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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in the Ames test, CHO/HPRT test and rat lymphocyte chromosomal aberration test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 July - 29 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations
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:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice.
Test concentrations with justification for top dose:
In experiment B1 (initial toxicity-mutation assay), the maximum concentration tested was 5000 µg per plate, which is the maximum concentration recommended by test guidelines. This concentration was achieved using a concentration of 100 mg/mL and a plating aliquot of 50 µL. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. No positive mutagenic responses were observed with tester strains TA100, TA1537 and WP2 uvrA in the presence of S9 activation. Precipitate was observed beginning at 50, 500, 1500 or at 5000 µg per plate. Toxicity was observed beginning at 1.5, 15, 50 or 150 µg per plate. Due to excessive toxicity (beginning at 1.5 or 15 µg per plate), all tester strains in the absence of S9 activation and tester strain TA98 in the presence of S9 activation were not evaluated for mutagenicity but were retested in experiment B2 based on the precipitate and toxicity profile observed. Tester strain TA1535 in the presence of S9 activation was not evaluated for mutagenicity due to an unacceptable positive control value and was also retested in experiment B2. Based on the findings of the initial toxicity mutation assay, the maximum doses plated in the retest and confirmatory mutagenicity assays were 2.5 µg per plate with all Salmonella tester strains in the absence of S9 activation and tester strain TA98 in the presence of S9 activation, 25 µg per plate with tester strain WP2 uvrA in the absence of S9 activation and 200 µg per plate with tester strains TA100, TA1535, TA1537 and WP2 uvrA in the presence of S9 activation.

In experiment B2 (retest of the initial toxicity-mutation assay), no positive mutagenic responses were observed with tester strains TA98, TA1535, TA1537 and WP2 uvrA in the absence of S9 activation or with tester strain TA98 in the presence of S9 activation. The concentrations tested were 0.0075, 0.025, 0.075, 0.25, 0.75 and 2.5 µg per plate with all Salmonella tester strains in the absence of S9 activation an
Vehicle / solvent:
The vehicle used to deliver BADGE-MXDA (#42) to the test system was DMSO (CAS No. 67 68-5, Lot No. 51098138, Purity: 99.95%, Expiration date: March 2015, obtained from EMD Chemicals Incorporated and Lot No. SHBB8179V, Purity: > 99.99%, Exp. Date: June 2015, obtained from Sigma-Aldrich).
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
With S9, TA98, TA100, TA1535, TA1537 and WP2 uvrA - 2 aminoanthracene, Without S9, TA98 - 2-nitrofluorene, TA100 and TA1535 - sodium azide, TA1537 - 9-aminoacridine, and WP2 uvrA - methyl methanesulfonate.
Details on test system and experimental conditions:
Test System
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2 °C for 11 to 15 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 (Lot No. 2960, Expiration date: 06 June 2014) was prepared by and purchased from Moltox. Upon arrival at BioReliance, the S9 was stored at -60 °C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2 aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose 6 phosphate, 4 mM ß nicotinamide adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar.

Solubility Test
A solubility test was conducted to determine the vehicle. The test was conducted using deionized water and DMSO to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents.

Initial Toxicity-Mutation Assay
The initial toxicity-mutation assay was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9. In the retests of the initial toxicity-mutation assay, six, seven or nine dose levels of test article were used based on precipitate and/or toxicity profile.

Confirmatory Mutagenicity Assay
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. Six or seven dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

Plating and Scoring Procedures
The test system was exposed to the test article via the preincubation methodology described by Yahagi et al. (1977).

On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L histidine, D biotin and L tryptophan solution to a final concentration of 50 µM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of deionized water for each 100 mL of minimal top agar. Bottom agar was Vogel Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).

Each plate was labeled with a code system that identified the test article, test phase, concentration, tester strain and activation, as described in detail in BioReliance's Standard Operating Procedures.

One half (0.5) milliliter of S9 or sham mix, 100 µL of tester strain (cells seeded) and 50 µL of vehicle or test article dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37±2 °C. After vortexing, these mixtures were incubated with shaking for 20±2 minutes at 37±2 °C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2 °C. Plates that were not counted immediately following the incubation period were stored at 2 - 8 °C until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification.

Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.
Evaluation criteria:
For the test article to be evaluated positive, it must cause a reproducible, concentration-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA98, TA1535, TA1537 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA100 were judged positive if the increase in mean revertants at the peak of the response was greater than or equal to 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative, if it was neither positive nor equivocal.

Criteria for a Valid Test (See below)
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Solubility Test
Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. After sonication at 31.9ºC for five minutes, the test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

Sterility Results
No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

Initial Toxicity-Mutation Assay
In experiment B1 (initial toxicity-mutation assay), the maximum concentration tested was 5000 µg per plate, which is the maximum concentration recommended by test guidelines. This concentration was achieved using a concentration of 100 mg/mL and a plating aliquot of 50 µL. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. No positive mutagenic responses were observed with tester strains TA100, TA1537 and WP2 uvrA in the presence of S9 activation. Precipitate was observed beginning at 50, 500, 1500 or at 5000 µg per plate. Toxicity was observed beginning at 1.5, 15, 50 or 150 µg per plate. Due to excessive toxicity (beginning at 1.5 or 15 µg per plate), all tester strains in the absence of S9 activation and tester strain TA98 in the presence of S9 activation were not evaluated for mutagenicity but were retested in experiment B2 based on the precipitate and toxicity profile observed. Tester strain TA1535 in the presence of S9 activation was not evaluated for mutagenicity due to an unacceptable positive control value and was also retested in experiment B2. Based on the findings of the initial toxicity mutation assay, the maximum doses plated in the retest and confirmatory mutagenicity assays were 2.5 µg per plate with all Salmonella tester strains in the absence of S9 activation and tester strain TA98 in the presence of S9 activation, 25 µg per plate with tester strain WP2 uvrA in the absence of S9 activation and 200 µg per plate with tester strains TA100, TA1535, TA1537 and WP2 uvrA in the presence of S9 activation.

In experiment B2 (retest of the initial toxicity-mutation assay), no positive mutagenic responses were observed with tester strains TA98, TA1535, TA1537 and WP2 uvrA in the absence of S9 activation or with tester strain TA98 in the presence of S9 activation. The concentrations tested were 0.0075, 0.025, 0.075, 0.25, 0.75 and 2.5 µg per plate with all Salmonella tester strains in the absence of S9 activation and tester strain TA98 in the presence of S9 activation, 0.075, 0.25, 0.75, 2.5, 7.5 and 25 µg per plate with tester strain WP2 uvrA in the absence of S9 activation and 0.25, 0.75, 2.5, 7.5, 25, 75 and 200 µg per plate with tester strain TA1535 in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 7.5 or at 200 µg per plate with several test conditions. Due to a lack of toxicity as compared to the initial trial, tester strain TA98 in the presence and absence of S9 activation and tester strains TA1535 and TA1537 in the absence of S9 activation were retested in experiment B4. Unacceptable positive control values were also observed with tester strain TA100 in the absence of S9 activation and tester strain TA1535 in the presence of S9 activation, and therefore, those test conditions were retested in experiment B4. In retrospect, the results from this assay with the Salmonella tester strains were most likely attributed to problems with the packaging of the minimal bottom agar plates that were used (i.e. erratic colony counts, inconsistent background lawn growth and unacceptable vehicle and/or positive control values).

In experiment B4 (second retest of the initial toxicity-mutation assay), no positive mutagenic responses were observed with any of the Salmonella tester strains in the absence of S9 activation or with tester strains TA98 and TA1535 in the presence of S9 activation. The concentrations tested were 0.025, 0.075, 0.25, 0.75, 2.5, 7.5, 25, 75 and 250 µg per plate. No precipitate was observed. Toxicity was observed beginning at 7.5, 25 or 75 µg per plate.

Confirmatory Mutagenicity Assay
In experiment B3 (confirmatory mutagenicity assay), no positive mutagenic responses were observed with tester strains TA98, TA1535 and WP2 uvrA in the absence of S9 activation or with any of the tester strains in the presence of S9 activation. The concentrations tested were 0.0075, 0.025, 0.075, 0.25, 0.75 and 2.5 µg per plate with all Salmonella tester strains in the absence of S9 activation and tester strain TA98 in the presence of S9 activation, 0.075, 0.25, 0.75, 2.5, 7.5 and 25 µg per plate with tester strain WP2 uvrA in the absence of S9 activation and 0.25, 0.75, 2.5, 7.5, 25, 75 and 200 µg per plate with tester strains TA100, TA1535, TA1537 and WP2 uvrA in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 25, 75 or at 200 µg per plate with several test conditions. Due to a lack of toxicity as compared to the initial trial, tester strain TA98 in the presence and absence of S9 activation and tester strain TA1535 in the absence of S9 activation were retested in experiment B5. Unacceptable positive control values were also observed with tester strains TA100 and TA1537 in the absence of S9 activation, and therefore, those test conditions were retested in experiment B5. The results from this assay with the Salmonella tester strains were most likely attributed to problems with the packaging of the minimal bottom agar plates that were used.

In experiment B5 (retest of the confirmatory mutagenicity assay), no positive mutagenic responses were observed with any of the Salmonella tester strains in the absence of S9 activation or with tester strain TA98 in the presence of S9 activation. The concentrations tested were 0.075, 0.25, 0.75, 2.5, 7.5 and 25 µg per plate with all Salmonella tester strains in the absence of S9 activation and 0.75, 2.5, 7.5, 25, 75 and 250 µg per plate with tester strain TA98 in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 7.5 or 75 µg per plate.

Dosing Formulation Analysis
Concentration analysis indicates that the actual mean concentrations of the analyzed dose levels were between 87.0 and 201.2% of their respective targets with < 20% RPD (relative percent difference). Although the actual concentrations of some of the analyzed formulations were higher than expected, the critical top concentrations were within 85 to 115% of target in each assay. This indicates that the regulatory-required top dose level was achieved. Therefore, the Study Director has concluded that the observed differences from nominal concentration had no adverse impact on the assessment of the test article’s mutagenicity and the results support the validity of the study conclusion. No test article was detected in the vehicle control samples.

Formulation stability analysis was conducted by the Sponsor. The results of the analysis indicate that BADGE-MXDA (#42) was stable in DMSO at a concentration of 25 mg/mL for four days when stored at -80 ºC and at ambient temperature, at a concentration of 11 mg/mL for 12 days when stored at -80 ºC and at ambient temperature, and at a concentration of 4.7 µg/mL for three hours when stored at ambient temperature.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1

Initial Toxicity-Mutation Assay without S9 activation

 Test Material  Dose Level per plate  Mean Revertants per plate (St. Dev.)
 TA98
   BADGE-MXDA (#42)  5000 ug  0 (0)
   1500 ug  0 (0)
   500 ug  0 (0)
   150 ug  0 (0)
   50 ug  0 (0)
   15 ug  0 (0)
   5.0 ug  0 (0)
 1.5 uL  33 (1)
 DMSO  50 uL   32 (8)
      TA100
 BADGE-MXDA (#42)  5000 ug  0 (0)
   1500 ug  0 (0)
   500 ug  0 (0)
   150 ug  0 (0)
   50 ug  0 (0)
   15 ug  0 (0)
   5.0 ug  0 (0)
   1.5 ug  82 (57)
 DMSO  50 uL  150 (44)
   TA1535   
 BADGE-MXDA (#42)  5000 ug  0 (0)
   1500 ug  0 (0)
   500 ug  0 (0)
   150 ug  0 (0)
   50 ug  0 (0)
   15 ug  0 (0)
   5.0 ug  0 (0)
   1.5 ug  16 (2)
 DMSO  50 uL  17 (2)
   TA1537   
 BADGE-MXDA (#42)  5000 ug  0 (0)
   1500 ug  0 (0)
   500 ug  0 (0)
   150 ug  0 (0)
   50 ug  0 (0)
   15 ug  0 (0)
   5.0 ug  0 (0)
   1.5 ug  11 (3)
 DMSO  50 uL  14 (1)
   WP2uvrA    
 BADGE-MXDA (#42)  5000 ug  0 (0)
   1500 ug  0 (0)
   500 ug  0 (0)
   150 ug  0 (0)
   50 ug  0 (0)
   15 ug  27 (7)
   5.0 ug  27 (4)
   1.5 ug  35 (4)
 DMSO  50 uL  30 (2)
 2NF  TA98 (1.0 ug)  570 (25)
 SA  TA100 (1.0 ug)  585 (19)
 SA  TA1535 (1.0 ug)  225 (28)
 9AAD  TA1537 (75 ug)  1438 (71)
 MMS  WP2uvrA (1000 ug)  285 (64)

2NF

SA

9AAD

MMS

2-nitrofluorene

sodium azide

9-Aminoacridine

methyl methanesulfonate

Table 2

Initial Toxicity-Mutation Assay with S9 activation

 Test Material  Dose Level per plate  Mean Revertants per plate (St. Dev.)
 TA98
   BADGE-MXDA (#42)  5000 ug  0 (0)
   1500 ug  0 (0)
   500 ug  0 (0)
   150 ug  0 (0)
   50 ug  0 (0)
   15 ug  27 (0)
   5.0 ug  47 (15)
 1.5 uL  53 (30)
 DMSO  50 uL   50 (0)
      TA100
 BADGE-MXDA (#42)  5000 ug  0 (0)
   1500 ug  0 (0)
   500 ug  0 (0)
   150 ug  0 (0)
   50 ug  27 (11)
   15 ug  141 (6)
   5.0 ug  190 (6)
   1.5 ug  179 (16)
 DMSO  50 uL  136 (88)
   TA1537   
 BADGE-MXDA (#42)  5000 ug  0 (0)
   1500 ug  0 (0)
   500 ug  0 (0)
   150 ug  0 (0)
   50 ug  19 (11)
   15 ug  14 (6)
   5.0 ug  14 (1)
   1.5 ug  20 (4)
 DMSO  50 uL  16 (2)
  WP2uvrA   
 BADGE-MXDA (#42)  5000 ug  0 (0)
   1500 ug  0 (0)
   500 ug  0 (0)
   150 ug  18 (6)
   50 ug  38 (6)
   15 ug  39 (9)
   5.0 ug  41 (1)
   1.5 ug  36 (0)
 DMSO  50 uL  38 (0)
 2AA  TA98 (1.0 ug)  361 (16)
 2AA  TA100 (2.0 ug)  412 (93)
2AA  TA1537 (1.0 ug)  51 (2)
 2AA  WP2uvrA (15 ug)  260 (83)

2AA

2-aminoanthracene

Table 3

Retest of the Initial Toxicity-Mutation Assay without S9 activation

 Test Material  Dose level per plate  Mean Revertants per plate (St. Dev.)
   TA98   
 BADGE-MXDA (#42)  2.5 ug  18 (1)
   0.75 ug  29 (10)
   0.25 ug  35 (4)
   0.075 ug  33 (1)
   0.025 ug  36 (7)
   0.0075 ug  31 (0)
 DMSO  50 uL  33 (14)
   TA1535   
 BADGE-MXDA (#42)  2.5 ug  10 (14)
   0.75 ug  22 (2)
   0.25 ug  20 (3)
   0.075 ug  10 (7)
   0.025 ug  16 (2)
   0.0075 ug  20 (9)
 DMSO  50 uL  13 (6)
   TA1537   
 BADGE-MXDA (#42)  2.5 ug  6 (3)
   0.75 ug  6 (3)
   0.25 ug  9 (1)
   0.075 ug  7 (2)
   0.025 ug  5 (0)
   0.0075 ug  6 (4)
 DMSO  50 uL  3 (2)
   WP2uvrA   
 BADGE-MXDA (#42)  25 ug  0 (0)
   7.5 ug  20 (0)
   2.5 ug  23 (4)
   0.75 ug  19 (7)
   0.25 ug  22 (13)
   0.075 ug  22 (4)
 DMSO  50 uL  18 (1)
 2NF  TA98 (1.0 ug)  422 (78)
 SA  TA1535 (1.0 ug)  172 (37)
 9AAD  TA1537 (75 ug)  471 (47)
 MMS  WP2uvrA (1000 ug)  263 (47)

2NF

SA

9AAD

MMS

2-nitrofluorene

sodium azide

9-Aminoacridine

methyl methanesulfonate

Table 4

Retest of the Initial Toxicity-Mutation Assay with S9 activation

 Test Material  Dose level per plate  Mean Revertants per plate (St. Dev.)
   TA98   
 BADGE-MXDA (#42)  2.5 ug  39 (16)
   0.75 ug  27 (7)
   0.25 ug  32 (6)
   0.075 ug  34 (4)
   0.025 ug  38 (13)
   0.0075 ug  29 (10)
 DMSO  50 uL  32 (6)
 2AA  TA98 (1.0 ug)  358 (127)

2AA

2-aminoanthracene

Table 5

Second Retest of the Initial Toxicity-Mutation Assay without S9 activation

 Test Material  Dose level per plate  Mean Revertants per plate (St. Dev.)
   TA98   
 BADGE-MXDA (#42)  250 ug  0 (0)
   75 ug  0 (0)
   25 ug  0 (0)
   7.5 ug  7 (1)
   2.5 ug  37 (1)
   0.75 ug  26 (2)
   0.25 ug  32 (6)
   0.075 ug  25 (6)
   0.025 ug  23 (0)
 DMSO  50 uL  27 (1)
   TA100   
 BADGE-MXDA (#42)  250 ug  0 (0)
   75 ug  0 (0)
   25 ug  0 (0)
   7.5 ug  72 (10)
   2.5 ug  90 (2)
   0.75 ug  94 (19)
   0.25 ug  86 (4)
   0.075 ug  89 (20)
   0.025 ug  102 (21)
 DMSO  50 uL  92 (10)
   TA1535   
 BADGE-MXDA (#42)  250 ug  0 (0)
   75 ug  0 (0)
   25 ug  0 (0)
   7.5 ug  9 (13)
   2.5 ug  11 (0)
   0.75 ug  18 (9)
   0.25 ug  16 (2)
   0.075 ug  11 (4)
   0.025 ug  12 (2)
 DMSO  50 uL  12 (1)
   TA1537   
 BADGE-MXDA (#42)  250 ug  0 (0)
   75 ug  0 (0)
   25 ug  0 (0)
   7.5 ug  5 (0)
   2.5 ug  9 (4)
   0.75 ug  5 (6)
   0.25 ug  6 (3)
   0.075 ug  8 (3)
   0.025 ug 5 (0)
 DMSO  50 uL  9 (1)
 2NF  TA98 (1.0 ug)  567 (88)
 SA  TA100 (1.0 ug)  500 (41)
 SA  TA1535 (1.0 ug)  468 (35)
 9AAD  TA1537 (75 ug)  217 (42)

2NF

SA

9AAD

2-nitrofluorene

sodium azide

9-Aminoacridine

Table 6

Second Retest of the Initial Toxicity-Mutation Assay with S9 activation

 Test Material  Dose Level per plate  Mean revertants per plate (St. Dev.)
   TA98   
 BADGE-MXDA (#42)  250 ug  0 (0)
   75 ug  22 (0)
   25 ug  36 (6)
   7.5 ug  35 (10)
   2.5 ug  39 (4)
   0.75 ug  30 (17)
   0.25 ug  46 (8)
   0.075 ug  33 (6)
   0.025 ug  30 (3)
 DMSO  50 uL  29 (12)
   TA1535   
 BADGE-MXDA (#42)  250 ug  0 (0)
   75 ug  0 (0)
   25 ug  4 (1)
   7.5 ug  13 (6)
   2.5 ug  11 (3)
   0.75 ug  7 (5)
   0.25 ug  15 (5)
   0.075 ug  8 (4)
   0.025 ug  7 (4)
 DMSO  50 uL  15 (8)
 2AA  TA98 (1.0 ug)  545 (24)
 2AA  TA1535 (1.0 ug)  58 (16)

2AA

2-aminoanthracene

Table 7

Confirmatory Mutagenicity Assay without S9 activation

 Test Material  Dose Level per plate  Mean Revertants per plate (St. Dev.)
   TA98   
 BADGE-MXDA (#42)  2.5 ug  14 (5)
   0.75 ug  24 (6)
   0.25 ug  11 (10)
   0.075 ug  26 (4)
   0.025 ug  29 (13)
   0.0075 ug  13 (6)
 DMSO  50 uL  19 (5)
   TA1535   
 BADGE-MXDA (#42)  2.5 ug  8 (2)
   0.75 ug  14 (4)
   0.25 ug  8 (3)
   0.075 ug  12 (3)
   0.025 ug  8 (4)
   0.0075 ug  13 (1)
 DMSO  50 uL  15 (4)
   WP2uvrA   
 BADGE-MXDA (#42)  25 ug  22 (4)
   7.5 ug  23 (6)
   2.5 ug  24 (5)
   0.75 ug  17 (2)
   0.25 ug  21 (7)
   0.075 ug  23 (3)
 DMSO  50 uL  21 (7)
 2NF  TA98 (1.0 ug)  439 (42)
 SA  TA1535 (1.0 ug)  265 (5)
 MMS WP2uvrA (1000 ug)  397 (21)

2NF

SA

MMS

2-nitrofluorene

sodium azide

methyl methanesulfonate

Table 8

Confirmatory Mutagenicity Assay with S9 activation

 Test Material  Dose Level per plate  Mean revertants per plate (St. Dev.)
   TA98   
 BADGE-MXDA (#42)  2.5 ug  49 (11)
   0.75 ug  46 (5)
   0.25 ug  35 (15)
   0.075 ug  34 (11)
   0.025 ug  29 (5)
   0.0075 ug  28 (11)
DMSO  50 uL  30 (6)
   TA100   
 BADGE-MXDA (#42)  200 ug  0 (0)
   75 ug  18 (16)
   25 ug  48 (3)
   7.5 ug  92 (7)
   2.5 ug  105 (15)
   0.75 ug  79 (15)
   0.25 ug  101 (15)
 DMSO  50 uL  93 (29)
   TA1535   
 BADGE-MXDA (#42)  200 ug  0 (0)
   75 ug  5 (3)
   25 ug  16 (6)
   7.5 ug  10 (4)
   2.5 ug  10 (7)
   0.75 ug  11 (5)
   0.25 ug  13 (4)
 DMSO  50 uL  12 (2)
   TA1537   
 BADGE-MXDA (#42)  200 ug  0 (0)
   75 ug  14 (4)
   25 ug  14 (5)
   7.5 ug  6 (3)
   2.5 ug  11 (3)
   0.75 ug  13 (5)
   0.25 ug  11 (1)
 DMSO  50 uL  13 (1)
   WP2uvrA  
 BADGE-MXDA (#42)  200 ug  0 (0)
   75 ug  25 (8)
   25 ug  35 (5)
   7.5 ug  25 (11)
   2.5 ug  29 (11)
   0.75 ug  30 (3)
   0.25 ug  25 (3)
 DMSO  50 uL  26 (7)
 2AA  TA98 (1.0 ug)  393 (69)
 2AA  TA100 (2.0 ug)  318 (30)
 2AA  TA1535 (1.0 ug)  38 (23)
 2AA  TA1537 (1.0 ug)  52 (18)
 2AA  WP2uvrA (15 ug)  155 (32)

2AA

2-aminoanthracene

Table 9

Retest of the Confirmatory Mutagenicity Assay without S9 activation

 Test Material  Dose Level per plate  Mean revertants per plate (St. Dev.)
   TA98   
 BADGE-MXDA (#42)  25 ug  0 (0)
   7.5 ug  0 (0)
   2.5 ug  19 (4)
   0.75 ug  26 (9)
   0.25 ug  28 (3)
   0.075 ug  34 (3)
 DMSO  50 uL  25 (4)
   TA100   
 BADGE-MXDA (#42)  25 ug  0 (0)
   7.5 ug  34 (29)
   2.5 ug  64 (6)
   0.75 ug  82 (10)
   0.25 ug  74 (10)
   0.075 ug  81 (12)
 DMSO  50 uL  81 (3)
   TA1535   
 BADGE-MXDA (#42)  25 ug  0 (0)
   7.5 ug  4 (4)
   2.5 ug  8 (2)
0.75 ug   11 (7)
   0.25 ug  10 (5)
   0.075 ug  10 (4)
 DMSO  50 uL  8 (2)
   TA1537   
 BADGE-MXDA (#42)  25 ug  0 (0)
   7.5 ug  2 (3)
   2.5 ug  7 (2)
   0.75 ug  6 (2)
   0.25 ug  7 (2)
   0.075 ug  6 (3)
 DMSO  50 uL  3 (3)
 2NF  TA98 (1.0 ug)  250 (4)
 SA  TA100 (1.0 ug)  503 (48)
 SA  TA1535 (1.0 ug)  466 (25)
 9AAD  TA1537 (75 ug)  222 (37)

2NF

SA

9AAD

2-nitrofluorene

sodium azide

9-Aminoacridine

Table 10

Retest of the Confirmatory Mutagenicity Assay with S9 activation

 Test Material  Dose Level per plate  Mean Revertants per plate (St. Dev.)
   TA98   
 BADGE-MXDA (#42)  250 ug  0 (0)
   75 ug  15 (8)
   25 ug  27 (6)
   7.5 ug  20 (6)
   2.5 ug  18 (9)
   0.75 ug  14 (5)
 DMSO  50 uL  18 (5)
 2AA  TA98 (1.0 ug)  424 (24)

2AA

2-aminoanthracene

Conclusions:
Interpretation of results (migrated information):
negative

All criteria for a valid study were met as described in the protocol. The results of the bacterial reverse mutation assay indicate that, under the conditions of this study, BADGE-MXDA (#42) was negative (non-mutagenic) both in the presence and absence of Aroclor induced rat liver S9.
Executive summary:

The test article, BADGE-MXDA (#42)Reaction product of m-phenylenebis(methylamine) with oligomerisation products of 4,4’-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane (ECnr. 500-302-7), hereafter referred to as BADGE-MXDA (#42),was tested in the bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and Escherichia coli tester strain WP2 uvrA in the presence or absence of Aroclor‑induced rat liver S9. The assay was performed in two phases using the preincubation method. The first phase, the initial toxicity‑mutation assay, was used to establish the dose‑range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. After sonication at 31.9 ºC for five minutes, the test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

In the initial toxicity-mutation assay, the maximum concentration tested was 5000 µg per plate, which is the maximum concentration recommended by test guidelines. This concentration was achieved using a concentration of 100 mg/mL and a plating aliquot of 50 µL. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. No positive mutagenic responses were observed with tester strains TA100, TA1537 and WP2 uvrA in the presence of S9 activation. Precipitate was observed beginning at 50, 500, 1500 or at 5000 µg per plate. Toxicity was observed beginning at 1.5, 15, 50 or 150 µg per plate. Due to excessive toxicity (beginning at 1.5 or 15 µg per plate), all tester strains in the absence of S9 activation and tester strain TA98 in the presence of S9 activation were not evaluated for mutagenicity but were retested based on the precipitate and toxicity profile observed. Tester strain TA1535 in the presence of S9 activation was not evaluated for mutagenicity due to an unacceptable positive control value and was also retested. Based on the findings of the initial toxicity‑mutation assay, the maximum doses plated in the retest and confirmatory mutagenicity assays were 2.5 µg per plate with allSalmonellatester strains in the absence of S9 activation and tester strain TA98 in the presence of S9 activation, 25 µg per plate with tester strain WP2uvrA in the absence of S9 activation and 200 µg per plate with tester strains TA100, TA1535, TA1537 and WP2uvrA in the presence of S9 activation.

In the retest of the initial toxicity-mutation assay, no positive mutagenic responses were observed with tester strains TA98, TA1535, TA1537 and WP2uvrA in the absence of S9 activation or with tester strain TA98 in the presence of S9 activation. The concentrations tested were 0.0075, 0.025, 0.075, 0.25, 0.75 and 2.5 µg per plate with allSalmonellatester strains in the absence of S9 activation and tester strain TA98 in the presence of S9 activation, 0.075, 0.25, 0.75, 2.5, 7.5 and 25 µg per plate with tester strain WP2uvrA in the absence of S9 activation and 0.25, 0.75, 2.5, 7.5, 25, 75 and 200 µg per plate with tester strain TA1535 in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 7.5 or at 200 µg per plate with several test conditions. Due to a lack of toxicity as compared to the initial trial, tester strain TA98 in the presence and absence of S9 activation and tester strains TA1535 and TA1537 in the absence of S9 activation were retested. Unacceptable positive control values

were also observed with tester strain TA100 in the absence of S9 activation and tester strain TA1535 in the presence of S9 activation, and therefore, those test conditions were retested. In retrospect, the results from this assay with theSalmonellatester strains were most likely attributed to problems with the packaging of the minimal bottom agar plates that were used (i.e. erratic colony counts, inconsistent background lawn growth and unacceptable vehicle and/or positive control values).

In the second retest of the initial toxicity-mutation assay, no positive mutagenic responses were observed with any of the Salmonella tester strains in the absence of S9 activation or with tester strains TA98 and TA1535 in the presence of S9 activation. The concentrations tested were 0.025, 0.075, 0.25, 0.75, 2.5, 7.5, 25, 75 and 250 µg per plate. No precipitate was observed. Toxicity was observed beginning at 7.5, 25 or 75 µg per plate.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with tester strains TA98, TA1535 and WP2uvrA in the absence of S9 activation or with any of the tester strains in the presence of S9 activation. The concentrations tested were 0.0075, 0.025, 0.075, 0.25, 0.75 and 2.5 µg per plate with allSalmonellatester strains in the absence of S9 activation and tester strain TA98 in the presence of S9 activation, 0.075, 0.25, 0.75, 2.5, 7.5 and 25 µg per plate with tester strain WP2uvrA in the absence of S9 activation and 0.25, 0.75, 2.5, 7.5, 25, 75 and 200 µg per plate with tester strains TA100, TA1535, TA1537 and WP2 uvrA in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 25, 75 or at 200 µg per plate with several test conditions. Due to a lack of toxicity as compared to the initial trial, tester strain TA98 in the presence and absence of S9 activation and tester strain TA1535 in the absence of S9 activation were retested. Unacceptable positive control values were also observed with tester strains TA100 and TA1537 in the absence of S9 activation, and therefore, those test conditions were retested. The results from this assay with theSalmonellatester strains were most likely attributed to problems with the packaging of the minimal bottom agar plates that were used.

In the retest of the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the Salmonella tester strains in the absence of S9 activation or with tester strain TA98 in the presence of S9 activation. The concentrations tested were 0.075, 0.25, 0.75, 2.5, 7.5 and 25 µg per plate with allSalmonellatester strains in the absence of S9 activation and 0.75, 2.5, 7.5, 25, 75 and 250 µg per plate with tester strain TA98 in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 7.5 or 75 µg per plate.

Dosing formulations were analyzed by the Sponsor. The analytically-determined concentrations ofBADGE‑MXDA (#42)in the confirmatory and retest dose formulations ranged from87.0 to 201.2% of their respective targets with < 20% RPD. Although the actual concentrations of some of the analyzed formulations were higher than expected, the critical top concentrations were within 85 to 115% of target. This indicates that the regulatory-required top dose level was achieved and the results support the validity of the study conclusion. Stability analysis conducted by the Sponsor indicated that BADGE-MXDA (#42) was stable in DMSO at a concentration of 25 mg/mL for four days when stored at -80 ºC and at ambient temperature, at a concentration of 11 mg/mL for 12 days when stored at -80 ºC and at ambient temperature, and at a concentration of 4.7 µg/mL for three hours when stored at ambient temperature.

All criteria for a valid study were met as described in the protocol. The vehicle controls and positive controls in the initial toxicity-mutation and confirmatory mutagenicity assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay except as indicated above in the initial toxicity-mutation, retest and confirmatory mutagenicity assays, and were retested as appropriate. The positive control values for several tester strains were lower than the acceptable ranges. Those tester strains were retested, and the positive controls were within the acceptable historical range and fulfilled the requirements for a valid assay.

Under the conditions of this study, test article BADGE-MXDA (#42) was negative (non‑mutagenic) in the bacterial reverse mutation assay.

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:
9 August - 30 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
The in vitro mammalian cell forward gene mutation (CHO/HPRT) assay was designed to select for mutant cells which have become resistant to such purine analogues as 6-thioguanine (TG) and 8-azaguanine as a result of a mutation at the X-chromosome-linked HPRT locus (O'Neill et al., 1977; Hsie et al., 1981; Machanoff et al., 1981; Li et al., 1987). HPRT is a purine salvage enzyme that allows cells to utilize hypoxanthine and guanine for DNA synthesis. It catalyzes the reaction between 5 phosphoribosyl-1 pyrophosphate and hypoxanthine or guanine to form inosine or guanosine monophosphate, respectively. If the purine analog TG is included in the growth medium, it will be phosphorylated by HPRT and incorporated into nucleic acids, resulting in cell death.

The hprt locus is located on the X chromosome. Since only one of the two X chromosomes is functional in these female-derived cells, a single-step forward mutation from hprt+ to hprt in the functional X chromosome results in loss of HPRT activity and renders the cell unable to utilize purine analogs. Such mutant cells are TGr and are assumed to have mutated, either spontaneously or as the result of treatment, to the hprt genotype. However, these mutants still are viable, since DNA synthesis can proceed by the de novo purine biosynthetic pathway that does not involve hypoxanthine or guanine.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO-K1-BH4 cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14 hours, and a cloning efficiency generally greater than 80% (Li et al., 1987). The CHO-K1-BH4 cells used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories (Oak Ridge, TN) and were stored frozen in liquid nitrogen.
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenate (S9; Lot No. 2878; expiration 07 March 2014) was obtained from Molecular Toxicology, Inc. and prepared from male Sprague Dawley rats that have been injected (intraperitoneally) with Arochlor 1254.
Test concentrations with justification for top dose:
In the cytotoxicity assay, ten two-fold dilutions from 0.391 to 200 µg/mL with and without S9 (the maximum concentration was selected based on precipitation of BADGE-MXDA (#42) upon addition to the treatment medium) were examined.

In the definitive mutagenicity assay, concentrations of 1.25, 2.5, 5, 10, 15, and 20 µg/mL with S9, and at concentrations of 1.25, 2.5, 5, 10, 15, 17.5, and 20 µg/mL without S9 were examined.

Dose formulations were analyzed by the Sponsor and were within 87.1 to 103.5% of target with a relative percent difference < 20%. No test article was detected in the vehicle control samples. Formulation stability analysis was conducted by the Sponsor. The results of the analysis indicate that BADGE-MXDA (#42) was stable in DMSO at concentrations bracketing those used herein for at least three hours at ambient temperature.
Vehicle / solvent:
The test article was diluted for use in dimethyl sulfoxide (DMSO; CAS No. 67-68-5; Lot No. SHBB7660V; purity 99.95% anhydrous; expiration February 2015; and Lot No. MKBF8188V; purity 99.98%; expiration July 2014), obtained from Sigma-Aldrich. Test article dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light. The highest dose formulation used in the preliminary solubility assessment and in the cytotoxicity assay was sonicated for approximately five minutes and gently warmed for one minute to achieve complete solubility.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: EMS with and BP with S9
Remarks:
EMS is a direct acting mutagen; it was evaluated in the absence of S9 at a concentration 0.2 µL/mL (final). B(a)P requires metabolic activation to become mutagenic; it was evaluated in the presence of S9 at a concentration of 4 µg/mL.
Details on test system and experimental conditions:
Frequency and Route of Administration
Cells were treated for 4 hours in the presence and absence of S9, by adding the test and control article formulations into the treatment medium. This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et al., 1981; Li et al., 1987).

Selection of Vehicle and Treatment Concentrations for Dose Formulations
To determine the treatment concentrations for the cytotoxicity assay, a preliminary assessment of test article solubility in vehicle, precipitate in treatment medium, pH, and osmolality was conducted and shared with the Sponsor. Dose levels for the cytotoxicity assay were selected in consultation with the Sponsor.

Solubility in Vehicle
A solubility test was conducted to determine the vehicle. The test was conducted using deionized water and DMSO to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents.

Precipitation of Dose Formulations in Treatment Medium
Test article precipitation in the treatment medium was evaluated by adding the appropriate volume of dose formulation (1% v/v) in 5.0 mL culture medium. Ten concentrations, at two-fold dilutions from 10 to 5000 µg/mL, were evaluated in duplicate vented culture flasks during a 4 hour incubation period.

pH of Dose Formulations in Treatment Medium
Concurrent with the precipitation test, the pH of the test article in treatment medium was measured. Concentrations ≥2500 µg/mL were neutralized using 1N HCl (Lot No. HC084476; expiration July 2013) obtained from EMD Chemicals Incorporated.

Osmolality of Dose Formulations in Treatment Medium
The osmolality of the vehicle, the highest dose formulation, the lowest precipitating dose formulation and the highest soluble dose formulation was measured. It has been demonstrated that alterations in the pH and osmolality of the culture medium can result in false positive responses for in vitro assays (Thilagar et al., 1984; Galloway et al., 1985).

Cytotoxicity Assay
The cytotoxicity of the test article to the test system was determined in order to allow the selection of appropriate doses to be tested in the definitive mutagenicity assay.

Design
The test article was evaluated using a 4 hour treatment with and without S9. Ten concentrations of test article, as well as the vehicle control, were evaluated in single cultures with and without S9. The test article was evaluated for cytotoxicity up to 200 µg/mL, based on solubility limitations in treatment medium. Lower test article concentrations were based on 2 fold serial dilutions from the maximum evaluated concentration.
Dose formulations were prepared on the day of use. Neutralization of the dose formulations was not necessary.

Treatment
Cells for treatment were seeded (on Day 1) in 25 cm2 flasks at a density of approximately 1 x 10(6) in 5 mL F12FCM5. Following an overnight incubation (on Day 0), the cultures were washed twice with Hank's Balanced Salt Solution (HBSS) and re-fed with 5 mL treatment medium containing test or control article and S9, as appropriate. Following addition of the test or control articles, the cultures were incubated for 4 hours.

Determination of Initial Survival (Cloning Efficiency)
After the 4-hour treatment, the treatment medium was removed, the cultures were washed twice with Ca/Mg-free HBSS (CMF-HBSS) and then trypsinized and counted (two counts were made per culture whenever counting was required throughout the study). An aliquot of cells from each culture was immediately plated at a density of 200 cells/60-mm dish in triplicate using HX F12FCM5 medium. These cloning efficiency dishes were incubated for 7 days, and the resulting colonies were fixed in methanol, stained with Giemsa and counted. The cell survival of the test article-treated groups was expressed relative to the solvent control and reported as relative cloning efficiency.

The average cloning efficiency (%) was calculated for each culture, and the cytotoxicity was expressed as the initial survival (%; relative cloning efficiency as compared to the concurrent vehicle control). In this study, however, the test article also produced a marked decrease in cell numbers after treatment. Therefore, the relative cell density was also considered in the determination of initial survival (adjusted relative survival, calculated as relative cell density x relative survival).

CHO/HPRT Mutagenicity Assay
This assay procedure was based on that described by Hsie et al. (1975) and reviewed by Hsie et al. (1981) and Li et al. (1987).

Design
BADGE-MXDA (#42) was evaluated using a 4 hour treatment with and without S9. Although six or seven concentrations initially were treated with and without S9, respectively, only five concentrations were carried through the entire assay and selection. This procedure compensates for normal variations in cellular toxicity and helps to ensure the availability of representative concentrations over a wide cytotoxicity range. Some cultures at the upper end of the concentration range were discarded prior to selection due to excessive cytotoxicity, while some treated at lower concentrations were discarded because a sufficient number of higher concentrations was available.

All test and control article concentrations were evaluated in duplicate cultures. However, to ensure a sufficient number of surviving cells was available after treatment, the highest concentration evaluated without S9 in the fourth re-test mutagenicity assay was treated in quadruplicate flasks and subsequently combined into duplicate cultures.

In the initial definitive mutagenicity assay, the Day 0 vehicle control cloning efficiencies in the presence and absence of S9 failed to meet the acceptance criteria for a valid assay. Two re-tests were conducted with and without S9 and also failed due to contamination. Ultimately, a third and fourth re-test were conducted with and without S9, respectively.

Treatment
Exponentially growing CHO-K1-BH4 cells were seeded (on Day -1) in 75-cm2 flasks at a density of approximately 4 x 10(6) in 10 mL F12FCM5 medium.

Following an overnight incubation (on Day 0), the culture medium was drawn off (see Deviations) and the cultures were re-fed with 10 mL treatment medium containing test or control article and S9, as appropriate. Following addition of the test or control articles, the cultures were incubated for 4 hours.

Subculture for Phenotypic Expression and Initial Survival
After the 4-hour treatment, the treatment medium was removed, and the cultures were washed twice with CMF-HBSS, trypsinized and counted. Cells were reseeded at a density of 1.5 x 10(6) cells/150 mm dish in duplicate using F12FCM5 medium, and 200 cells/60-mm dish in triplicate using HX-F12FCM5 medium. The 60 mm dishes were incubated for 8 days for colony development and subsequent determination of cytotoxicity as described above.

The large 150 mm dishes were subcultured for 8 days, at 2 to 3 day intervals, to maintain logarithmic growth and permit expression of induced mutants. At each subculture, the duplicate dishes were trypsinized, combined, counted, and reseeded at 1.5 x 10(6) cells/150 mm dish using F12FCM5 medium (in duplicate; duplicate dishes were used for each culture to increase the population size as well as to serve as a back-up should one dish be lost due to technical issues).

Mutant Selection
At the end of the phenotypic expression period, 2.4 x 10(6) cells from each culture were reseeded, at a density of 2 x 10(5) cells/100-mm dish (12 dishes total) in selection medium. Three 60 mm dishes also were seeded, at 200 cells/dish in HX F12FCM5, to determine the cloning efficiency of each culture. The plates were incubated for 7 or 8 days under standard conditions to allow colony development.

After the 7- or 8-day incubation period, the colonies were fixed with methanol, stained with Giemsa and counted. Mutant frequencies were expressed as the number of TGr mutants/10(6) clonable cells. The number of clonable cells was determined from the number of cells plated, with adjustments for the cloning efficiency at the time of selection.

References:
Galloway, S. M., Bean, C. L., Armstrong, M. A., Deasy, D., Kraynak, A., and Bradley, M. O. (1985). False positive in vitro chromosome aberration tests with non mutagens at high concentrations and osmolalities. Environ. Mutagen. 7 (Suppl. 3), 48-49.

Hsie, A. W., Brimer, P. A., Mitchell, T. J., and Gosslee, D. J. (1975) Dose-response relationship for ethyl methanesulfonate induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese Hamster Ovary cells, Somatic Cell Genetics, 1:247-261.

Hsie, A. W., Casciano, D. A., Couch, D. B., Krahn, D. F., O'Neil, J. P., and Whitfield, B. L. (1981) The use of Chinese Hamster Ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals, Mutation Research, 8:193-214.

Li, A. P., Carver, J. H., Choy, W. N., Hsie, A. W., Gupta, R. S., Loveday, K. S., O’Neill, J. P., Riddle, J. C., Stankowski, Jr., L. F., and Yang, L. L. (1987) A guide for the performance of the Chinese Hamster Ovary cell/hypoxanthine-guanine phosphoribosyl transferase gene mutation assay, Mutation Research, 189:135-141.

Thilagar, A. K., Kumaroo, P. V., and Kott, S. (1984): Effects of low pH caused by glacial acetic acid and hydrochloric acid on chromosomal aberrations in CHO Cells. Toxicologist 4, 51.
Evaluation criteria:
Criteria for Determination of a Valid Test
An assay was considered acceptable for evaluation of test results only if all of the following criteria were satisfied.

Vehicle Control Values
The average absolute cloning efficiency of vehicle controls must be >60% (at initial survival and selection). In addition, the average spontaneous mutant frequency of the vehicle controls must be within reasonable limits of the laboratory historical control values and literature values. Spontaneous mutant frequencies were calculated separately for cultures treated with and without S9.

Positive Control Values
The positive controls must induce a significant increase in mutant frequency as compared to the concurrent vehicle controls (p 0.01). A significant increase in the absence of S9 indicates that the test system can identify a mutagen, while a significant increase in the presence of S9 was considered to have demonstrated the integrity of the S9 mix, as well as the ability of the test system to detect a mutagen.

Acceptable High Concentration
The highest concentration evaluated must be 5000 μg/mL, the maximum able to be administered based upon the limit of solubility in the vehicle and/or treatment medium, or that which induces 10 to 20% relative survival (whichever is lowest; however, if increasing cytotoxicity was observed at precipitating concentrations, cytotoxicity was the determining factor). There was no maximum concentration or toxicity requirement for test articles which clearly show mutagenic activity.

Continued below
Statistics:
Statistical analyses were performed using the method ofSnee and Irr (1981), with significance established at the 0.05 level.

Reference:
Snee, R.D. and Irr, J. D. (1981) Design of a statistical method for the analysis of mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase locus of cultured Chinese Hamster Ovary cells, Mutation Research, 85:77-93.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Excessive cytotoxicity was observed at concentrations ≥100 µg/mL with S9 and ≥25 µg/mL without S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Solubility Test
A preliminary assessment of test article solubility in the vehicle, precipitation in treatment medium, pH and osmolality was conducted to determine the treatment concentrations for the cytotoxicity assay. After sonication and gentle warming for five minutes in the solubility test, BADGE-MXDA (#42) was found to be freely soluble in DMSO at a concentration of ~500 mg/mL (the test article was insoluble in deionized water at concentrations of approximately 10 to 50 mg/mL). Therefore, DMSO was selected as the vehicle of choice based on the solubility of the test article and compatibility with the test system cells.

Solubility of the test article in treatment medium was determined in duplicate flasks at ten two fold dilutions from 10 to 5000 µg/mL during a 4 hour incubation period. BADGE-MXDA (#42) precipitated from solution in the aqueous treatment medium at concentrations ≥157 µg/mL at the beginning of treatment and at concentrations ≥ 40 µg/mL by the end of treatment. Concentrations of 2500 and 5000 µg/mL had a pH of 8.1 and 8.3, respectively, and were neutralized using 1N HCl; all other concentrations had pH values ranging from 7.0 to 7.4. There was no adverse effect on osmolality (osmolality of the duplicate vehicle controls averaged 447 mmol/kg, while the three test article concentrations analyzed averaged 378 450 mmol/kg).

Cytotoxicity Assay
Based on the preliminary solubility testing, BADGE-MXDA (#42) was evaluated in the cytotoxicity assay at ten two-fold dilutions from 0.391 to 200 µg/mL with and without S9 [the maximum concentration was selected based on precipitation of BADGE-MXDA (#42) in treatment medium]. All test article concentrations, as well as the vehicle control, were evaluated in single cultures. BADGE-MXDA (#42) again was found to precipitate from solution at a concentration of 200 µg/mL at the beginning and end of treatment. Excessive cytotoxicity was observed at concentrations ≥100 µg/mL with S9 and ≥25 µg/mL without S9 (Table 1). Relative cloning efficiencies at the remaining concentrations ranged from 39.7 to 108.7% with S9 and 70.3 to 107.6% without S9. Due to decreases in the cell densities at the end of treatment, adjusted relative survivals were also calculated and used to determine cytotoxicity. Adjusted relative survivals at the highest remaining concentrations (i.e., 50 µg/mL with S9 and 12.5 µg/mL without S9) were 20.8 and 27.0%, respectively.

Definitive Mutagenicity Assay
Based on the results of the cytotoxicity assay, the test article initially was evaluated in the definitive mutagenicity assay at concentrations of 5, 10, 20, 40, 50, 60, and 70 µg/mL with S9, and 1.25, 2.5, 5, 10, 12.5, 15, and 20 µg/mL without S9. All test article concentrations, as well as the positive and vehicle controls, were evaluated in duplicate cultures. BADGE-MXDA (#42) again was found to precipitate from solution at concentrations ≥10 µg/mL by the end of treatment. However, the Day 0 vehicle control cloning efficiencies in the presence and absence of S9 failed to meet the acceptance criteria for a valid assay (not shown), and the assay was repeated.

BADGE-MXDA (#42) was re-evaluated (twice) at concentrations of 1.25, 2.5, 5, 10, 15, and 20 µg/mL with S9, and 1.25, 2.5, 5, 10, 15, 17.5, and 20 µg/mL without S9 (the concentrations were adjusted slightly based upon precipitation observations in the previous trial). All test article concentrations, as well as the positive and vehicle controls, were evaluated in duplicate cultures. BADGE-MXDA (#42) again was found to precipitate from solution at concentrations ≥15 µg/mL by the end of treatment. However, both re-tests were terminated due to extensive microbial contamination.
BADGE-MXDA (#42) was re-evaluated under identical conditions as the mutagenicity assay above (independently with or without S9). All test article concentrations, as well as the positive and vehicle controls, were evaluated in duplicate cultures. The test article again was found to precipitate from solution at concentrations ≥10 µg/mL by the end of treatment. Those cultures treated at a concentration of 1.25 µg/mL with and without S9 were discarded prior to selection or excluded from evaluation of mutagenicity because a sufficient number of higher concentrations was available; cultures treated at a concentration of 20 µg/mL without S9 were excluded from evaluation of mutagenicity due to excessive cytotoxicity as assessed by adjusted relative survival (Table 2 and Table 3). Average relative cloning efficiencies at the highest remaining concentrations were 69.9% (at 20 µg/mL with S9) and 31.1% (at 17.5 µg/mL without S9). Corresponding average adjusted relative survivals were 74.8 and 9.3%, respectively. No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). In contrast, the benzo(a)pyrene and ethyl methanesulfonate positive controls induced significant increases in mutant frequencies (p< 0.01).

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met (the highest concentrations evaluated met or exceeded the cytotoxicity or solubility limits for this assay). In addition, all observed mutant frequencies in the test article-treated cultures were well within historical vehicle control ranges (all were below the historical means; Appendix I) up to a precipitating concentration. Based on the lack of mutagenic activity, the results of the study are considered to be conclusive and an independent confirmatory assay was not warranted.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Summary Results for BADGE-MXDA (#42) HPRT  

Cytotoxicity Assay ±S9

   Dose Level       Cloning Efficiency  Density  Adjusted Relative
 Treatment  (ug/ml)  S9  Average  %RCEa  Cells/ml (x106)  Survival (%)
 DMSO  10b  -  190.5  100.0  0.128  100.0
 BADGE-MXDA (#42)  0.391  134.0  70.3  0.102  56.3
   0.782  -  179.7  94.3  0.108  79.8
   1.56  -  205.0  107.6  0.123  103.4
   3.13  -  155.7  81.7  0.118  75.6
   6.25  -  163.0  85.6  0.096  64.0
   12.5  -  141.3  74.2  0.047  27.0
   25  -  5.3  2.8  0.020  0.4
   50  -  12.7  6.6  0.025  1.3
   100  -  3.0  1.6  0.026  0.3
   200p  -  0.7  0.3  0.030  0.1
             
 DMSO  10b  +  175.3  100.0  0.178  100.0
 BADGE-MXDA (#42)  0.391  +  152.7  87.1  0.130  63.7
   0.782  +  154.0  87.8  0.105  51.6
   1.56  +  163.0  93.0  0.105  54.7
   3.13  +  177.5  101.2  0.182  103.9
   6.25  +  170.7  97.3  0.132  72.5
   12.5  +  190.7  108.7  0.114  69.7
   25  +  117.0  66.7  0.145  54.4
   50  +  69.7  39.7  0.093  20.8
   100  +  0.0  0.0  0.099  0.0
   200p +  0.0  0.0  0.006  0.0

a %RCE = Percent relative cloning efficiency as compared to the solvent control.

b Dose level is expressed uL/ml.

p Test article precipitated from solution by end of treatment.

Table 2. Summary Results for BADGE-MXDA (#42) HPRT

Definitive Mutagenicity Assay –S9

 Treatment  Dose level     Cloning Efficiency (Inital Survival)  Cell Density  AdjustedRelative     Absolute Cloning Efficiency  Mutant  ReplicateAverage
   (ug/ml)  Average  %RCEa  Cells/ml (x 106)  Survival (%)  Mean  %ACEc  Frequencyd(x 106) Mutant Frequency(x 106
 DMSO  10e  130.3  91.0  0.369  81.3  125.3  62.7  0.66  
   10e  156.0  109.0  0.450  118.7  139.7  69.8  0.60  0.63
 BADGE-MXDA (#42)  1.25  113.3  79.2  0.398  76.2  106.7  53.3  0.00  
   1.25  128.0  89.4  0.466  100.9  118.7  59.3  0.70  0.35h
   2.5  115.3  80.6  0.337  65.7  131.3  65.7  1.27  
   2.5  113.7  79.4  0.364  69.9  94.3  47.2  1.77  1.52
   5  116.7  81.5  0.234  46.1  131.0  65.5  0.00  
   5  117.3  82.0  0.199  39.5  104.0  52.0  0.80  0.40
   10p  93.7  65.4  0.188  29.8  104.7  52.3  0.00  
   10p  94.0  65.7  0.142  22.6  87.3  43.7  1.91  0.95
   15p  78.0  54.5  0.212  27.9  114.0  57.0  0.73  
   15p  55.0  38.4  0.170  15.8  103.7  51.8  0.00  0.37
   17.5p  46.3  32.4  0.125  9.8  124.0  62.0  0.67  
   17.5p  42.7  29.8  0.121  8.7  110.7  55.3  3.01  1.84
   20p  34.3  24.0  0.072  4.2  92.3  46.2  0.90  
   20p  34.0  23.7  0.097  5.6  112.7  56.3  0.00  0.45g
 EMSf  0.2e  56.3  39.3  0.356  33.9  75.7  37.8  215.86  
   0.2e  64.0  44.7  0.363  39.2  83.7  41.8  144.42  180.14i

a %RCE = Percent relative cloning efficiency as compared to the solvent control.

c %ACE = Percent absolute cloning efficiency as compared to the number of cells plated at the time of selection

d Mutant Frequency = Number of TGf mutant/106 clonable cells.

e Dose level is expressed in ul/ml.

f EMS (positive control) = Ethylmethanesulfonate

g Excluded from evaluation of mutagenicity due to excessive cytotoxicity.

i Significant increase in mutant frequency as compared to the concurrent vehicle control values (p<0.01).

p Precipitate at end of treatment.

Table 3. Summary Results for BADGE-MXDA (#42) HPRT

Definitive Mutagenicity Assay +S9

Treatment  Dose level     Cloning Efficiency (Inital Survival)  Cell Density  AdjustedRelative     Absolute Cloning Efficiency  Mutant  ReplicateAverage
   (ug/ml)  Average  %RCEa  Cells/ml (x 106)  Survival (%)  Mean  %ACEc  Frequencyd(x 106) Mutant Frequency(x 106
 DMSO  10e  168.0  104.3  0.439  110.8  122.7  61.3  1.36  
   10e  154.0  95.7  0.386  89.2  152.0  76.0  0.55  0.95
 BADGE-MXDA (#42)  1.25  130.0  80.7  0.433  84.5  g  g  g  
   1.25  120.0  74.5  0.447  80.4  g  g  g  g
   2.5  144.0  89.4  0.404  87.3  102.0  51.0  0.82  
   2.5  141.7  88.0  0.419  89.0  124.3  62.2  1.34  1.08
   5  140.3  87.2  0.415  87.3  106.0  53.0  0.00  
   5  124.7  77.4  0.454  84.8  121.7  60.8  2.05  1.03
   10p  146.0  90.7  0.418  91.6  144.3  72.2  1.15  
   10p  151.7  94.2  0.414  94.2  120.7  60.3  1.38  1.27
   15p  154.3  95.9  0.478  110.6  102.7  51.3  0.00  
   15p  159.7  99.2  0.469  112.3  89.3  44.7  0.93  0.47
   20p  117.0  72.7  0.436 76.6  95.3  47.7  0.00  
   20p  108.0  67.1  0.450  73.0  115.3  57.7  0.00  0.00
 B(a)Pf  4  58.3  36.2  0.436  38.2  109.0  54.5  109.33  
   4  69.0  42.9  0.401  41.5  113.0  56.5  115 .04  112.19h

a%RCE = Percent relative cloning efficiency as compared to the solvent control.

c%ACE = Percent absolute cloning efficiency as compared to the number of cells plated at the time of selection

dMutant Frequency = Number of TGfmutant/106clonable cells.

eDose level is expressed in ul/ml.

fB(a)P (positive control) = Benzo (a) Pyrene

gExcluded from evaluation of mutagenicity due to excessive cytotoxicity.

iSignificant increase in mutant frequency as compared to the concurrent vehicle control values (p<0.01).

pPrecipitate at end of treatment.

Conclusions:
Interpretation of results (migrated information):
negative

These results indicate BADGE-MXDA (#42) was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures under the conditions, and according to the criteria of the test protocol.
Executive summary:

BADGE-MXDA (#42)Reaction product of m-phenylenebis(methylamine) with oligomerisation products of 4,4’-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane (ECnr. 500-302-7), hereafter referred to as BADGE-MXDA (#42),was evaluatedfor its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).

Based upon preliminary solubility testing,BADGE-MXDA (#42)was prepared in dimethyl sulfoxide (DMSO) and evaluated in the cytotoxicity assayat ten two-fold dilutions from 0.391 to 200 µg/mL with and without S9 (the maximum concentration was selected based on precipitation ofBADGE-MXDA (#42)upon addition to the treatment medium). All test article concentrations, as well as the vehicle control, were evaluated in single cultures. The test article was found to precipitate from solution at a concentration of 200 µg/mLat the beginning and end of treatment. Excessive cytotoxicity was observed at concentrations ≥100 µg/mL with S9 and ≥25 µg/mL without S9. Relative cloning efficiencies at the remaining concentrations ranged from 39.7 to 108.7% with S9 and 70.3 to 107.6% without S9. Due to decreases in the cell densities at the end of treatment, adjusted relative survivals were also calculated and used to determine cytotoxicity. Adjusted relative survivals at the highest remaining concentrations (i.e.,50 µg/mL with S9 and 12.5 µg/mL without S9) were 20.8 and 27.0%, respectively.

Based on these results and observations from an initial failed assay,BADGE‑MXDA (#42)was evaluated in the definitive mutagenicity assay at concentrations of 1.25, 2.5, 5, 10, 15, and 20 µg/mL with S9, and at concentrations of 1.25, 2.5, 5, 10, 15, 17.5, and 20 µg/mL without S9. All test article concentrations, as well as the positive and vehicle controls, were evaluated in duplicate cultures. BADGE‑MXDA (#42)again was found to precipitate from solution at concentrations ≥10 µg/mLby the end of treatment. Those cultures treated at a concentration of 1.25 µg/mL with and without S9 were discarded prior to selection or excluded from evaluation of mutagenicity because a sufficient number of higher concentrations was available; cultures treated at a concentration of 20 µg/mL without S9 were excluded from evaluation of mutagenicity due to excessive cytotoxicity as assessed by the adjusted relative survival. The average relative cloning efficiencies at the highest remaining concentrations were 69.9% (at 20 µg/mL with S9) and 31.1% (at 17.5 µg/mL without S9). Corresponding average adjusted relative survivals were 74.8 and 9.3%, respectively. No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). In contrast, thebenzo(a)pyrene andethyl methanesulfonate positive controls induced significant increases in mutant frequencies (p< 0.01). 

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met (the highest concentrations evaluated met or exceeded the cytotoxicity or solubility limits for this assay).

Analysis of the dose formulations indicated that all were within 87.1 to 103.5% of target with a relative percent difference <20%. 

Stability analysis conducted by the Sponsor indicated that BADGE-MXDA (#42) was stable in DMSO at concentrations bracketing those used herein for at least three hours at ambient temperature.

These results indicate BADGE-MXDA (#42) was negative in theIn VitroMammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures under the conditions, and according to the criteria of the test protocol.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August - 29 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: METI: Act No. 117, Ministerial Ordinance No.1-3, Enforced April 2004
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosome aberration
Species / strain / cell type:
lymphocytes: rat
Details on mammalian cell type (if applicable):
Male Sprague-Dawley rats (aged approximately 11 weeks) were obtained from Harlan (Frederick, MD) for whole blood collection. The rats were anesthetized by exposure to 70%CO2/30%O2 and blood was collected via cardiac puncture.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 (Lot No. 2877, expiration date: 24 January 2014) was obtained from Molecular Toxicology, Inc. (Boone, NC).
Test concentrations with justification for top dose:
In the Initial Chromosome Aberration Assay, concentrations of 1, 2.5, 4.5, 9, 20, 40, 80, 160, 200 µg/mL were tested in the non-activated (4 and 24 hour) and S9-activated assays.

In the Repeat Chromosome Aberration Test, concentrations of 10, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, and 40 µg/mL were tested in the non-activated (4 and 24 hour) and S9-activated assays.

Dose Formulation Analysis
Test article dose formulations were analyzed by The Dow Chemical Company (Midland, MI). The results of the analysis indicates that the actual mean concentrations of the analyzed dose levels were between 90.1% and 107.1% of their respective targets with < 20% RPD (relative percent difference). This indicates that the regulatory-required top dose level was achieved and the results support the validity of the study conclusion. No test article was detected in the vehicle control sample.

Formulation stability analysis was conducted by the Sponsor. The stability data indicate that BADGE-MXDA (#42) was stable in DMSO at a concentration of 25 mg/mL for four days when stored at -80 ºC and at ambient temperature, and at a concentration of 4.7 µg/mL for three hours when stored at ambient temperature.
Vehicle / solvent:
DMSO (CAS No.: 67-68-5, Lot No. 51098138, expiration date: January 2015 and Lot No. 51283202, expiration date: August 2015) obtained from EMD Chemicals.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Mitomycin C (MMC; CAS No. 50-07-7, Lot No. 089K0731, Expiration date: 31 Mar 2014) and cyclophosphamide (CP; CAS No. 6055-19-2, Lot No. 120M1253V, expiration date: 31 Dec 2013) was obtained from Sigma-Aldrich.
Remarks:
Mitomycin C was used in the non-activated test system at concentrations of 50 and 75 µg/mL for the non-activated 4-hour treatment or 5, 7.5 or 11.25 µg/mL for the 24-hour treatment. Cyclophosphamide was used at 0.2, 0.4, and 0.6 mg/mL in S9 test.
Details on test system and experimental conditions:
Solubility Test
A solubility test was conducted to determine the vehicle. The test was conducted using water and DMSO to determine the vehicle, selected in order of preference that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents.

Solubility Assessment in Treatment Medium, Osmolality and pH Measurements
Test article precipitate in the treatment medium was evaluated by adding appropriate volume of stock dosing formulations (1% v/v) to 5.0 mL treatment medium at a target dose level of 5000 µg/mL and nine 2-fold dilutions. Each concentration was tested in duplicate using loosely fitted non vented culture flasks. Precipitate was evaluated at the beginning and at the end of the 4 hour and 24-hour time period by visual observation.
The pH of each test article dose level in the treatment medium was measured. When necessary, pH was adjusted using 1N HCl to maintain a neutral pH in the treatment medium. The osmolality in treatment medium of the vehicle, the highest test article dose level, the lowest precipitating test article dose level and the highest soluble test article dose level was measured. The osmolality of the test article dose levels was compared to that of the vehicle control to confirm non-excessive values (> 20% of vehicle control culture). It has been demonstrated that alterations in the pH and osmolality of the culture medium can result in false positive responses for in vitro chromosomal aberrations assays (Thilagar et al., 1984; Galloway et al., 1985).

Chromosome Aberration Assays
The initial and repeat chromosome aberration assays were performed by exposing duplicate cultures of rat lymphocytes to nine or ten concentrations of the test article as well as positive and vehicle controls. The dividing cells were harvested at approximately 24 hours from the initiation of treatment.

For the initial chromosome aberration assay, 40 mL of whole blood (of the 40 mL obtained) was mixed with 400 mL of RPMI-1640 complete medium [RPMI-1640 containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 25 mM HEPES, 100 units penicillin/mL, 100 µg streptomycin/mL] in proportion to 0.5 mL of whole blood per 5 mL of RPMI-1640 complete medium supplemented with 20 µg/mL phytohemagglutinin (PHA). For the repeat assay, 45 mL of whole blood (of the 45 mL obtained) was mixed with 450 mL of RPMI-1640 complete medium in proportion to 0.5 mL of whole blood per 5 mL of RPMI 1640 complete medium supplemented with 20 µg/mL PHA. The cultures were inoculated into flasks and then incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air for 44-48 hours. At the time of test article treatment, all treatment cultures were transferred to 15 mL tubes labeled with the study number, treatment condition, and concentration. The tubes were centrifuged at approximately 1200 rpm for about 5 minutes and the supernatant was carefully aspirated with care taken not to disturb the cell pellet. The cells were resuspended in either 5 mL of fresh RPMI-1640 serum-free medium for the non activated 4 hour treatment, 5 mL of fresh RPMI-1640 complete medium for the non activated 24-hour treatment, or 5 mL of the S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 µL of the test article dosing solution or positive control in vehicle or vehicle alone. The cultures for the non-activated 24 hour treatment were then transferred to the corresponding flasks for incubation. The pH of the highest test article dosing solution was measured in the treatment medium.
In the non-activated studies, the cells were exposed for 4 or 24 hours at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air. For the 4-hour exposure group, after the exposure period, the treatment medium was removed, the cells were washed with PBS-CMF (Phosphate buffered saline – Calcium Magnesium free), re-fed with complete medium, added to the corresponding flask, and returned to the incubator for an additional 20 hours. Two hours prior to the scheduled cell harvest at 24 hours after treatment initiation, Colcemid® was added to the cultures at a final concentration of 0.2 µg/mL. The 24-hour exposure group treatment was continuous until the time of cell collection and two hours prior to the scheduled cell harvest (24 hours after treatment initiation), Colcemid® was added to the cultures at a final concentration of 0.2 µg/mL.

For the S9-activated studies, the cells were exposed for 4 hours at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air. After the exposure period, prior to harvest, the treatment medium was removed and the cells were washed with PBS-CMF, re-fed with complete medium, added to the corresponding flask, and returned to the incubator for an additional 20 hours. Two hours prior to the scheduled cell harvest at 24 hours after treatment initiation, Colcemid® was added to the cultures at a final concentration of 0.2 µg/mL.

Collection of Metaphase Cells
Two hours after the addition of Colcemid®, metaphase cells were harvested for both the activated and non-activated studies. At the end of the 2 hour Colcemid® treatment, the cultures were transferred to 15 mL centrifuge tubes labeled with study number, treatment condition, test phase, dose level, and activation system. The cells were collected by centrifugation at approximately 1200 rpm for about 5 minutes. The cell pellet was resuspended in 5 mL 0.075 M KCl and incubated at 37 ± 1°C for 20 minutes. At the end of the KCl treatment and immediately prior to centrifuging, the cells were gently mixed, and approximately 0.5 mL of fixative (methanol:glacial acetic acid, 3:1 v/v) was added to each tube. The tubes were centrifuged for about 5 minutes, the supernatant was aspirated, the cell pellet was disrupted and 3-5 mL of fixative was added slowly to each tube while maintaining gentle agitation by flicking the tube with the fingers. The cells were centrifuged again for about 5 minutes, the supernatant was aspirated, the cells were resuspended in 3-5 mL of fresh fixative, centrifuged again for 5 minutes at 1200 rpm. The supernatant was aspirated and the cells were resuspended in 3-5 mL of fresh fixative and stored in fixative overnight or longer at approximately 2-8°C.

Slide Preparation
To prepare slides, the fixed cells in 15 mL centrifuge tubes were centrifuged for about 5 minutes at approximately 1200 rpm. The supernatant was aspirated, leaving approximately 0.2 mL of fixative above the cell pellet, and 1 mL of cold fresh fixative was added to each tube. The cells were collected by centrifugation, and the supernatant was aspirated, leaving 0.1 to 0.3 mL of fixative above the cell pellet (the volume depended upon size of the cell pellet). One to two drops of the cell suspension were dropped from an appropriate distance by means of a Pasteur pipette on clean microscope slides, and the slides were allowed to air dry at room temperature. Each slide was identified by the study number, treatment condition, activation system, harvest date, and duplicate tube designation. The dried slides were stained with 5% Giemsa, air dried, and permanently mounted.

Selection of Dose Levels for Analysis
The highest dose selected for microscopic analysis was the one which induced approximately 60 ± 10% reduction in the mitotic index. Two additional lower dose levels were included in the evaluation.

Evaluation of Metaphase Cells
The mitotic index was recorded as the percentage of cells in mitosis per 1000 cells counted. Slides were coded using random numbers by an individual not involved with the scoring process. Initially, slides from the short treatment (with and without S9) were evaluated for cytogenetic analysis. Slides from the continuous treatment without S9 were evaluated only when the results with short treatment yielded negative findings. Metaphase cells with 42 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate treatment condition) were examined and scored for structural abnormalities (Buckton and Evans, 1973; Sinha et al., 1984; Gollapudi et al., 1986; Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate tube was reduced to 50 metaphases when the percentage of aberrant cells reached a significant level (at least 20%) before 100 cells were scored. The microscopic coordinates of metaphases containing aberrations were recorded. Only those metaphases that contain 42 ± 2 centromeres were scored with the exception of cells with multiple aberrations, in which case accurate counts of the centromeres may not be possible. Structural chromosomal abnormalities included chromatid and chromosome gaps, chromatid breaks and exchanges, chromosome breaks and exchanges, and miscellaneous (chromosomal disintegration, chromosomal pulverization, etc.). Cells having ten or more aberrations/cell were classified as cells with multiple aberrations or severely damaged cells. Chromatid gaps and chromosome gaps were not included in calculations of total cytogenetic aberrations. The percentage of cells with numerical aberrations (polyploidy and endoreduplicated cells) was evaluated per 100 cells per duplicate culture.

Controls
MMC was used as the positive control at final concentrations of 0.5 and 0.75 µg/mL in the non activated 4-hour exposure group; and 0.05, 0.075, or 1.125 µg/mL in the non-activated 24 hour exposure group. CP was used as the positive control in the S9-activated study at final concentrations of 2, 4, and 6 µg/mL. For both positive controls, one dose level exhibiting a sufficient number of scorable metaphase cells was selected for analysis. The vehicle for the test article was used as the vehicle control at the same concentration as that used for the test article treated groups.

References:
Buckton, K.E. and Evans, H.J. (eds.) (1973). Methods for the analysis of human chromosome aberrations. World Health Organization, Geneva.

Galloway, S. M., Bean, C. L., Armstrong, M. A., Deasy, D., Kraynak, A., and Bradley, M. O. (1985). False positive in vitro chromosome aberration tests with non mutagens at high concentrations and osmolalities. Environ. Mutagen. 7 (Suppl. 3), 48-49.

Gollapudi, B.B., Sutcliffe, D.J., and Sinha, A.K. (1986). Assessment of cytogenetic response to folic acid deprivation in rat lymphocytes. In Vitro, 22, 681-684.

Scott, D., Dean, B.J., Danford, N.D., and Kirkland, D.J. (1990). Metaphase Chromosome aberration assays. In Basic Mutagenicity Tests: U.K. EMS Recommended Procedures, (D.J. Kirkland Ed.), Cambridge University Press, New York, NY.

Sinha, A.K., Linscombe, V.A., Gollapudi, B.B., McClintock, M.L., Flake, R.E. and Bodner, K.M. (1984). The incidence of spontaneous cytogenetic aberrations in lymphocytes cultured from normal humans for 48 and 72 hours, Can. J. Genet. Cytol, 26, 528-531.

Thilagar, A. K., Kumaroo, P. V., and Kott, S. (1984): Effects of low pH caused by glacial acetic acid and hydrochloric acid on chromosomal aberrations in CHO Cells. Toxicologist 4, 51.
Evaluation criteria:
The toxic effects of treatment were based upon mitotic inhibition relative to the vehicle treated control and are presented for the chromosome aberration assays. The number and types of aberrations per cell, the percentage of structurally and numerically damaged cells (percent aberrant cells), and the frequency of structural aberrations per cell (mean aberrations per cell) in the total population of cells examined was calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.

All conclusions were based on sound scientific judgment; however, as a guide to interpretation of the data, a test article would be considered to induce a positive response when the percentage of cells with aberrations was increased in a dose-responsive manner, with one or more concentrations being statistically significant and clearly outside the historical vehicle control data (p
In the event of a positive response only at the high dose level(s) with approximately 60 ± 10% reduction in the mitotic index with adequate scorable cells relative to the respective vehicle control and demonstrates no evidence of dose response in one or more treatment conditions, the test article was considered to induce positive response at the cytotoxic dose level.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher's Exact test. Fisher's Exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. In the event of a positive Fisher's Exact test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness (Armitage, 1971).

Reference
Armitage, P. (1971). Statistical Methods in Medical Research. John Wiley & Sons, Inc., New York, NY.
Species / strain:
lymphocytes: rat
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Solubility Test
Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test article and compatibility with the target cells. Upon sonication for five minutes at 31.9 ºC, the test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested for solubility.

Solubility Assessment in Vehicle and Treatment Medium
In the solubility assessment conducted by the Cytogenetic lab at BioReliance, the test article was soluble in DMSO at all concentrations tested. Visible precipitate was observed at dose levels >/=156 µg/mL, while the dose levels /=39 µg/mL, while the dose levels
The osmolality in treatment medium of the highest dose formulation assessed for solubility, 5000 µg/mL, was 397 mmol/kg (Flasks A) and 394 (Flask B). The osmolality in treatment medium of the lowest precipitating dose, 156 µg/mL, was 393 mmol/kg (Flask A) and 395 mmol/kg (Flask B). The osmolality in the treatment medium of the highest soluble dose formulation, 78 µg/mL, was 411 mmol/kg (Flask A) and 409 mmol/kg (Flask B). The osmolality of the vehicle (DMSO) in the treatment medium was 408 mmol/kg (Flask A) and 410 mmol/kg (Flask B). The osmolality of the test article dose formulations in the treatment medium are acceptable because they did not exceed the osmolality of the vehicle by more than 20%. The pH of the solvent and that of all dose levels were measured as follows in Table 1.

The pH of the three highest doses was adjusted using 1N HCl to maintain a neutral pH in the treatment medium. Based on the solubility, pH, osmolality, and precipitate tests, the dose levels tested in the initial chromosome aberration assay were as follows in Table 2.

Initial Chromosome Aberration Assay
In the initial chromosome aberration assay, the test article was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels >/= 80 µg/mL, while dose levels /= 80 µg/mL in all three treatment conditions. The pH of the highest dose level of test article in treatment medium was 7.0. Substantial toxicity (approximately 60 ± 10% reduction in mitotic index relative to the vehicle control) was observed at dose levels >/= 40 µg/mL in all three exposure groups. However, cytotoxicity as required by the testing guidelines was not achieved; therefore, the chromosome aberration assay was repeated in its entirety at dose levels of 10, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, and 40 µg/mL. Cytotoxicity data from the initial assay are presented in Tables 3, 4, and 5.

Repeat Chromosome Aberration Assay
In the repeat assay, the test article was soluble in DMSO and in the treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. At the conclusion of the treatment period, hemolysis was observed at dose levels >/= 20 µg/mL in the non activated 24-hour exposure group. The pH of the highest dose level of test article in treatment medium was 7.0

The findings of the cytogenetic analysis of the non-activated 4-hour exposure group are summarized by group in Table 6. At the highest test concentration evaluated microscopically for chromosome aberrations, 25 µg/mL, mitotic index was 58% reduced, relative to the vehicle control. The frequency of cells with structural aberrations in the vehicle control was 0.5% and the corresponding values at doses of 10, 22.5, and 25 µg/mL were 0.0%, 0.5%, and 0.0%, respectively. Statistical analyses of these data did not identify significant differences between the vehicle control and any of the treated cultures without S9 (p > 0.05, Fisher's Exact test). The percentage of structurally damaged cells in the MMC (positive control) group (20.0%) was statistically significant (p 0.05, Fisher’s Exact test).

The findings of the cytogenetic analysis of the S9-activated 4-hour exposure group are summarized by group in Table 6. At the highest test concentration evaluated microscopically for chromosome aberrations, 27.5 µg/mL, mitotic index was 57% reduced, relative to the vehicle control. The frequency of cells with structural aberrations in the vehicle control was 0.0% and the corresponding values at doses of 10, 20, and 27.5 µg/mL were 0.0%, 0.0%, and 0.5%, respectively. Statistical analyses of these data did not identify significant differences between the vehicle control and any of the treated cultures with S9 (p > 0.05, Fisher's Exact test). The percentage of structurally damaged cells in the CP (positive control) group (17.0%) was statistically significant (p 0.05, Fisher’s Exact test).

The findings of the cytogenetic analysis of the non-activated 24-hour exposure group are summarized by group in Table 6. At the highest test concentration evaluated microscopically for chromosome aberrations, 25 µg/mL, mitotic index was 54% reduced, relative to the vehicle control. The frequency of cells with structural aberrations in the vehicle control was 0.5% and the corresponding values at doses of 10, 20, and 25 µg/mL were 0.5%, 0.0%, and 1.0%, respectively. Statistical analyses of these data did not identify significant differences between the vehicle control and any of the treated cultures without S9 (p > 0.05, Fisher's Exact test). The percentage of structurally damaged cells in the MMC (positive control) group (17.0%) was statistically significant (p 0.05, Fisher’s Exact test).

Thus, the chromosome aberration assay indicates no significant increase in structural or numerical chromosome aberrations in the test article-treated groups relative to the respective vehicle controls in both the absence and the presence of the S9 metabolic activation system. The percentages of aberrant cells in the test article-treated groups were within the historical vehicle control range. Based on these criteria, the negative result is justified and does not require conduct of a confirmatory assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 pH of the solvent and that of all dose levels

Dose Level

(µg/mL)

Original pH

Amount of 1N HCl (µL)

9.75

7.5

-

19.5

7.5

-

39

7.5

-

78

7.5

-

156

7.5

-

312.5

7.5

-

625

7.5

-

1250

8.0

30

2500

8.5

90

5000

9.0

140

Table 2 Dose Levels Tested

Treatment

Condition

Treatment

Time

Recovery

Time

Dose levels

(µg/mL)

 Non-activated

4 hr

20 hr

1, 2.5, 4.5, 9, 20, 40, 80, 160, 200

24hr

0 hr

1, 2.5, 4.5, 9, 20, 40, 80, 160, 200

 S9-activated

4 hr

20 hr

1, 2.5, 4.5, 9, 20, 40, 80, 160, 200

TABLE 3

CYTOTOXICITY OFBADGE-MXDA (#42)
IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION

4-HOUR TREATMENT, 16-HOUR RECOVERY PERIOD (INITIAL ASSAY)

Treatment

Mitotic

Average

Mitotic

 (µg/mL)

Flask

Index

Mitotic Index

Inhibition

 

 

DMSO

A

12.5%

B

13.2%

12.9%

BADGE-MXDA (#42)

1

A

11.2%

B

12.2%

11.7%

9%

2.5

A

11.0%

B

10.7%

10.9%

16%

4.5

A

11.1%

B

11.6%

11.4%

12%

9

A

11.3%

B

11.8%

11.6%

10%

20

A

12.0%

B

11.5%

11.8%

9%

40

A

1.2%

B

0.9%

1.1%

92%

80

A

0.0%

B

0.0%

0.0%

100%

160

A

0.0%

B

0.0%

0.0%

100%

200

A

0.0%

B

0.0%

0.0%

100%

MMC

A

6.5%

0.5

B

6.8%

6.7%

48%

MMC

A

5.1%

0.75

B

4.7%

4.9%

62%

Treatment:Rat lymphocytes were treated in the absence of an exogenous source of metabolic activation for 4 hours at 37 ± 1°C.

Mitotic Index= (Average cells in mitosis/1000 cells scored per culture) x 100.

Mitotic Inhibition= (Treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage.

TABLE 4

CYTOTOXICITY OFBADGE-MXDA (#42)
IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION

4-HOUR TREATMENT, 16-HOUR RECOVERY PERIOD (INITIAL ASSAY)

Treatment

Mitotic

Average

Mitotic

 (µg/mL)

Flask

Index

Mitotic Index

Inhibition

 

 

DMSO

A

12.6%

B

11.7%

12.2%

BADGE-MXDA (#42)

1

A

10.9%

B

11.5%

11.2%

8%

2.5

A

9.8%

B

10.5%

10.2%

16%

4.5

A

9.0%

B

9.4%

9.2%

24%

9

A

9.4%

B

10.1%

9.8%

20%

20

A

10.3%

B

9.8%

10.1%

17%

40

A

0.0%

B

0.0%

0.0%

100%

80

A

0.0%

B

0.0%

0.0%

100%

160

A

0.0%

B

0.0%

0.0%

100%

200

A

0.0%

B

0.0%

0.0%

100%

CP,2

A

8.5%

B

8.7%

8.6%

29%

CP,4

A

6.2%

B

6.7%

6.5%

47%

CP,6

A

2.5%

B

2.1%

2.3%

81%

Treatment:Rat lymphocytes were treated in the presence of an exogenous source of metabolic activation for 4 hours at 37 ± 1°C.

Mitotic Index= (Average cells in mitosis/1000 cells scored per culture) x 100.

Mitotic Inhibition= (Treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage.

TABLE 5

CYTOTOXICITY OFBADGE-MXDA (#42)
IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION

4-HOUR TREATMENT, 20-HOUR RECOVERY PERIOD (INITIAL ASSAY)

Treatment

Mitotic

Average

Mitotic

 (µg/mL)

Flask

Index

Mitotic Index

Inhibition

 

 

DMSO

C

10.8%

D

10.1%

10.5%

BADGE-MXDA (#42)

1

C

11.3%

D

11.1%

11.2%

-7%

2.5

C

10.1%

D

10.0%

10.1%

4%

4.5

C

10.7%

D

10.3%

10.5%

0%

9

C

11.0%

D

10.6%

10.8%

-3%

20

C

10.5%

D

11.1%

10.8%

-3%

40

C

0.0%

D

0.0%

0.0%

100%

80

C

0.0%

D

0.0%

0.0%

100%

160

C

0.0%

D

0.0%

0.0%

100%

200

C

0.0%

D

0.0%

0.0%

100%

MMC

C

9.6%

0.05

D

8.3%

9.0%

14%

 Treatment:Rat lymphocytes were treated in the absence of an exogenous source of metabolic activation for 24 hours at 37 ± 1°C.

Mitotic Index= (Average cells in mitosis/1000 cells scored per culture) x 100.

Mitotic Inhibition= (Treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage.

TABLE 6

SUMMARY (REPEAT ASSAY)

 Treatment  MeanMitotic     Cells Scored     Aberrations/Cell     Cells with Aberration
 (ug/ml)  Index  Numberical  Structural  Mean  St. Dev.  Numberical (%)  Structural (%)
 Without S9 4 hour treatment                     
 DMSO  7.7  200  200  0.005  0.071  0.0  0.5
 BADGE-MXDA (#42)              
 10  7.7  200  200  0.000  0.000  0.0  0.0
 22.5  4.9  200  200  0.005  0.071  0.0  0.5
 25  3.3  200  200  0.000  0.000  0.0  0.0
 MMC (0.75)  3.9  200  100  0.540  1.374  0.0  20.0**
 With S9 4 hour treatment                     
 DMSO  9.8  200  200  0.000  0.000  0.0  0.0
 BADGE-MXDA (#42)              
 10  8.7  200  200  0.000  0.000  0.0  0.0
 20  6.8  200  200  0.000  0.000  0.0  0.0
 27.5  4.2  200  200  0.005  0.071  0.0  0.5
 CP (2)  5.6  200  100  0.370  0.950  0.0  17.00**
 Without S9 24 hour treatment                     
 DMSO  9.9  200  200  0.005  0.071  0.0  0.5
 BADGE-MXDA (#42)              
 10  10.4  200  200  0.005  0.071  0.0  0.5
 20  9.3  200  200  0.000  0.000  0.0  0.0
 25  4.6  200  200  0.010  0.100  0.0  1.0
 MMC (0.05)  4.7  200  100  0.220  0.543  0.0  17.00**

Treatment:Cells from all treatment conditions were harvested at 24 hours after the initiation of the treatments.

Aberrations per Cell:Severely damaged cells were counted as 10 aberrations.

      ** p<0.01(highly significant); results of statistical analysis using the Fisher's Exact test.

       DMSO = Dimethyl sulfoxide; MMC = Mitomycin C; CP = Cyclophosphamide

Conclusions:
Interpretation of results (migrated information):
negative

All criteria for a valid study were met as described in the protocol.

Under the conditions of the assay described in this report, BADGE-MXDA (#42) was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9 activated test systems in the in vitro rat lymphocytes chromosome aberration assay
Executive summary:

The test article, BADGE-MXDA (#42)Reaction product of m-phenylenebis(methylamine) with oligomerisation products of 4,4’-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane (ECnr. 500-302-7), hereafter referred to as BADGE-MXDA (#42), was tested in thein vitrochromosome aberration assay utilizing cultured rat lymphocytes in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. The chromosome aberration assay was used to evaluate the clastogenic potential of BADGE-MXDA (#42) in cultured rat lymphocytes.

Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test article and compatibility with the target cells. Upon sonication for five minutes at 31.9 ºC, the test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested for solubility.

In the solubility assessment conducted by the cytogenetic lab at BioReliance, the test article was soluble in DMSO at all concentrations tested. Visible precipitate was observed at dose levels >156 µg/mL, while the dose levels <78 µg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed at dose levels >39 µg/mL, while the dose levels < 9.5 µg/mL were soluble in treatment medium. Based on these solubility, pH, osmolality, and precipitate tests, the definitive chromosome aberration assay was conducted at dose levels of 1, 2.5, 4.5, 9, 20, 40, 80, 160, and 200 µg/mL.

In the initial chromosome aberration assay, rat lymphocytes were treated for 4 and 24 hours in the non‑activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation. The test article was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels >80 µg/mL, while dose levels <40 µg/mL were soluble in treatment medium at the beginning and conclusion of the treatment period. At the conclusion of the treatment period, hemolysis was observed at dose levels³80 µg/mL in all three treatment conditions. Substantial toxicity (approximately 60 ± 10% reduction in mitotic index relative to the vehicle control) was observed at dose levels >40 µg/mL in all three exposure groups.  However, cytotoxicity as required by the testing guidelines was not achieved in any treatment condition; therefore, the chromosome aberration assay was repeated in its entirety at dose levels of 10, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, and 40 µg/mL.

In the repeat assay, the test article was soluble in DMSO and in the treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. At the conclusion of the treatment period, hemolysis was observed at dose levels >20 µg/mL in the non-activated 24-hour exposure group. Substantial toxicity (approximately 60 ± 10% reduction in mitotic index relative to the vehicle control) was observed at dose levels >25 µg/mL in all three exposure groups. Based on these acceptable levels of cytotoxicity, the test article was evaluated for induction of chromosome aberrations at dose levels of 0 (vehicle control), 10, 22.5, and 25 µg/mL in the non-activated 4-hour exposure group, at dose levels of 10, 20, and 27

.5 µg/mL in the S9-activated 4-hour exposure group, and at dose levels of 0 (vehicle control), 10, 20, and 25 µg/mL in the non-activated 24-hour exposure group.

The percentages of cells with structural or numerical (polyploidy and endoreduplication) aberrations in the test article-treated groups were not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results of the assay are summarized in the following table:

 

Mean

 

 

 

Mitotic

Mitotic

Cells With Aberrations

Treatment

Index1

Inhibition2

Numerical3

Structural

µg/mL

(%)

(%)

(%)

(%)

4 hours treatment in the absence of S9

DMSO

7.7

NA

0.0

0.5

BADGE-MXDA (#42)

 

10

7.7

0

0.0

0.0

22.5

4.9

37

0.0

0.5

25

3.3

58

0.0

0.0

MMC

 

 

 

 

0.75

3.9

49

0.0

20.0**

4 hours treatment in the presence of S9

DMSO

9.8

NA

0.0

0.0

BADGE-MXDA (#42)

 

10

8.7

11

0.0

0.0

20

6.8

31

0.0

0.0

27.5

4.2

57

0.0

0.5

CP

 

 

 

 

2

5.6

43

0.0

17.0**

24 hours treatment in the absence of S9

DMSO

9.9

NA

0.0

0.5

BADGE-MXDA (#42)

 

10

10.4

-5

0.0

0.5

20

9.3

6

0.0

0.0

25

4.6

54

0.0

1.0

MMC

 

 

 

 

0.05

4.7

53

0.0

17.0**

 

 

 

 

 

1Mitotic Index is the percentage of cells in mitosis per 1000 cells counted.

2 Mitotic Inhibition is relative to DMSO vehicle control.

3Numerical aberrations include both polyploidy and endoreduplicated cells.

   ** p£0.01 (highly significant); results of statistical analysis using the Fisher's Exact test.

NA = Not applicable

Test article dose formulations were analyzed by The Dow Chemical Company (Midland, MI). The results of the analysis indicates that the actual mean concentrations of the analyzed dose levels were between 90.1% and 107.1% of their respective targets with < 20% RPD (relative percent difference). This indicates that the regulatory-required top dose level was achieved and the results support the validity of the study conclusion. No test article was detected in the vehicle control sample.

Based on the findings of this study, BADGE-MXDA (#42) was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9‑activated test systems in thein vitrorat lymphocyteschromosome aberration assay. 

DMSO = Dimethyl sulfoxide;MMC = Mitomycin C; CP = Cyclophosphamide

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Negative in the Ames test, CHO/HPRT test and rat lymphocyte chromosomal aberration test.





Justification for classification or non-classification

Based on reliable, relevant and adequate data the substance is considered to be not mutagenic and not clastogenic. According to Regulation EC No 1272/2008 no classification and labelling for mutagenicity is required.