 50 metaphases suitable for scoring but since there was a statistically significant response demonstrated this was considered to be acceptable. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
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.

From the polyploid cell frequency data 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.

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to 64 µg/ml in the presence of S9. In
the absence of S9 the maximum test item dose level with metaphases suitable for scoring was 48 µg/ml. No precipitate of the test item was observed at the end of exposure in either exposure group. Haemolysis was seen at the end of the exposure period at and above 24 µg/ml in the presence of S9
and at and above 48 µg/ml in the 24-hour exposure group in the absence of S9.

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 IFF TM 11-212 (μg/ml)
24-hour without S9 0*, 2, 4, 8, 16*, 24*, 32*, 48*, 64, MMC 0.2*
4(20)-hour with S9 (1%) 0*, 2, 4, 8, 16, 24*, 32*, 48*, 64*, CP 5*

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to 64 μg/ml in the presence of S9. In
the absence of S9 the maximum test item dose level with metaphases suitable for scoring was 48 μg/ml. No precipitate of the test item was observed at the end of exposure in either exposure group. Haemolysis was seen at the end of the exposure period at and above 24 μg/ml in the presence of S9
and at and above 48 μg/ml in the 24-hour exposure group in the absence of S9.

The results of the mitotic indices (MI) from the cultures after their respective treatments confirm the qualitative observations in that a dose-related inhibition of mitotic index that 57% mitotic inhibition was achieved at 48 μg/ml in the absence of S9. In the presence of S9 60% mitotic inhibition was achieved at 64 μg/ml. The maximum dose level selected for metaphase analysis for the 4(20)-hour exposure group in the presence of S9 was the same as in Experiment 1 where the toxicity was very similar and was 64 μg/ml. The 24-hour exposure group appeared to be less toxic than the 4(20)-hour exposure group in the absence of S9 in Experiment 1, however the mitotic index for the vehicle control group of the 24-hour exposure group was low, particularly the ‘A’ culture resulting in the toxicity possibly being underestimated. The maximum dose selected for metaphase analysis in the 24-hour exposure group was 48 μg/ml.

The chromosome aberration data were recorded. 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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.

From polyploid cell frequency data, 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.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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 metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

TM11 -212 was assessed for in vitro cytogenicity using human lymphcytes according to the OECD Test Guideline 473. In the first test metaphases could be scored up to 32 µg/ml in the absence of S9 and up to at 64 µg/ml in the presence S9, where mitotic inhibition of 76% and 62% was achieved, respectively. In the second test metaphases were scored up to 48 µg/ml in the absence of S9 and up to at 64 µg/ml in the presence S9, achieving mitotic inhibition of 57% and 60%, respectively.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced greater than 50% mitotic inhibition. The test item was considered to be non-clastogenic to human lymphocytesin vitro.