Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item showed positive results in Ames Test (three test strains of bacteria, TA98, TA100, and TA1537, under certain experimental conditions). However, an in vitro gene mutation test showed that the test substance is non-mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-01-11 until 2012-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
According to OECD 476. Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation
third experiment: 4 hours treatment with metabolic activation
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase Locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: Clone 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I (4 hours treatment):
without S9 mix: 78.1; 156.3; 312.5; 625.o; 937.5; 1250.0 µg/mL
with S9 mix: 312.5; 625.0; 1250.0; 2500.0; 3750.0; 5000.0 µg/mL
Experiment II (24 hours treatment)
without S9 mix: 75.0; 150.0; 300.0; 400.0; 500.0; 600.0 µg/mL
Experiment II (4 hours treatment)
with S9 mix: 300.0; 600.0; 1200.0; 2400.0; 3000.0; 3600.0 µg/mL
Experiment III (4 hours treatment):
with S9 mix: 300.0; 600.0; 1200.0; 2400.0; 3000.0 µg/mL
Following the expression phase of 48 hours the cultures (printed in bold letters) at 78.1 µg/mL without metabolic activation in experiment I and at 300 µg/mL with metabolic activation in experiment II were not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures at 5000 µg/mL with metabolic activation in experiment I and 500 and 600 µg/mL without metabolic activation in experiment II were not continued due to exceedingly severe cytotoxic effects.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the
corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Statistics:
Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.39 in the solvent control versus pH 7.27 at 5000 µg/mL)
- Effects of osmolality: No relevant increase (273 in the solvent control versus 308 at 5000 µg/mL)
- Evaporation from medium: Not examined
- Water solubility: Miscible in water
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 39.1 µg/mL and 5000 µg/mL were used with respect to the current guidelines.
Toxic effects leading to RSG values below 50% were observed at 625 µg/mL and above in the absence (4 and 24 hours treatment) and at 2500 µg/mL and above in the presence of metabolic activation (4 hours treatment).
The test medium was checked for precipitation or phase separation at the end of the treatment period (4 hours) before the test item was removed. No precipitation or phase separation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
Both, pH value and osmolarity were determined in the pre-experiment at the highest concentrations of the test item and in the solvent control without metabolic activation. There was no relevant shift of both parameters.
The dose range of the main experiments was set according to the cytotoxicity data of the test item. In the main experiments the individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 625 µg/mL and above with and at 1250 µg/mL without metabolic activation. In the second experiment cytotoxic effects as described were noted at 600 µg/mL and above with and at 150 µg/mL and above without metabolic activation. In the third experiment cytotoxic effects occurred at 2400 µg/mL and above. The recommended cytotoxic range of approximately 10-20% relative total growth was covered with and without metabolic activation. The data generated in experiment I at 3750 µg/mL with metabolic activation and in experiment II at 3000 and 3600 µg/mL with metabolic activation are not considered valid since the relative total growth fell even short of the 1% limit set by the IWGT exception criteria.
Remarks on result:
other: strain/cell type: in vitro gene mutation assay with L5178Y cells
Remarks:
Migrated from field 'Test system'.
Summary Table
      relative mutant   relative mutant  
  conc. µg S9 total colonies/   total colonies/  
  per mL mix growth 106cells threshold growth 106cells threshold
Column 1 2 3 4 5 6 7 8
Experiment I / 4 h treatment   culture I culture II
Solv. control with water - 100.0  93 219 100.0 130 256
Pos. control with MMS  19.5 -  39.7 434 219  21.7 689 256
Test item  78.1 - culture was not continued# culture was not continued#
Test item  156.3 - 102.9 119 219  38.8 400 256
Test item  312.5 -  90.6 134 219  48.9 292 256
Test item  625.0 - 130.2 118 219  95.9 197 256
Test item  937.5 -  78.2 197 219  76.2 223 256
Test item 1250.0 -  2.1 258 219  3.0 347 256
       
Solv. control with water + 100.0 100 226 100.0 117 243
Pos. control with CPA   3.0 +  53.7 245 226 110.0 250 243
Pos. control with CPA   4.5  +   27.8 415 226  53.0 444 243
Test item  312.5  +   66.9  71 226 103.5 142 243
Test item  625.0  +   49.5 157 226 105.0 148 243
Test item 1250.0  +   46.3 119 226  95.0 150 243
Test item 2500.0  +   17.0 165 226  44.3 126 243
Test item 3750.0  +   0.6 231 226  2.0 267 243
Test item 5000.0  +  culture was not continued## culture was not continued##
Experiment II / 24 h treatment   culture I culture II
Solv. control with water - 100.0  87 213 100.0  88 214
Pos. control with MMS  13.0 -  19.5 341 213  23.8 477 214
Test item  75.0 -  78.0 116 213  64.9 134 214
Test item  150.0 -  31.9 169 213  44.9 140 214
Test item  300.0 -  15.2 189 213  12.1 182 214
Test item  400.0 -  2.8 132 213  1.7 132 214
Test item  500.0 - culture was not continued## culture was not continued##
Test item  600.0 - culture was not continued## culture was not continued##
Experiment II / 4 h treatment   culture I culture II
Solv. control with water + 100.0  94 220 100.0 124 250
Pos. control with CPA   3.0 +  35.8 356 220  33.6 691 250
Pos. control with CPA   4.5 +  24.4 469 220  37.9 663 250
Test item  300.0 + culture was not continued# culture was not continued#
Test item  600.0 +  39.1 125 220  71.8 158 250
Test item 1200.0 +  18.1 125 220  26.7 170 250
Test item 2400.0 +  5.5 126 220  10.4 155 250
Test item 3000.0 +  2.0  74 220  0.5 235 250
Test item 3600.0 +  0.3  47 220  0.4 369 250
Experiment III / 4 h treatment   culture I culture II
Solvent control with water + 100.0 201 327 100.0  83 209
Pos. control with CPA   3.0 +  64.2 259 327 126.2 162 209
Pos. control with CPA   4.5 +  43.3 447 327  41.8 365 209
Test item  300.0 +  82.9 138 327 139.6  53 209
Test item  600.0 +  69.2 191 327  98.0  93 209
Test item 1200.0 +  65.6 136 327  78.6  87 209
Test item 2400.0 +  33.1 166 327  41.0 174 209
Test item 3000.0 +  5.6 338 327  15.4 116 209

threshold = number of mutant colonies per 106cells of each solvent control plus 126

#   culture was not continued since a minimum of only four analysable concentrations is required

##   culture was not continued due to exceedingly severe cytotoxic effects

The values printed in bold are judged as invalid, since even the exception criteria of the IWGT are not met (RTG < 1% in at least one of both parallel cultures).

 

Conclusions:
Under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

The study was performed to investigate the potential of 2,4-Pentanedione, peroxide (CAS 37187-22-7), 30% in solvent mixture to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in three independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation. As only three analysed concentrations remained within the acceptable cytotoxic range in the second experiment with metabolic activation this experimental part was repeated as experiment three. So, the third experiment was performed with a treatment period of 4 hours solely in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I

without S9 mix:                      156.3: 312.5; 625.0; 937.5; 1260.0 µg/mL
with S9 mix:                       312.5; 625.0; 1250.0; 2500.0; 3750.0 µg/mL

Experiment II

without S9 mix:                                     75.0; 150.0; 300.0; 400.0 µg/mL
with S9 mix:                     600.0; 1200.0; 2400.0; 3000.0; 3600.0 µg/mL

Experiment III

with S9 mix:                       300.0; 600.0; 1200.0; 2400.0; 3000.0 µg/mL

Relevant toxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 625 µg/mL and above with and at 1250 µg/mL without metabolic activation. In the second experiment cytotoxic effects as described were noted at 600 µg/mL and above with and at 150 µg/mL and above without metabolic activation. In the third experiment cytotoxic effects occurred at 2400 µg/mL and above. The recommended cytotoxic range of approximately 10-20% relative total growth was covered with and without metabolic activation. The data generated in experiment I at 3750 µg/mL with metabolic activation and in experiment II at 3000 and 3600 µg/mL with metabolic activation are not considered valid since the relative total growth fell even short of the 1% limit set by the IWGT exception criteria.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation at acceptable levels of cytotoxicity. The isolated increase of the mutation frequency noted at a severely cytotoxic concentration of 1250 µg/mL in both cultures of the first experiment without metabolic activation was not reproduced in the second experiment without metabolic activation at about the same cytotoxic level generated at 400 µg/mL. Furthermore, this increase was not dose dependent as indicated by the lacking statistical significance in one of the parallel cultures and consequently judged as biologically irrelevant. The increased mutation frequency exceeding the threshold in just one of both parallel cultures (culture II of the first experiment without metabolic activation at 156.3 and 312.5 µg/mL and culture I of the third experiment at 3000 µg/mL) was not considered relevant as the increase was not reproduced in the parallel cultures.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency using SYSTATâstatistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the first culture of the first experiment without metabolic activation. However, this trend was not reproduced in the parallel culture and therefore, not considered biologically relevant as discussed above.

The highest solvent control value (201 colonies per 106cells) exceeded the recommended 50 – 170 x 106control range as stated under paragraph 10.12,acceptability of the assay of this report. The mean value of both parallel cultures however, (201 and 83 equal to 142 colonies per 106cells) is fully acceptable. The cloning efficiency (viability) slightly exceeded the upper limit of 120% (127%) in the solvent control of the first culture of the first experiment with metabolic activation. This minor deviation was judged as irrelevant as it was rather minor and the cloning efficiency of the parallel culture remained within the acceptable range.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies with at least one of the concentrations.

Under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A well performed GLP study done according to OECD 471 guideline..
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Test System Salmonella typhimurium bacteria
Rationale Recommended test system in international guidelines
(e.g. EPA, OECD, EEC).
Source Dr. Bruce N. Ames, University of California at Berkeley, U.S.A.
(Salmanella typhimurium strains)
TA100 received 18-02-1993, used batch:TA100.150900
TA98 received 21-02-1991, used batch: TA98.231100
TA1535 received 14-12-1994, used batch: TA1535.231100
TA1537 received 14-12-1994, used batch: TA1537.231100
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutetion Mutation type
TA 1537 hisC3076 Frameshift
TA98 hls030521R-factor* Frameshift
TA1535 hisG46 Base-pair sustitutions
TA100 hisG46/R-faclor* Base-pair substitutions
*: R-factor =plasmid pKM101 (Increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cell coat)
gal: mutation in the galactose metabolism
chl: mutation in nitrate reductase
bio: defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were regularly checked to confirm their histidine requirement, crystal violet senssitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the four strains were stored In liquid nitrogen (-196'C).
Test concentrations with justification for top dose:
Negative control:
The vehicle of the test article, being MHIi-Q-water.

Positive controls:
Without metabolic activation (-S9-mjx):

Solvents for reference substances
Saline = physiological saline (NPBI, Emmer Compascuum, The Netherlands)
DMSO = dimethyl sulfoxide of spectroscopic quality (Merck).
Details on test system and experimental conditions:
TRIGONOX 44B
CELL CULTURE
Preparation of
Bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxold no. 2) and Incubated In a shaking Incubator (37•C, 150 spm), until the cultures reached an optical density of 1.0:1: 0.1 at 700 nm (109 Cells/ml). Freshly grown cultures of each strain were used for a test.
Agar plates: Agar plates (9 cm) contained 25 ml glucose agar medium. Glucose agar medium oontained per liter: 18 g purified agar (Oxoid, code l28) in Vogel-Bonner Medium E, 20 g glucose. N.B. The agar plates also contained 12.5 ug/plate biotin and 15 ug/plate histidine.

Top agar:; Top agar medium, contalned 0.6% (wlv) purified agar and 0.5% (wlv) NaCl, was heated to dissolve the agar Samples of 3 ml top agar were transferred into 10 mI glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 + 1 •C.

Environmental conditions: All Incubations were carried out in the dark at 37+1oC. The temperature was monitored during the experiment.

Negative control:
The vehicle of the test article, being MHIi-Q-water.

Positive controls:
Without metabolic activation (-S9-mjx):

Solvents for reference substances
Saline = physiological saline (NPBI, Emmer Compascuum, The Netherlands)
DMSO = dimethyl sulfoxide of spectroscopic quality (Merck).

Preparation of S9 fraction;
Rat liver microsomal enzymes were routinely prepared from adult male Wlstar rats, which were obtained from Charles River, Sulzfeld, Germany.
The animals were housed at NOTOX In a special room under standard laboratory conditions, as described in the Standard Operating Procedures. The rats were Injected intraperitoneally with a solution (20% (wlv» of Arodor 1254 (500 mglkg body weight) in oem 011. FIve days later, !hey were knled by decapitation; (they were denied access to foDd for at least 12 hours preceding sacrifice). The livers Df the rats were removed aseptically, and washed In cold (O·C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na,-EOTA. Subsequently the livers were minced In a blender and homogenized In 3 volumes of phosphate buffer with a Potler homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supematant (59) was transferred into sterile ampules, which were stored In liquid nitrogen (-196"C) and identified by the day of preparatlon.
Before use, all 59-batches were characterized with the metabolic activation requiring positive control; benzo[a]pyrene (Sigma) in tester strain TA96 at the concentration of 5ug/plate.

Preoparatlon of S9-mix
S9-mix was prepared immediately before use and kept on ice. 59·mlx contained per 10 ml: 30 mg NADP and 15.2 mg glucose..e·phosphate in 5.5 ml MiIIi·Q water; 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl, solution; 1 ml 0.33 M KCI solution. The above solution was filter (0.22 IJm}-sterUized. To 9.5 ml of 59-mix components 0.5 ml 59-fraction added (5% (vlv) 59-frac1ion) to complete the S9-mix. The 59-batch used was no. 01·2

Dose range finding tes!
Selection of an adequate range of doses was based on a dose range finding test with strain TA100, with and without 59-mix. Bght concentrations were tested in triplicate. This dose range finding test was reported as a part of the mutation assay. The highest concentration of TRIGONOX 446 used In the subsequent mutation assay was the recommended 5 mg/plate or the level at which the test substance Inhibited bacterial growth.

Mutation Assay
At least five di!ferent doses (Increasing with approximately haif-log steps) of the test substance were tested in triplicate In each strain. The test substance was tested both in the absence and presence of S9-mix In each strain Top agar in top agar tubes was molten and heated to 45'C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacteria! culture (1 billionce!ls/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance In MIIII·Q.water and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (In case of non·activation assays). The Ingredients were mixed on a Vortex and the cootent of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated In the dark at 37 ± 1 'C for 48 h. After this period revertant colonies were counted.

Colony counting: The revertant colonies were counted automatically with a Protos model 50000 colony counter or manually, If less than 40 colonies per plate were present.

Evaluation criteria:
Acceptance Criteria:
The assay is considered acceptable if it meets the following criteria:
a) The negative control dala (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses In all tester strains which are within the laboratory historical range documented for each positive control substance.
c) The selected dose range should include a clearly toxic concentration or should exhibit Iimited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 ug/plate.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
The 3 strains that reacted positive were: TA100, TA1537, and TA98; Negative for TA1535.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

REFERENCES

1. Leonardo, J.M., Dornfeld, 5.5. and Peak, M.J., 1984, Evaluation of E. coil K12 343 \ 13 and derived strains for microbial mutagenicity assays. Mutation Res., 130, 87-95.

2 Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmone/mammalian microsome mutagenicity test. Mutation Res., 31, 347-364.

3. Maron, D.M. and Ames, B.N., 1983, Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173 -215. Erratum, 1983, Mutation Res., 113, 533.

Conclusions:
TRIGONOX 44B was tested in theSalmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonellatyphimurium (TA1535, TA1537, TA100 and TA98). Based on the results of this study, it is concluded that TRIGONOX 44B is mutagenic in the Salmonella typhimurium reverse mutation assay
Executive summary:

TRIGONOX 44B was tested in theSalmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonellatyphimurium (TA1535, TA1537, TA100 and TA98). In the dose range finding test, TRIGONOX 44B was tested up to concentrations of 5000 ug/plate in the absence and presence of S9-mix in the strain TA100.TRIGONOX 44B did not precipitate on the plates at this dose level. Toxicity was observed at test substance concentrations of 3330 and 5000 ug/plate In the absence of S9-mix only.

In the mutation assay, test substance (TRIGONOX 44B) was tested up to concentrations of 3330 and 5000 ug/plate in the absence and presence of S9-mix, respectively. In the presence of S9-mix, the bacterial background lawn was not reduced at all concentrations tested and no decrease In the

number of revertants was observed. In the absence of S9 -mix, toxicity was observed at the test substance concentration of 3330 ug/plate in the strains TA1535, TA1537 and TA98.

In the absence of S9-mix in tester strain TAl 00, TRIGONOX 44B induced an up to 2.0-fofd, Dose-related increase in the number of revertant colonies compared to the solvent control. In tester strain TA1537, TRIGONOX 44B induced an up to 4.0 -fold, dose-related increase in the number of revertant colonies compared to the solvent control. In the tester strains TA1535 and TA98, TRIGONOX 44B did not induce a dose-related, two-fold increase in the number of revertant (HIS+) colonies.

 

In the presence of S9 -mix in tester strain TA98, TRIGONOX 44B induced an up to 2.8 -foId, dose-related increase in the number of revertant colonies compared to the solvent control. In tester strain TA1537, TRIGONOX 44B induced an up to 2.8 -fold increase in the number of revertant colonies compared to the solvent control (however, any mean plate count of less than 20 is considered to be not significant).In the tester strains TA1535 and TA100,TRIGONOX 44B did not Induce a dose-related, two-fold increase in the number of revertant (HIS+) colonies.

Based on the results of this study, it is concluded that TRIGONOX 44B is mutagenic in the Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo mammalian cell micronucleus test (mice): The test substance was tested in vivo in the Micronucleus Test in mice, to evaluate its potential genotoxic effect on erythrocytes in bone marrow (Meerts, 2004). The procedures were based on the OECD testing guideline No. 474. Mice were used in 6 treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. Bone marrow of the groups was harvested 24 or hours after dosing. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item. It is concluded that the test item is not mutagenic in vivo in the micronucleus test under the conditions described in this report.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A GLP study done according to OECD guideline 474. Although the certificate of analysis for the test item is not included, sufficient basic details including the composition are provided.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS

NMRI BR mice (SPF) were used as test system. These mice are recommended by international
guidelines (e.g. EPA, FDA, OECD, EEC). Females were nulliparous and non-pregnant. The
animals were provided by Charles River, Sulzfeld, Germany.

In the micronucleus main test 5 male and 5 female mice were treated per sampling time In each
treatment group. Young adult animals were selected (6-8 weeks old).

The body weights of the mice at the start of the treatment are presented in table 2. An unique
number on the tail identified the mice. The animals were allocated to treatment groups as they
came to hand from the delivery boxes.

ANIMAL HUSBANDRY

Environmental Conditions
The animals were housed in an air-conditioned room with approximately 15 air changes per hour
and a conlrolled environment with a temperature of 21 ± 3°C and a relative humidity of 30-70%.
Due to cleaning procedures or performance of functional observations in the room, temporary
deviations from the maximum level for humidity (with max. 20%) occurred. Based Qn laboratory
historical data these deviations are considered not to affect the study integrity. The room was
illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.

Accommodation
On arrival and at the start of the treatment, all animals were clinically examined to ensure
selected animals were in a good state of health. Group housing of 5 animals per sex per cage
In labelled polycarbonate cages containing purified sawdust as bedding material (Sawi, Jelu Werk,
Rosenberg, Germany). Paper bedding was provided as nest material (B.M.I. Helmond, The Netherlands).
Certificates of analysis were examined and then retained in the NOTOX archives. Acclimatisation period
was at least 5 days before start of treatment under laboratory conditions.

Diet
Free access to standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany).
Certificates of analysis were examined and then retained in the NOTOX archives.

Water
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and then
retained in the NOTOX archives.

Study Plan Start: 20 October 2003; Completion: 7 December 2003
Route of administration:
intraperitoneal
Vehicle:
0.9% NaCl
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Test Substance preparation
TRIGONOX 44B was dissolved in physiological saline (B. Braun, Melsungen, Germany).
TRIGONOX 44B concentrations were dosed within 4 hours after preparation.

Reference substances:
1. Negative Control: Negative control animals were treated with physiological saline.
2. Positive Control: The positive conlrol used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0;
Endoxan, Asta-Werke, F.R.G.) dissolved in physiological saline (B. Braun, Melsungen, Gennany)
dosed as a single intraperitoneal injection of 50 mg saltlkg body weight.
Duration of treatment / exposure:
Single injection
Frequency of treatment:
Treatment
The mice received an intraperitoneal Injection of a maximum tolerated (high), an intermediate and a low dose of TRIGONOX 44B.
Ttie route of administration was chosen to maximize the chance of the test article reaching the target tissue.
The dosing volume was 10 ml/kg body weight.
The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test article.
Post exposure period:
Micronucleus main test
Five male and five female mice were used per sampling time in each treatment group. The animals were dosed once and sampled according to the following scheme.

Treatment Dose (mg/kg body weight) Sampling time (hrs) Group

0.9% NaCI -- 24 A
TRIGONOX 44B 1500 24,48 B,C
TRIGONOX 44B 750 24 D
TRIGONOX 44B 375 24 E
Cyclophosphamide 50 48 F
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
No. of animals per sex per dose:
Five male and five female mice were used per sampling time in each treatment group. The animals were dosed once and sampled according to the table described under the section, "post exposure period".
Positive control(s):
Reference substances:

The positive conlrol used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0;
Endoxan, Asta-Werke, F.R.G.) dissolved in physiological saline (B. Braun, Melsungen, Gennany)
dosed as a single intraperitoneal injection of 50 mg saltlkg body weight.

Negative control animals were treated with physiological saline.
Tissues and cell types examined:
Bone marrow of TRIGONOX 44B-treated groups was sampled 24 or 48 hrs after dosing. Bone marrow of the negative and positive control groups were isolated 24 and 48 hrs after dosing, respectively. The bone was flushed and the cell suspension prepared. Bone marrow smears were prepared from these suspensions.The slides were automatically stained using the "Wright-stain-procedure". The dry slides were dipped in xylene, embedded in MicroMount and mounted with a coverslip. Slides were scored at 1000X magnification. The number of micronuclealed polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by simultaneously counting and differentiating the first 1000 erythrocytes. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

In-life Observations:
The systemic toxic signs were recorded at least once a day. The animals were weighed just prior to dosing.
Details of tissue and slide preparation:
Bone marrow cells: Bone marrow of TRIGONOX 44B-treated groups was sampled 24 or 48 hrs after dosing. Bone marrow of the negative and positive control groups were isolated 24 and 48 hrs after dosing, respectively. Animals were sacrificed, both femurs removed and both ends of the bone were shortened until the marrow canal became visible. The bone was flushed with fetal calf serum (2 mL), and the cell suspension centrifuged at 1000 rpm (~100 g) for 5 min.

Bone marrow smears: The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum with the remaining serum. A drop of the cell suspension was placed on the end of a slide (2 slides / animal), which was previously cleaned (24
h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether) and marked with study identification and the animal numbers. The drop was spread by a clean slide at an angle of ~45· over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight.

Bone marrow smears: The slides were automatically stained using the "Wright-stain-procedure". The dry slides were dipped in xylene, embedded in MicroMount and mounted with a coverslip.

Analysis of the micronuctei: All slides were randomly coded before examination. An adhesive identification label (with a code) was stuck over the marked slide. At first the slides were screened at 100x magnification for regions of suitable technical quality (cells well spread, undamaged and well stained). Slides were scored at 1000X magnification. The number of micronuclealed polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by simultaneously counting and differentiating the first 1000 erythrocytes. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
Evaluation criteria:
Acceptability of the assay:
A micronucleus test is considered acceptable if it meets the following criteria:

a) The positive control substance induced a statistically signifIcant (Wilcoxon Rank Sum Test,
two-sided test at P < .0.05) increase In the frequency of micronucleated polychromatic
erythrocytes.

b) The incidence of micronucleated polychromatic erythrocytes in the control animals should
reasonably be within the laboratory historical control data range (mean ± three times the
standard deviation): Males: 1.2%o:i: 3.6%0 indicated are means for n=229. Females: 1.3% ±
3.9% indicated are means for n=150).

Statistics:
Data evatuatlon and statistical procedures:

Equivocal results should be clarified by further testing using modification of experimental
conditions.
A test substance Is considered positive In the micronucfeus test if:
It induced a biologically as well as a statistically signWicant (Wilcoxon Rank Sum Test; twosided
test at P < 0.05) increase In the frequency of micronucleated polychromatic erythrocytes
(at any dose or at any sampling time) In the combined data for both sexes or in the data for
male or female groups separately.

A test Substance is considered negative In the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P <
0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the
combined data for both sexes nor In the data for male or female groups separately.
The preceding criteria are not absolute and other modifying factors may enter into the final
evaluation decision.

Please see Appendix III (Statitics) under "Any other results" section.
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: In a dose range finding study 8 animals (group A: 1 male and 1 female, group B: 3 males and 3 females) were dosed intraperitoneally (single dose) with 2000 and 1500 mglkg body weight (groups A and B respectively).

- Solubility: No data provided.
- Clinical signs of toxicity in test animals: The following toxic signs were observed in the animals dosed with 2000 mg/kg body weight: lethargy, ataxia, no reaction to stimulus (only observed at one hour after dosing), ventre-lateral recumbency, a hunched posture and a rough coat. At the dose level of 1500 mg/kg body weight the same toxic signs were observed as in Ihe animals treated with 2000 mg/kg, but the time that the animals showed no reaction to stimulus was shorter in these animals. Therefore, a dose level of 1500 mg/kg body weight was used as the highest dose level for the main study.

- Rationale for exposure: The group comprising 3 males and 3 females were dosed with the highest concentration that was used for the main study. The study duration per dosing was one to four days. During this period mortality and physical condition were recorded at least once daily. At the dose level of 1500 mg/kg body weight the same toxic signs were observed as in Ihe animals treated with 2000 mg/kg, but the time that the animals showed no reaction to stimulus was shorter in these animals. Therefore, a dose level of 1500 mg/kg body weight was used as the highest dose level for the main study.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay) is described

Micronucleus main test results:
Based on the results of the dose range finding study dose levels of 375, 750 and 1500 mg/kg, body weight were selected as appropriale doses for the micronucleus main test. Five male and five female animals were used in each treatment group. The mean body weights per group recorded immediately prior to dosing are presented in Table 2.

Mortality and systemic toxic signs - In-life Observations:
The systemic toxic signs were recorded at least once a day. The animals were weighed just prior to dosing. The animals of the groups treated with 375 mg TRIGONOX 44B /kg body weight and the animals of the negative and positive control groups showed no abnormalities. (Appendix I (Tables 1 and 2). The following clinical observations were made in the groups treated with 750 and 1500 mg TRIGONOX 44B /kg body weight:
During the first hour after dosing all animals of the groups treated wilh 1500 mg/kg body weight were lethargic, showed ventre-lateral recumbency and no reaction to stimulus. In the group treated with 750 mg!kg body weight all animals were lethargic and showed ataxia. Within 2 hours after dosing in the groups treated with 1500 mg/kg body weight all animals were lethargic. Thirteen animals additionally had a rough coat. Six animals also showed a hunched posture. The animals dosed with 750 mg/kg body weight showed no abnormalities. Within 42 hours after dosing all animals had recovered from the treatment.

Micronucleated polychromatic erythrocytes
The mean number of micronucleated polychromatic erythrocyles per group and the mean ratio of polychromatic to normochromatic eryihrocyles are presented in Table 3 (Appendix I). The individual data are described in Appendix II. The mean number of micronucleated polychromatic erythrocytes scored in TRIGONOX 44B treated groups were compared with the corresponding solvent control group.

No increase in the frequency of micronucleated polychromatic erythrocyles was observed in the polychromatic erythrocytes of the bone marrow of TRIGONOX 44B treated animals compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.

APPENDIX I

 

TABLES

 

Table1. Mortality and systemic toxic signs after treatment with TRIGONOX44Bin the dose range finding study

Group 

 

 Sex 

 

 Animal 

number 

 Dose 

 mgIkg 

                                 Systemic toxic signs 

           day1 within 

 day· 2 

 day 3 

 day4 

10 min.

1 hr

4 hrs

 A 

 Male 

 1 

 2000 

 FC 

 FGX 

 FNY 

 B 

 B 

 B 

 A 

 Female 

 2 

 2000 

 FC 

 FGX 

 FX 

 B 

 B 

 B 

 B 

 Male 

 3 

 1500 

 FC 

 FGX 

 N 

 B 

 B 

 

 B 

Female 

 4 

 1500 

 FC 

 FGX 

 FNY 

 B 

 B 

 

 B 

 Male 

 5 

 1500 

 FC 

 FGX 

 FNY 

 B 

 B 

 

 B 

 Male 

 6 

 1500 

 FC 

 FGX 

 FNY 

 B 

 B 

 

 B 

 Female 

 7 

 1500 

 FC 

 FGX 

 FN 

 B 

 B 

 

 B 

 Female 

 8 

 1500 

 FC 

 FGX 

 NY 

 B 

 B 

 

• Legend 'Mortality and systemic toXic signs':

B=showed no abnormalities; C=ataxia; Flethargy; Gno reaction to stimulus; N=rough coat; X=

ventro-lateral recumbency; Y=hunched posture

Table 2 Meanbody weightimmediately prior to dosingwithTRIGONOX44B

Group

Body weight (g) (mean±S.D.)(1)

 

MALES                                  FEMALES

A

32.8± 1.5

26.4±1.5

B

31.8±1.8·

27.2±1.3

C

32.0 ± 2.2

27.4±1.1

D

32.4±2.3

26.6±0.9

E

32.0±2.0

27.2±1.3

F

32.0±2.4

25.6:t0.9

(1) Five animals per treatment group

Table 3. Mean number of micronucleated polychromatic erythrocytesper2000

polychromatic erythrocyte and ratio of polychromatic/normochromatic erythrocytes

Group 

 

 

 

 Treatment 

 

 

 

 Dose (mg/kg 

 body weight) 

 

 

Sampling

time 

 (hours) 

 

 No. of micronucleatedpolychromatic erythrocytes  per 2000 polychromatic erythrocytes (mean±SD)(1) 

 Ratio polychromatic/ 

 nonnochromatic 

 erythrocytes 

 (mean±S.D.)(1) 

MALES

 A 

 Solvent control 

 0 

 24 

 1.6±0.5 

 0.97 :to.02 

 B 

 TRIGONOX 44B 

 1500 

 24 

 2.0 :t 1.6 

 0.95 ±0.05 

 C 

 TRIGONOX 44B 

 1500 

 48 

 2.4 :t 1.5 

 0.96 :to.06 

 D

TRIGONOX 44B 

 750 

 24 

 2.4 ± 1.1 

 0.96 :to.03 

 E 

 TRIGONOX 44B

 375 

 24 

 1.4 ± 0.5 

 0.96 :to.05 

 F 

 CP 

 50 

 48 

 24.8 ± 8.0(2) 

 0.51 to.08 

FEMALES

 A 

 Solvent control 

 0 

 24 

 1.2 ± 0.8 

 0.95 ±0.01 

 8 

TRIGONOX 44B

 1500 

 24 

 3.4±1.1 

 0.97 ±0.04 

 C 

TRIGONOX 44B

 1500 

 48 

 1.4 ± 1.5 

 0.91 :to.05 

 D

TRIGONOX 44B

 750 

 24 

 3.2 ± 1.5 

 0.96 :to.04 

 E 

TRIGONOX 44B 

 375 

 24 

 1.6±0.5 

 0.97 :to.03 

 F 

 CP 

 50 

 48 

 23.0 ± 4.7(2) 

 0.51 :to.10 

Solvent control=physiological saline

CP=Cyclophosphamide

(1) Five animalspertreatment group

(2) Significantiy different from corresponding control group (Wilcoxon Rank Sum Test, P<0.01).

APPENDIX II INDIVIDUAL DATA

Individual data (males)

(groupA :intraperitoneal injection of physiological saline)

(group B & C: intraperitoneal injection of TRIGONOX 44B at 1500mg/kgbody weight)

(group D:intraperitoneal injection of TRIGONOX44Bat 750mglkgbody weight)

(groupE :intraperitoneal injection ofTRIGONOX44Bat 375 mg/kg body weight)

(groupF :intraperitoneal injection of cyclophosphamide)

Group

 

 

Animal number 

Number of mlcronucleated 

 polychromatic erythrocytes / 2000 polychromatic erythrocytes 

 Ratio polychromatic/normochromatlc 

 erythrocytes 

 

 A 

 1 

 2 

 0.99 

 A 

 2 

 1 

 0.98 

 A 

 3 

 2 

 0.97 

 A 

 4 

 1 

 0.97 

 A 

 5 

 2 

 0.94 

 B 

 11 

 1 

 0.90 

 B 

 12 

 2 

 0.96 

 B 

 13 

 4 

 0.93 

 B 

 14 

 0 

 0.91 

 B 

 15 

 3 

 1.03 

 C 

 21 

 3 

 0.96 

 C 

 22 

 2 

 1.03 

 C 

 23 

 3 

 0.99 

 C 

 24 

 0 

 0.91 

 C 

 25 

 4 

 1.00 

 0 

 31 

 2 

 0.95 

 0 

 32 

 4 

 0.99 

 0 

 33 

 2 

 0.93 

 0 

 34 

 3 

 0.93 

 0 

 35 

 1 

 0.98 

 E 

 41 

 1 

 0.88 

 E 

 42 

 2 

 1.01 

 E 

 43 

 1 

 0.98 

 E 

 44 

 2 

 0.92 

 E 

 45 

 1 

 0.99 

 F 

 51 

 34 

 0.43 

 F 

 52 

 19 

 0.56 

 F 

 53 

 18 

 0.45 

 F 

 54 

 20 

 0.63 

 F 

 55 

 33 

 0.48 

Individual data (females)

(groupA :intraperitoneal injection of physiological saline)

(group B & C: intraperitoneal injection of TRIGONOX 44B at 1500mg/kgbody weight)

(group D:intraperitoneal injection of TRIGONOX44Bat 750mglkgbody weight)

(groupE :intraperitoneal injection ofTRIGONOX44Bat 375 mg/kg body weight)

(groupF :intraperitoneal injection of cyclophosphamide)

Group 

Animal number 

Number of mIcronucleated 

 polychromatic erythrocytes / 2000 polychromatic erythrocytes 

 Ratio polychromatic/normochromatlc 

 erythrocytes 

 

 A 

 6 

 1 

 0.95 

 A 

 7 

 2 

 0.94 

 A 

 8 

 0 

 0.94 

 A 

 9 

 2 

 0.97 

 A 

 10 

 1 

 0.95 

 B 

 16 

 5 

 0.96 

 B 

 17 

 3 

 0.94 

 B 

 18 

 4 

 1.01 

 B 

 19 

 3 

 0.93 

 B 

 20 

 2 

 1.03 

 C 

 26 

 1 

 0.B9 

 C 

 27 

 0 

 0.B5 

 C 

 2B 

 0 

 0.B9 

 C 

 29 

 3 

 0.97 

 C 

 30 

 3 

 0.96 

 D 

 36 

 3 

 0.90 

 D 

 37 

 1 

 0.99 

 D 

 36 

 5 

 0.96 

 D 

 39 

 4 

 0.96 

 D 

 40 

 3 

 0.95 

 E 

 46 

 1 

 0.94 

 E 

 47 

 2 

 0.96 

 E 

 4B 

 2 

 1.00 

 E 

 49 

 2 

 1.01 

 E 

 50 

 1 

 0.94 

 F 

 56 

 31 

 0.47 

 F 

 57 

 23 

 0.39 

 F 

 5B 

 21 

 0.59 

 F 

 59 

 19 

 0.46 

 F 

 60 

 21 

 0.63 

APPENDIX III  STATISTICS

Wilcoxon Rank Sum Test.

 

Number of micronucleated polychromatic erythrocytes per2000polychromatic erythrocytes;

Treatment/conlrol comparison.

Group 

 

 

 Treatment 

 

 

 Dose 

 mg/kgbody weight 

 Sex 

 

 

P-value  (two-sided) 

 

Decision at 95%  confidence level 

 

 F 

cyclophosphamide 

 50 

males 

<0.01 

 significant 

 F 

cyclophosphamide 

 50 

females 

<0.01 

 significant 

Conclusions:
Interpretation of results (migrated information): negative
II is concluded that this test Is valid and that TRIGONOX 44B is not mutagenic in the
micronucleus test under the experimental conditions described in this report.
Executive summary:

TRIGONOX 44B was tested in the Micronucleus Test in mice, to evaluale its genotoxic effect on erythrocytes in bone marrow. The study procedures described in this report were based on the following guidelines:

• European Economic Community (EEC), Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.12: "Mutagenicity: In Vivo Mammalian Erythrocyte Micronucleus Test" .

• OECD Guidelines for the Testing of Chemicals, Guideline No. 474: Mammalian Erythrocyte Micronucleus Test (adopted 21st July 1997).

Batch 0410306045344 of TRIGONOX 44B was a clear colourless liquid. The test substance was dissolved in 0.9% NaCI. Five male and fIve female animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with TRIGONOX 44B at 1500 (two groups), 750 (one group), and 375 (one group) mg/kg body weight. After dosing the animals of the dose level of 1500 mglkg body weight showed the following toxic signs: lethargy, no reactlon to stimulus, ventra-lateral recumbency, rough coat and a hunched posture. All animals of lhe dose level of 750 mglkg body weight were lethargic and showed ataxia after dosing. The animals of the dose level of 375 mglkg body weight showed no abnormalities after dosing. Bone marrow of the groups treated with TRIGONOX 44B was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and postive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with TRIGONOX 44B. The incidence of micro nucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromalic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met. The groups that were treated wilh TRIGONOX 448 showed no decrease in the ratio of polychromatic to normochromalic erythrocytes compared to the vehicle conlrols, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromallc erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

It is cencluded that TRIGONOX 44B is not mutagenic in the micronucleus test under the experimental conditions described in this report.

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

Additional information

Additional information from genetic toxicity in vivo:

In vivo mammalian cell micronucleus test (mice): The test substance was tested in vivo in the Micronucleus Test in mice, to evaluate its potential genotoxic effect on erythrocytes in bone marrow (Meerts, 2004). The procedures were based on the OECD testing guideline No. 474. Mice were used in 6 treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. Bone marrow of the groups was harvested 24 or hours after dosing. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item. It is concluded that the test item is not mutagenicin vivoin the micronucleus test under the conditions described in this report.

 

In vitro Mouse Lymphoma Assay;The GLP study was performed (Wollny, 2012) according to OECD test guideline No. 476 to investigate the potential of 2,4-Pentanedione, peroxide (CAS 37187-22-7), 30% in solvent mixture to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. Thein vitroassay was performed in 3 independent experiments, using 2 parallel cultures each. No substantial and reproducible dose dependent increase in the mutation frequency was observed with and without metabolic activation at acceptable levels of cytotoxicity. It was concluded that under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

 

In vitro bacterial reverse mutation assay: The test item was tested (Verspeek-Rip, 2001) in theSalmonella typhimuriumreverse mutation assay with 4 histidine-requiring strains of Salmonellatyphimurium(TA1535, TA1537, TA100 and TA98) according to OECD test guideline 471. In the dose range finding test, the test item was tested up to 5000 ug/plate with or without S9-mix in the strain TA100 with no precipitate on the plates at this dose level. Toxicity was observed at test substance concentrations of 3330 and 5000 ug/plate in the absence of S9-mix only. In the mutation test, in the absence of S9-mix in tester strains TAl 00 and TA1537, the test item induced up to 2.0- and 4-fold dose-related increases, respectively, in the number of revertant colonies compared to the solvent control. In the presence of S9-mix along with the tester strains TA1537 and TA98, the test substance induced a dose-related, 2-fold increase in the number of revertant (HIS+) colonies. In the tester strain TA1535 there was no induction of revertant (HIS+) colonies with or without the S9-mix. Based on these results, it is concluded that the test item is mutagenic in the Salmonellatyphimuriumreverse mutation assay.

Overall discussion of the endpoint summary: Although the test item, 2,4-Pentanedione, peroxide (CAS 37187-22-7), 30% in solvent mixture, was mutagenic to bacteria (three strains of bacteria in Ames test, TA98, TA100, and TA1537), the test item was shown to be non-mutagenic in L5178Y cells (no toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells, with or without metabolic activation) as well as non-mutagenic in anin vivo micronucleus test in mice. In accordance with Endpoint Specific Guidance Chapter R.7A, Figure R.7.7 -1 "Flow chart of the mutagenicity testing strategy", no further testing (i.e., no in vivo testing) need be proposed in the event of a negative mouse lymphoma assay or hprt assay, regardless of whether or not the gene mutation test in bacteria is positive or negative. This, therefore, implies that when considering whether an in vivo gene mutation request is required for substances requiring Annex IX test proposals due to their volume bands, a negative mouse lymphoma assay is sufficient evidence to waive the need for an in vivo gene mutation test. Furthermore, in this case, a reliable negative in vivo mutation test result in mice is available to support the conclusion that the test item is not mutagenic.


Justification for selection of genetic toxicity endpoint
An in vivo gene mutation study, conducted in compliance with OECD 474 test guidelines is available. The GLP-compliant study report results were conclusive and were done to a valid guideline. Therefore, the test item was considered to be non mutagenic in mammalian cells.

Justification for classification or non-classification

The test item showed positive results in Ames Test (three test strains of bacteria, TA98, TA100, and TA1537, under certain experimental conditions). However, an in vitro gene mutation test, and an in vivo mammalian gene mutation test showed that the test substance is non-mutagenic. Overall, the data from the genetic toxicity tests are conclusive but not sufficient for classification.