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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear colorless thick liquid
Details on test material:
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) (lot# ZH1650NZ08). The test material composition was determined to be 35.3 wt% m-xylene diamine by gas chromatography and 64.7 wt% BADGE-MXDA (#42) by difference (100-wt% residual m-xylene diamine). Identification was by nuclear magnetic resonance and liquid chromatography mass spectrometry

Method

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
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).
Controls
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.

Results and discussion

Test results
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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.