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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- in a bacterial reverse mutation assay according to OECD test guideline 471, GLP, Reliability 1, the test item did not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism

- in an in vitro micronucleus assay according to OECD test guideline 487, GLP, Reliability 1, the test item did not induce micronuclei in human lymphocytes after in vitro treatment

- in a mouse lymphoma assay according to OECD test guideline 490, GLP, Reliability 1, the test item did not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation

- in a read-across study according to Notification on Guidelines for Genotoxicity Studies of Drugs (Notification No. 1604 of the Evaluating and Licensing Division, PMSB dated November 1, 1999), Reliability 2, the source substance did not induce reverse mutation in the absence or presence of S9 metabolism

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experimental phase: 04 July 2017; End of experimental phase: 31 July 2017; Study completion: 06 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 1997
Deviations:
yes
Remarks:
- Test item formulation in the preliminary toxicity test was carried out without correction for the test item displacement - In the Preincubation method, the test item was assayed at a maximum concentration of 10000 µg/plate, instead of 5000 µg/plate
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
The E.coli used for this study was the strain WP2 uvrA.
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate from rats pre-treated with Phenobarbital and 5,6-Benzoflavone.
Test concentrations with justification for top dose:
Preliminary toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate

Main Assay I:
- TA1535, WP2 uvrA, TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate
- TA1537: ± S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate
- TA100 : ± S9: 2500, 1250, 625, 313 ,156 and 78.1 µg/plate

Main Assay II:
- TA1535, WP2 uvrA: ± S9: 10000, 5000, 2500, 1250 and 625 µg/plate
- TA100: :+ S9: 10000, 5000, 2500, 1250 and 625 µg/plate
- TA1537, TA100: - S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate
- TA1537: +S9: 10000, 5000, 2500, 1250, 625 and 313 µg/plate
- TA98: ± S9: 10000, 5000, 2500, 1250, 625 and 313 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile water for injection.
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 metabolic activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Remarks:
Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
Details on test system and experimental conditions:
The preliminary toxicity test and the first experiment were performed using a plate-incorporation method.
The second experiment was performed using a pre-incubation method.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
Doubling rate (Chu et al. 1981); Regression line.
Key result
Species / strain:
other: S.typhyimurium TA1535, TA1537, TA98 and TA100; E.coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item did not induce two-fold increases in the number of revertant colonies, in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.
Conclusions:
It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.
Executive summary:

The test item HH-2015-623 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

The test item was used as a solution in sterile water for injection.

Toxicity test

The test item HH-2015-623 was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration tested in the absence or presence of S9 metabolic activation. Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies, was observed with TA1537 and TA100 tester strains at the highest or two highest dose levels both in the absence and presence of S9 metabolic activation. No increase in revertant numbers was observed at any concentration tested with any tester strain/activation condition combinations.

Main Assays

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

- TA1535, WP2 uvrA, TA98: ± S9:  5000, 2500, 1250, 625 and 313 µg/plate

- TA1537:   ± S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

- TA100 :    ± S9: 2500, 1250, 625, 313 ,156 and 78.1 µg/plate

Toxicity was observed at the highest or two highest dose levels with TA1537 tester strain both in the absence and presence of S9 metabolism and with TA100 only in its absence. As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed including a pre-incubation step for all treatments.The following dose levels were used:

- TA1535, WP2 uvrA: ± S9: 10000, 5000, 2500, 1250 and 625 µg/plate

- TA100: :+  S9: 10000, 5000, 2500, 1250 and 625 µg/plate

- TA1537, TA100: - S9: 5000, 2500, 1250, 625, 313  and 156 µg/plate

- TA1537:  +S9: 10000, 5000, 2500, 1250, 625  and 313 µg/plate

- TA98:  ± S9: 10000, 5000, 2500, 1250, 625 and 313 µg/plate

Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at the highest dose level tested with TA1537 and TA100 tester strains both in the absence and presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period in any experiment, at any concentration tested, in the absence or presence of S9 metabolic activation.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Conclusion

It is concluded that the test item HH-2015-623 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study start: 16 January 2017; End of experimental phase: 12 June 2017; Final Report signed: 12 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
Adopted July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
The test item was assay for the ability to induce micronuclei in human lymphocytes cultured in vitro, after treatment in the absence and presence of S9 metabolism.
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
Two batches of human whole blood, provided by Biopredic International (France), were used in this study and had the following characteristics:

Sex Female
Age 35 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 644 IU/mL of whole blood
Collection date 05-Apr-2017
Batch Number SAG025A1E019


Sex Female
Age 35 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 556 IU/mL of whole blood
Collection date 05-Apr-2017
Batch Number SAG025A1E020
Cytokinesis block (if used):
the inhibitor of actin polymerisation: cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9 tissue fraction, provided by Trinova Biochem GmbH, from Rats pretrerated with Phenobarbital and 5,6-Benzoflavone
Test concentrations with justification for top dose:
Dose levels for treatment were selected on the basis of the cytotoxicity observed in previous experiments where unacceptable values of frequency of micronucleated cells were observed or the required cytotoxicity was not achieved to select dose levels for scoring micronuclei.

Dose levels of 528, 440, 367, 306, 255, 212, 177, 147, 123, 102 and 85.3 µg/mL were used for the three hour treatment in the absence of S9 metabolism.

Dose levels of 684, 570, 475, 396, 330, 275, 229 and 191 µg/mL were used for the three hour treatment in the presence of S9 metabolism.

Dose levels of 191, 159, 133, 111, 92.1, 76.7, 64.0, 53.3 and 44.4 were used for the continuous treatment in the absence of S9 metabolism.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.
Vehicle / solvent:
Solutions of the test item were prepared in culture medium.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Colchicine 0.080, 0.040 and 0.020 μg/mL
Details on test system and experimental conditions:
Culture media:
The culture medium for the lymphocytes had the following composition:
RPMI 1640 1x (Dutch modification) 500mL
Foetal Calf Serum 100mL
L-Glutamine (200mM) 6.25mL
Antibiotic solution 1.25mL

The foetal calf serum was heat-inactivated at 56°C for 20 minutes before use. For the initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used in the following proportion: 10mL of PHA was added to 500mL of medium.

Preparation of the test cultures and treatment:

Two main experiments were performed including negative and positive controls. Two cultures were prepared at each test point. Lymphocyte cultures were treated approximately fourty-eight hours after they were initiated. Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.

Since the test item was solubilized in culture medium, the composition of the treatment media was as follows:

Treatment medium in the presence of S9 metabolism
Test item solution 0.50mL
S9 mix 1.00mL
Culture medium (without PHA) 3.50mL

Treatment medium in the absence of S9 metabolism
Test item or control solution 0.50mL
Culture medium (without PHA) 4.50mL

For the first main experiment, the treatment media was added to the tubes and the cultures were incubated for 3 hours at 37°C. At the end of treatment time, the cell cultures were centrifuged and washed twice with Phosphate Buffered Saline Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time Cytochalasin-B was added to achieve a final concentration of 6 µg/mL.

For the second main experiment, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting.

Harvesting and slide preparation:
The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed up to approximately 5 mm from the pellet. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.
A few drops of the cell suspension obtained in this way were dropped onto clean, wet, greasefree glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

Slide evaluation:
The cytokinesis-block proliferation index CBPI was calculated as follows:
CBPI = mononucleated + (2×binucleated) + (3×multinucleated) / total number of cells counted where mononucleated, binucleated and multinucleated are respectively the number of mononucleated cells, binucleated cells and multinucleated cells.
CBPI was used to measure the cytotoxic effect. Five hundred cells per cell culture were analysed. The highest concentration for genotoxicity assessment was selected on the basis of the cytotoxicity as calculated by the CBPI.

The percentage cytotoxicity was evaluated according to the following formula:
% Cytotoxicity = 100 − [100 x (CBPIt-1/CBPIc −1)]

where:
t = test item treated culture
c = vehicle /solvent control culture

The highest concentration for genotoxicity assessment was selected as a dose which produces a substantial cytotoxicity compared with the solvent control. Ideally the cytotoxicity should be between 50 % and 60 %. In the absence of cytotoxicity, the highest treatment level is selected as the highest concentration for scoring.
Two lower concentrations were also selected for the scoring of micronuclei. For the three selected concentrations, for the untreated and for the positive controls (Cyclohosphamide),
1000 binucleated cells per cell culture were scored to assess the frequency of micronucleated cells.
Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was
observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.

The criteria for identifying micronuclei were as follows:
1. the micronucleus diameter was less than 1/3 of the nucleus diameter;
2. the micronucleus diameter was greater than 1/16 of the nucleus diameter;
3. no overlapping with the nucleus was observed;
4. the aspect was the same as the chromatin.
Evaluation criteria:
Acceptance criteria:
The assay is considered valid if the following criteria are met:
– The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
– Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
– Adequate cell proliferation is observed in solvent control cultures.
– The appropriate number of doses and cells is analysed.

Criterion for outcome:
In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the concentrations shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data.
Statistics:
Statistical analysis:
For the statistical analysis, a modified X^2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
On the basis of the above results, it is concluded that HH-2015-623 does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.
Executive summary:

The test item HH-2015-623 was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation.

Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 32 hours corresponding to approximately 2.0 cell cycles was used. A second experiment was performed in the absence of S9 metabolism using a continuous treatment until harvest at 31 hours.

Solutions of the test item were prepared in culture medium.

Dose levels for treatment were selected on the basis of the cytotoxicity observed in previous experiments where unacceptable values of frequency of micronucleated cells were observed or the required cytotoxicity was not achieved to select dose levels for scoring micronuclei.

Dose levels of 528, 440, 367, 306, 255, 212, 177, 147, 123, 102 and 85.3 µg/mL were used for the three hour treatment in the absence of S9 metabolism.

Dose levels of 684, 570, 475, 396, 330, 275, 229 and 191 µg/mL were used for the three hour treatment in the presence of S9 metabolism.

Dose levels of 191, 159, 133, 111, 92.1, 76.7, 64.0, 53.3 and 44.4 µg/mL were used for the continuous treatment in the absence of S9 metabolism.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.

The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

Dose levels were selected for the scoring of micronuclei on the basis of the cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI).

For both experiments, the following dose levels were selected for scoring:

Experiment No.: S9    Treatment time (hours)  Harvest time (hours)  Concentration (µg/mL)
 1  -  3 31-32   212, 147 and 102
 1  +  3  31-32  570, 396 and 275
 2  - 31  31  

133, 92.1 and 64.0

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series.

All results were within the distribution of historical negative control data.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

It is concluded that HH-2015-623 does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2018- March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
July 2016
GLP compliance:
yes
Type of assay:
other: gene mutation in mammalian cells
Target gene:
The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT).
This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells. TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence.
In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/−) may undergo a single step forward mutation to the TK−/− genotype in which little or no TK activity remains.
The cells used, L5178Y TK+/−, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive et al., 1979) and is accepted by most of the regulatory authorities.
The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI medium supplemented with Horse serum.
- Properly maintained: yes; Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Prior to use, cells were cleansed of pre-existing mutants.
Metabolic activation:
with and without
Metabolic activation system:
S9 tissue fraction: Species: Rat; Strain: Sprague Dawley; Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number: 3878
Test concentrations with justification for top dose:
A concentration of 5000 µg/mL, which is the maximum dose level stated in the Study Protocol, was selected as the top dose level to be used. In the preliminary cytotoxicity test, both in the absence and presence of S9 metabolic activation, the test item was assayed at this concentration and at a
wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1, and 19.5 µg/mL.

Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the following dose levels:
Main Assay I (-S9, 3 hour treatment): 150, 125, 104, 86.8, 72.3 and 60.3 μg/mL.
Main Assay I (+S9, 3 hour treatment): 600, 480, 384, 307, 246 and 197 μg/mL.
Main Assay II (+S9, 3 hour treatment): 480, 440, 400, 367, 333 and 305 μg/mL.
Vehicle / solvent:
Test item solutions were prepared using dimethylsulfoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.
Mutation assay
Treatment of cell cultures
The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). In the presence of S9 metabolism, the steep decline of Relative Total Growth did not allow to evaluate mutagenic effects at the appropriate level of cytotoxicity. Therefore, a second experiment in the presence of S9 metabolism was performed using a different range of concentrations. Since clearly negative results were obtained, no further experiment was
performed.
Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2×105 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
Expression period
During the expression period (two days after treatment), the cell populations were subculturedin order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×105 cells/mL.
Plating for 5-trifluorothymidine resistance
After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2 × 103 cells were plated in each well of four 96-well plates. Plateswere incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for
14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.
Plating for viability
After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.
Evaluation criteria:
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10^−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Survival after treatment
In the first experiment, in the absence of S9 metabolic activation, no cell survived treatment at the highest dose level, marked toxicity reducing relative total growth (RTG) to 20% of the concurrent negative control was noted at 125 µg/mL, slight toxicity was observed at 104 µg/mL, while no relevant toxicity was noted over the remaining concentrations tested.
In the presence of S9 metabolism, no cell survived treatment at the two highest dose levels; treatment with the test item at 384 µg/mL yielded mild toxicity reducing RTG to 57% of the concurrent negative control value, while no relevant toxicity was observed over the remaining concentrations tested.
In the second experiment, in the presence of S9 metabolic activation, no cell survived after treatment at the highest dose level; the next concentration (440 µg/mL) yielded moderate toxicity reducing RTG to 26% of the concurrent negative control value. Mild toxicity was observed at 400 µg/mL (RTG = 54%), while no relevant toxicity was seen over the remaining dose levels tested.
Mutation results
No statistically significant increase in mutant frequency was observed at any concentration, in any experiment, in the absence or presence of S9 metabolism.
A very small percentage of chemicals are uniquely positive using the 24 hour treatment; moreover the longer treatment becomes necessary when a chemical’s insolubility precludes testing at adequate level of cytotoxicity. Since no precipitation of the test item in the treatment mixture was noted and adequate levels of cytotoxicity were achieved both in the absence and presence of S9 metabolism, data obtained were considered sufficient to provide evidence of negative results.
For the negative and positive controls, the small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequate recovery of small colony mutants was observed following treatment with the positive control.
Conclusions:
It is concluded that the test item does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Executive summary:

The test item was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 5000 µg/mL using dimethylsulfoxide (DMSO) as solvent (the upper limit to testing indicated in the test guideline OECD No. 490 for Chemical Substances of Unknown or Variable Composition). On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic

activation, the test item was assayed at a maximum dose level of 5000 µg/mL and at a wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1, and 19.5 µg/mL. In the absence of S9 metabolic activation, no cells survived treatment from 156 µg/mL onwards. No relevant toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived treatment from 313 µg/mL onwards, mild toxicity was noted at the next lower concentration (RS=53%), while no relevant toxicity was observed over the remaining dose levels tested. Following treatment in the presence of S9 metabolic activation, no cells survived treatment from 625 µg/mL onwards. Test item treatment at the two next lower dose levels (313 and 156 µg/mL) yielded slight toxicity reducing RS to 60% and 73%, respectively. No relevant toxicity was observed over the remaining dose levels tested.

Based on the results obtained in the preliminary trial, a first Main Assay for mutation at the TK locus was performed using the following dose levels:

Main Assay I (-S9, 3 hour treatment): 150, 125, 104, 86.8, 72.3 and 60.3 μg/mL.

Main Assay I (+S9, 3 hour treatment): 600, 480, 384, 307, 246 and 197 μg/mL.

Adequate levels of cytotoxicity, covering a range from the maximum to little or no cytoxicity, were observed in the absence of S9 metabolism. In its presence, test item treatments did not meet appropriate cytotoxicity levels reducing relative total growth (RTG) to 57% at the highest analysable concentration. Based on these results, a second experiment (Main Assay 2) was performed in the presence of S9 metabolism using the following concentrations:

Main Assay II (+S9, 3 hour treatment): 480, 440, 400, 367, 333 and 305 μg/mL.

Adequate levels of cytotoxicity were obtained.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. It is concluded that HH-2015-623 does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
November 12, 2004 - November 27, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study with no deviation from the guideline followed.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The toxicological profile for systemic endpoints is expected to be similar for target substance Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed when compared to the source substance Sophorolipid, SLH solution. Read-across is expected to be reliable including the data on acute oral toxicity, sensitization and gene toxicity in vitro.
For further information, please see the attached justification for read-across.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Notification on Guidelines for Genotoxicity Studies of Drugs (Notification No. 1604 of the Evaluating and Licensing Division, PMSB dated November 1, 1999)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Dose range finding test: 0, 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate
Main study:
10, 20, 39, 78, 156 µg/plate in TA 1537 strain
313, 625, 1250, 2500, 5000 µg/plate in TA 100, Ta 1535, TA 98, TA 1537 strains and E. coli strain
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2 HCl; 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
No cytotoxicity observed
Evaluation criteria:
The results were judged as positive if both of the following conditions were met: 1) the number of revertant colonies in the culture treated with the test substance exceeded considerably (i.e. 2-fold increase from the negative control) that of spontaneous revertant colonies; and 2) the increase showed dose dependancy and reproducibility. No specific statistic treatment was performed for the judgement.
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The number of colonies that had reverted by treatment with the test substance did not increase by 2-fold or above when compared with the negative controls nor show dose dependency in both base pair substitution and frameshift strains.

COMPARISON WITH HISTORICAL CONTROL DATA: all values were within the range of historical controls

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed

Study results (main study)

Test period

November 24, 2004 – November 27, 2004

Presence or absence of metabolic activation system

Dose of test substance (µg/plate)

Number of revertant colonies/plate

Base pair substitution strains

Frameshift strains

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

-S9 mix

Negative control (water for injection)

125

148 (137)

15

20 (18)

24

28 (26)

18

23 (21)

10

18 (14)

10

NT

NT

NT

NT

9

15 (12)

20

NT

NT

NT

NT

9

14 (12)

39

NT

NT

NT

NT

15

16 (16)

78

NT

NT

NT

NT

15

16 (16)

156

NT

NT

NT

NT

21*

21*(21)*

313

125

130 (128)

14

12 (13)

30

24 (27)

17

13 (15)

11*

8* (10)*

625

122

113 (118)

11

14 (13)

24

24 (24)

17

14 (16)

NT

1250

106

108 (107)

12

9 (11)

25

29 (27)

19

12 (16)

NT

2500

102

101 (102)

9

10 (10)

26

24 (25)

13

21 (17)

NT

5000

107

91 (99)

14

14 (14)

33

22 (28)

19

13 (16)

NT

+S9 mix

Negative control (water for injection)

155

150 (153)

15

14 (15)

39

31 (35)

38

40 (39)

30

28 (29)

156

NT

NT

NT

NT

22

24 (23)

313

168

181 (175)

20

18 (19)

30

34 (32)

31

22 (27)

17

19 (18)

625

140

177 (159)

10

14 (12)

37

34 (36)

32

37 (35)

11

21 (16)

1250

168

134 (151)

10

11 (11)

36

39 (38)

38

38 (38)

22

16 (19)

2500

149

123 (136)

20

14 (17)

22

32 (27)

27

32 (30)

12*

16* (14)*

5000

103

127 (115)

15

10 (13)

21

34 (28)

26

19 (23)

13*

14* (14)*

Positive control

S9 mix not required

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Dose (µg/plate)

0.01

0.5

0.01

0.1

1.0

Number of colonies/plate

672

616 (644)

463

515 (489)

152

167 (160)

552

586 (569)

2276

2278 (2277)

S9 mix required

Name

B[a]P

2AA

2AA

B[a]P

B[a]P

Dose (µg/plate

5.0

2.0

10.0

5.0

5.0

Number of colonies/plate

720

687 (704)

321

361 (341)

410

463 (437)

163

189 (176)

89

68 (79)

AF-2:     2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3:     Sodium azide

ICR 191: 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine x 2 HCl

2AA:      2-Aminoanthracene

B[a]P:    Benzo[a]pyrene

 

*: Growth inhibition by the test substance observed

NT: Not tested

Figures in () indicate mean values of the measurements of two plates

Conclusions:
The test substance was not mutagenic in this bacterial reverse mutation assay.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
November 12, 2004 - November 27, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study with no deviation from the guideline followed.
Qualifier:
according to
Guideline:
other: Notification on Guidelines for Genotoxicity Studies of Drugs (Notification No. 1604 of the Evaluating and Licensing Division, PMSB dated November 1, 1999)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Dose range finding test: 0, 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate
Main study:
10, 20, 39, 78, 156 µg/plate in TA 1537 strain
313, 625, 1250, 2500, 5000 µg/plate in TA 100, Ta 1535, TA 98, TA 1537 strains and E. coli strain
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2 HCl; 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
No cytotoxicity observed
Evaluation criteria:
The results were judged as positive if both of the following conditions were met: 1) the number of revertant colonies in the culture treated with the test substance exceeded considerably (i.e. 2-fold increase from the negative control) that of spontaneous revertant colonies; and 2) the increase showed dose dependancy and reproducibility. No specific statistic treatment was performed for the judgement.
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The number of colonies that had reverted by treatment with the test substance did not increase by 2-fold or above when compared with the negative controls nor show dose dependency in both base pair substitution and frameshift strains.

COMPARISON WITH HISTORICAL CONTROL DATA: all values were within the range of historical controls

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed

Study results (main study)

Test period

November 24, 2004 – November 27, 2004

Presence or absence of metabolic activation system

Dose of test substance (µg/plate)

Number of revertant colonies/plate

Base pair substitution strains

Frameshift strains

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

-S9 mix

Negative control (water for injection)

125

148 (137)

15

20 (18)

24

28 (26)

18

23 (21)

10

18 (14)

10

NT

NT

NT

NT

9

15 (12)

20

NT

NT

NT

NT

9

14 (12)

39

NT

NT

NT

NT

15

16 (16)

78

NT

NT

NT

NT

15

16 (16)

156

NT

NT

NT

NT

21*

21*(21)*

313

125

130 (128)

14

12 (13)

30

24 (27)

17

13 (15)

11*

8* (10)*

625

122

113 (118)

11

14 (13)

24

24 (24)

17

14 (16)

NT

1250

106

108 (107)

12

9 (11)

25

29 (27)

19

12 (16)

NT

2500

102

101 (102)

9

10 (10)

26

24 (25)

13

21 (17)

NT

5000

107

91 (99)

14

14 (14)

33

22 (28)

19

13 (16)

NT

+S9 mix

Negative control (water for injection)

155

150 (153)

15

14 (15)

39

31 (35)

38

40 (39)

30

28 (29)

156

NT

NT

NT

NT

22

24 (23)

313

168

181 (175)

20

18 (19)

30

34 (32)

31

22 (27)

17

19 (18)

625

140

177 (159)

10

14 (12)

37

34 (36)

32

37 (35)

11

21 (16)

1250

168

134 (151)

10

11 (11)

36

39 (38)

38

38 (38)

22

16 (19)

2500

149

123 (136)

20

14 (17)

22

32 (27)

27

32 (30)

12*

16* (14)*

5000

103

127 (115)

15

10 (13)

21

34 (28)

26

19 (23)

13*

14* (14)*

Positive control

S9 mix not required

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Dose (µg/plate)

0.01

0.5

0.01

0.1

1.0

Number of colonies/plate

672

616 (644)

463

515 (489)

152

167 (160)

552

586 (569)

2276

2278 (2277)

S9 mix required

Name

B[a]P

2AA

2AA

B[a]P

B[a]P

Dose (µg/plate

5.0

2.0

10.0

5.0

5.0

Number of colonies/plate

720

687 (704)

321

361 (341)

410

463 (437)

163

189 (176)

89

68 (79)

AF-2:     2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3:     Sodium azide

ICR 191: 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine x 2 HCl

2AA:      2-Aminoanthracene

B[a]P:    Benzo[a]pyrene

 

*: Growth inhibition by the test substance observed

NT: Not tested

Figures in () indicate mean values of the measurements of two plates

Conclusions:
The test substance was not mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria according to the guideline for Genotoxicity Studies of Drugs (Notification No. 1604 of the Evaluating and Licensing Division, PMSB dated November 1, 1999), strains TA 1535, TA 1537, TA 98, TA 100 of S. typhimurium and E. coli WP2 were exposed to the substance in water at concentrations of 0, 10, 20, 39, 78, 156, 313, 625, 1250, 2500 and 5000 μg/plate (Salmonella strains) and 0, 313, 625, 1250, 2500 and 5000 µg/plate (E. coli strain) in the presence and absence of mammalian metabolic activation (S9 mix).  

growth inhibition was observed at 156 and 313 µg/plate (TA 1537; without metabolic activation) and 2500 and 5000 µg/plate (TA 1537; with metabolic activation). The positive controls induced the appropriate responses in the corresponding strains.There was no evidence of induced mutant colonies over background.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay (Ames-Test)

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone. The test item was used as a solution in sterile water for injection.

Toxicity test

The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration tested in the absence or presence of S9 metabolic activation. Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies, was observed with TA1537 and TA100 tester strains at the highest or two highest dose levels both in the absence and presence of S9 metabolic activation. No increase in revertant numbers was observed at any concentration tested with any tester strain/activation condition combinations.

Main Assays

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

- TA1535, WP2 uvrA, TA98: ± S9:  5000, 2500, 1250, 625 and 313 µg/plate

- TA1537:   ± S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

- TA100 :    ± S9: 2500, 1250, 625, 313 ,156 and 78.1 µg/plate

Toxicity was observed at the highest or two highest dose levels with TA1537 tester strain both in the absence and presence of S9 metabolism and with TA100 only in its absence. As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed including a pre-incubation step for all treatments.The following dose levels were used:

- TA1535, WP2 uvrA: ± S9: 10000, 5000, 2500, 1250 and 625 µg/plate

- TA100: :+  S9: 10000, 5000, 2500, 1250 and 625 µg/plate

- TA1537, TA100: - S9: 5000, 2500, 1250, 625, 313  and 156 µg/plate

- TA1537:  +S9: 10000, 5000, 2500, 1250, 625  and 313 µg/plate

- TA98:  ± S9: 10000, 5000, 2500, 1250, 625 and 313 µg/plate

Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at the highest dose level tested with TA1537 and TA100 tester strains both in the absence and presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period in any experiment, at any concentration tested, in the absence or presence of S9 metabolic activation.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Conclusion

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Micronucleus assay

The test item was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation. Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 32 hours corresponding to approximately 2.0 cell cycles was used. A second experiment was performed in the absence of S9 metabolism using a continuous treatment until harvest at 31 hours. Solutions of the test item were prepared in culture medium.

Dose levels for treatment were selected on the basis of the cytotoxicity observed in previous experiments where unacceptable values of frequency of micronucleated cells were observed or the required cytotoxicity was not achieved to select dose levels for scoring micronuclei.

Dose levels of 528, 440, 367, 306, 255, 212, 177, 147, 123, 102 and 85.3 µg/mL were used for the three hour treatment in the absence of S9 metabolism.

Dose levels of 684, 570, 475, 396, 330, 275, 229 and 191 µg/mL were used for the three hour treatment in the presence of S9 metabolism.

Dose levels of 191, 159, 133, 111, 92.1, 76.7, 64.0, 53.3 and 44.4 µg/mL were used for the continuous treatment in the absence of S9 metabolism.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.

The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

Dose levels were selected for the scoring of micronuclei on the basis of the cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI). For both experiments, the following dose levels were selected for scoring:

Experiment No.: S9    Treatment time (hours)  Harvest time (hours)  Concentration (µg/mL)
 1  -  3 31-32   212, 147 and 102
 1  +  3  31-32  570, 396 and 275
 2  - 31  31  

133, 92.1 and 64.0

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. All results were within the distribution of historical negative control data.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

It is concluded that the test itemdoes not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Mouse lymphoma cell assay

The test item was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 5000 µg/mL using dimethylsulfoxide (DMSO) as solvent (the upper limit to testing indicated in the test guideline OECD No. 490 for Chemical Substances of Unknown or Variable Composition). On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 5000 µg/mL and at a wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1, and 19.5 µg/mL. In the absence of S9 metabolic activation, no cells survived treatment from 156 µg/mL onwards. No relevant toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived treatment from 313 µg/mL onwards, mild toxicity was noted at the next lower concentration (RS=53%), while no relevant toxicity was observed over the remaining dose levels tested. Following treatment in the presence of S9 metabolic activation, no cells survived treatment from 625µg/mL onwards. Test item treatment at the two next lower dose levels (313 and 156µg/mL) yielded slight toxicity reducing RS to 60% and 73%, respectively. No relevant toxicity was observed over the remaining dose levels tested.

Based on the results obtained in the preliminary trial, a first Main Assay for mutation at the TK locus was performed using the following dose levels:

Main Assay I (-S9, 3 hour treatment): 150, 125, 104, 86.8, 72.3 and 60.3 μg/mL.

Main Assay I (+S9, 3 hour treatment): 600, 480, 384, 307, 246 and 197 μg/mL.

Adequate levels of cytotoxicity, covering a range from the maximum to little or no cytoxicity, were observed in the absence of S9 metabolism. In its presence, test item treatments did not meet appropriate cytotoxicity levels reducing relative total growth (RTG) to 57% at the highest analysable concentration. Based on these results, a second experiment (Main Assay 2) was performed in the presence of S9 metabolism using the following concentrations:

Main Assay II (+S9, 3 hour treatment): 480, 440, 400, 367, 333 and 305 μg/mL.

Adequate levels of cytotoxicity were obtained.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. It is concluded that the test item does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Further supporting data are available which gave the same result. In a read-across study according to Notification on Guidelines for Genotoxicity Studies of Drugs (Notification No. 1604 of the Evaluating and Licensing Division, PMSB dated November 1, 1999), Reliability 2, the source substance did not induce reverse mutation in the absence or presence of S9 metabolism.

Justification for classification or non-classification

The genotoxic potential of the test item was assayed in three OECD guideline studies (471, 487 and 490). The test item did not show any genotoxicity in any of these studies. Therefore, the test item is not classified for genotoxicity.