Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

IN VITRO

A bacterial reverse mutation assay conducted in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation in accordance with OECD 471 concluded that BPA-DA is non-mutagenic.

An in vitro mammalian chromosome aberration test conducted in Chinese hamster Ovary (CHO) cells with and without metabolic activation in accordance with OECD 473 concluded that BPA-DA is non-mutagenic.

An in vitro mammalian cell gene mutation assay conducted in mouse lymphoma L5178Y cells with and without metabolic activation in accordance with OECD 476 concluded that BPA-DA is non-mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg per plate (Preliminary assay)
100, 333, 1000, 3333 and 5000 µg per plate (Mutagenicity assays)
Vehicle / solvent:
Dimethyl sulfoxide (DMSO; CAS No. 67-68-5); from Fisher Scientific
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Remarks:
All strains; With and without activation
Positive controls:
yes
Remarks:
With S9 activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains; 1.0 µg/plate all Salmonella strains; 10 µg/plate WP2 uvrA
Positive controls:
yes
Remarks:
Without S9 activation
Positive control substance:
sodium azide
Remarks:
TA100, TA1535; 1.0 µg/plate
Positive controls:
yes
Remarks:
Without S9 activation
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA; 1,000 µg/plate
Positive controls:
yes
Remarks:
Without S9 activation
Positive control substance:
9-aminoacridine
Remarks:
TA1537; 75 µg/plate
Positive controls:
yes
Remarks:
Without S9 activation
Positive control substance:
2-nitrofluorene
Remarks:
TA98; 1.0 µg/plate
Details on test system and experimental conditions:
Preliminary Toxicity Assay: The preliminary toxicity assay was used to establish the dose range over which the test article would be assayed. Vehicle control and ten dose levels of the test article were plated, one plate 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.
Mutagenicity Assay: The mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test article. Five dose levels of test article along with vehicle control and appropriate positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA 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: On the day of its use, minimal top agar was melted and supplemented. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by the Milli Q Reagent Water System. Bottom agar was Vogel Bonner minimal medium E.
Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain and activation.
One half (0.5) milliliter of S9 or Sham mix, 100 uL of tester strain and 50 uL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2 degrees C. After vortexing, the mixture was 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 approximately 48 to 72 hours at 37±2 degrees C. Plates that were not counted immediately following the incubation period were stored at 2 8 degrees 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 assay was the preliminary toxicity assay or the plate exhibited toxicity.
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-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 TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA1537, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
All validity criteria were met

Initial Mutagenicity Assay - Mean Number of Revertants Per Plate

Activation:  None

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle (DMSO)

10 ± 1

137 ± 12

12 ± 1  

11 ± 5  

11 ±   3

100

9 ± 2

137 ± 14  

13 ± 4

10 ± 2  

10 ±   1

333

11± 4

133 ± 15  

12 ± 2  

10 ± 2  

11 ±   1

1000

13 ± 1

135 ± 14

8 ± 1  

12 ± 1  

10 ±   5

3333

14 ± 5

125 ± 3

12 ± 0   

9 ± 3   

9 ±   2

5000

15 ± 3

113 ± 15

7 ± 2   

9 ± 1   

9 ±   1

Positive Control

128 ± 12

582 ± 28

211 ± 27  

68 ± 6  

74 ±   3

Initial Mutagenicity Assay - Mean Number of Revertants Per Plate

Activation:  S9

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle (DMSO)

18 ± 2

151 ± 17

11 ± 3

11 ± 3

10 ± 1

100

13 ± 2

129 ± 3

12 ± 3

10 ± 2

8 ± 1

333

13 ± 3

118 ± 14

9 ± 1

10 ± 1

11 ± 2

1000

9 ± 2

116 ± 17

10 ± 2

9 ± 3

9 ± 1

3333

13 ± 2

120 ± 9

12 ± 3

7 ± 2

9 ± 1

5000

14 ± 2

117 ± 5

10 ± 4

10 ± 3

12 ± 4

Positive Control

713 ± 51

678 ± 42

185 ± 9

246 ± 19

770 ± 90

Confirmatory Mutagenicity Assay - Mean Number of Revertants Per Plate

Activation:  None

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle (DMSO)

15 ± 5 

177 ± 19

15 ± 4

14 ± 1

18 ± 3

100

12 ± 4

187 ± 6

13 ± 2  

19 ± 2  

18 ± 7

333

14 ± 2

202 ± 25

11 ± 1

16 ± 6

14 ± 4

1000

12 ± 1

186 ± 10

13 ± 3

12 ± 3

9 ± 1

3333

15 ± 2

180 ± 34

15 ± 7

9 ± 2

11 ± 5

5000

15 ± 5

148 ± 17

10 ± 3

15 ± 5   

11 ± 2

Positive Control

133 ± 7

636 ± 27

73 ± 9 

83 ± 4 

157 ± 3

Confirmatory Mutagenicity Assay - Mean Number of Revertants Per Plate

Activation:  S9

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle (DMSO)

27 ± 7

168 ± 11  

11 ± 1  

11 ± 4  

20 ± 5

100

27 ± 4 

183 ± 15

9 ± 5  

11 ± 1

21 ± 2

333

17 ± 5

181 ± 7

11 ± 7  

10 ± 6  

16 ± 3

1000

14 ± 6

166 ± 16

8 ± 3  

11 ± 4  

14 ± 1

3333

16 ± 6

164 ± 4

11 ± 4

13 ± 4

15 ± 3

5000

15 ± 3

173 ± 24

13 ± 3

10 ± 3  

11 ± 1

Positive Control

920 ± 6

1051 ± 25

235 ± 4

234 ± 34 

791 ± 40

Conclusions:
Under the conditions of this study, the test material did not cause a positive response in either the presence or absence of Aroclor induced rat liver S9 and is therefore non-mutagenic.
Executive summary:

The study was conducted in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A in accordance with OECD 471 under GLP conditions (BioReliance, 2002).

Bacteria were exposed to the test material at concentrations up to 5000 µg/plate in DMSO both with and without S9 metabolic activation.

Under the conditions of this study, the test material did not cause a positive response in either the presence or absence of Aroclor induced rat liver S9 and is therefore non-mutagenic.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254-induced rat liver S-9
Test concentrations with justification for top dose:
Up to 1500 µg/mL without activation
Up to 750 µg/mL with activation
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C (MMC; without metabolic activation) and Cyclophosphamide (CP; with metabolic activation)
Details on test system and experimental conditions:
Description of test procedure: 
A preliminary toxicity assay was performed for the purpose of selecting doses for the chromosome aberration assay and consisted of an evaluation of test article effect on cell growth.  CHO cells were seeded for each treatment condition at approximately 5x105 cells/25 cm² flask and were incubated at 37 ± 1 °C in a humidified atmosphere of 5 ± 1% CO2 in air for 16-24 hours.  Treatment was carried out by refeeding the flasks with complete medium McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS), 100 units penicillin and 100 µg streptomycin/mL, and 2 mM L-glutamine) for the non-activated study or S9 reaction mixture for the activated study, to which was added 50 µL dosing solution of test article in solvent or solvent alone.  The osmolality in treatment medium of the solvent and of the highest test article concentration, the lowest precipitating test article concentration and the highest soluble test article concentration were measured.  The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.  The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9.  At completion of the 4-hour exposure period, the cells were washed with buffered saline, refed with complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment.  At 20 hours after the initiation of treatment, the cells were harvested by trypsinization and counted using a Coulter counter.  The presence of test article precipitate was assessed using the unaided eye.  Cell viability was determined by trypan blue dye exclusion.  The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.  In the preliminary toxicity assay, the maximum dose tested was 2000 ug/mL.  The test article formed a workable suspension in DMSO at a concentration of 2000 µg/mL, the maximum concentration prepared in the assay.  Visible precipitate was observed in treatment medium at dose levels greater than or equal to 200 µg/mL. Dose levels less than or equal to 60 µg/mL were soluble in treatment medium.  The osmolality of the test article concentrations in treatment medium were acceptable because they did not exceed the osmolality of the solvent by more than 20%.  The pH of the highest concentration of test article in treatment medium was approximately 7.0.  Based on the toxicity study, the doses chosen for the chromosome aberration assay were: 0, 62.5, 125, 250, 500, 750, 1000, 1250 and 1500 µg/mL (4-hr treatment w/o activation); 0, 62.5, 125, 250, 500, 750, 1000, 1250 and 1500 µg/mL (20-hr treatment w/o activation); and 0, 62.5, 125, 250, 350, 500, 600 and 750 µg/mL (4-hr treatment with activation).  Samples were run in duplicate, with and without metabolic activation. Selection of doses for microscopic analysis:  The first criterion, specified by the Guideline, is to select the highest dose with at least 50% reduction in cell growth or mitotic index relative to the solvent control with a sufficient number of scorable metaphase cells, regardless of test article precipitation in the treatment medium.  For this study, the mitotic index was used to select the highest dose for each test condition.  Two lower doses also were included. 
For the chromosome aberration assay, CHO cells were seeded and treated as described above.  The osmolality in treatment medium of the solvent and of the highest test article concentration, the lowest precipitating test article concentration and the highest soluble test article concentration were measured.  The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.  A concurrent toxicity test was conducted for each treatment.  After cell harvest the cells were counted, test article precipitate was assessed and cell viability was determined by trypan blue dye exclusion.  Cell counts and viability were used to determine cell growth inhibition.  The cells were exposed to the test article continuously for 4 or 20 hours in the non-activated study, and for 4 hours in the activated study.  After the exposure period for the 4-hour exposure groups, the cells were washed and returned to the incubator.  Two hours prior to the scheduled cell harvest, Colcemid® was added to duplicate flasks for each treatment condition.  Two hours after the addition of Colcemid®, metaphase cells were harvested.  Cells were collected approximately 20 hours after initiation of treatment.  Slides were prepared from cells of each treatment and the cells stained with 5% Giemsa. 

Evaluation of metaphase cells: 
The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group.  Initially, the non-activated and S9 activated 4-hour exposure groups were evaluated for chromosome aberrations and since a negative result was obtained in the non-activated 4-hour exposure group, the non-activated 20-hour continuous exposure group was then also evaluated for chromosome aberrations.  When possible, a minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations.  The number of metaphase spreads that were examined and scored per duplicate flask may have been reduced if the percentage of aberrant cells reached a statistically significant level before 100 cells are scored.  Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements.  Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings.  Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome).  Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange.  Pulverized chromosome(s), pulverized cells and severely damaged cells (greater than or equal to 10 aberrations) were also recorded.  Chromatid and isochromatid gaps were recorded but not included in the analysis.  Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.
Evaluation criteria:
Evaluation of test results: 
The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies.  The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated and reported for each group.  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 solvent control.  In the event of a positive Fisher's exact test at any test article dose group, the Cochran-Armitage test was used to measure dose-responsiveness.  As a guide to interpretation of the data, the test article was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p is less than or equal to 0.05).  However, values that are statistically significant but do not exceed the range of historic solvent controls may be judged as not biologically significant.  Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative. 

Criteria for a Valid Test: 
The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control.  The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p is less than or equal to 0.05, Fisher's exact test) relative to the solvent control.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the chromosome aberration assay, visible precipitate was observed in treatment medium at dose levels ≥ 250 µg/mL.  Dose levels ≤125 µg/mL were soluble in treatment medium. The osmolality in treatment medium of the highest concentration tested, 1500 µg/mL, was 396 mmol/kg.  The osmolality in treatment medium of the lowest precipitating concentration, 250 µg/mL, was 405 mmol/kg. The osmolality in treatment medium of the highest soluble concentration, 125 µg/mL, was 402 mmol/kg. The osmolality of the solvent (DMSO) in treatment medium was 398 mmol/kg. The osmolality of the test article concentrations in treatment medium are acceptable because they did not exceed the osmolality of the solvent by more than 20%. The pH of the highest concentration of test article in treatment medium was approximately 7.0.

4 hours in the absence of S9 activation: Toxicity of BPA-DA (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the absence of S9 activation was 17% at 500 µg/mL, the highest test concentration evaluated for chromosome aberrations.  The mitotic index at the highest dose level evaluated for chromosome aberrations, 500 µg/mL, was 54% reduced relative to the solvent control.  The dose levels selected for microscopic analysis were 125, 250 and 500 µg/mL.  The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control at any dose level (p > 0.05, Fisher's exact test). The percentage of structurally damaged cells in the MMC (positive control) treatment group (19.0%) was statistically significant.

4 hours in the presence of S9 activation: Toxicity of BPA-DA in CHO cells when treated for 4 hours in the presence of S9 activation was 52% at 350 µg/mL, the highest test concentration evaluated for chromosome aberrations.  The mitotic index at the highest dose level evaluated for chromosome aberrations, 350 µg/mL, was 52% reduced relative to the solvent control.  The dose levels selected for microscopic analysis were 125, 250 and 350 µg/mL.  The percentage of cells with structural or numerical aberrations in the test article-treated groups was not statistically increased above that of the solvent control at any dose level (p > 0.05, Fisher's exact test). The percentage of structurally damaged cells in the CP (positive control) treatment group (21.0%) was statistically significant.

20 hours without of S9 activation: Toxicity of BPA-DA was 39% at 500 µg/mL, the highest test concentration evaluated for chromosome aberrations in the non-activated 20 hour continuous exposure group.  The mitotic index at the highest dose level evaluated for chromosome aberrations, 500 µg/mL, was 51% reduced relative to the solvent control.  The dose levels selected for microscopic analysis were 125, 250 and 500 µg/mL.  The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control at any dose level (p > 0.05, Fisher's exact test). The percentage of structurally damaged cells in the MMC (positive control) treatment group (20.0%) was statistically significant.

The positive and solvent controls fulfilled the requirements for a valid test.
Remarks on result:
other: all strains/cell types tested

Summary of Test Results

Treatment

(µg/mL)

S9

Activation

Treatment

Time

Mean

Mitotic

Index

Cells

Scored

Aberrations

Per Cell

(Mean ± SD)

Cells with Numerical

Aberrations

(%)

Cells with Structural Aberrations

(%)

Vehicle (DMSO)

-

4

8.9

200

0.000 ± 0.000

1.5

0.0

125 BPA-DA

-

4

8.0

200

0.005 ± 0.071

2.5

0.5

250 BPA-DA

-

4

6.7

200

0.000 ± 0.000

3.0

0.0

500 BPA-DA

-

4

4.1

200

0.005 ± 0.071

4.5

0.5

Positive control (MMC)

0.2

-

4

7.6

100a

0.240 ± 0.588

2.5

19.0**

Vehicle (DMSO)

+

4

10.0

200

0.010 ± 0.100

3.0

1.0

125 BPA-DA

+

4

8.8

200

0.000 ± 0.000

2.0

0.0

250 BPA-DA

+

4

7.5

200

0.010 ± 0.100

1.0

1.0

350 BPA-DA

+

4

4.8

200

0.015 ± 0.122

5.0

1.5

Positive control (CP)

10

+

4

9.4

100a

0.310 ± 0.775

2.0

21.0**

Vehicle (DMSO)

-

20

8.2

200

0.000 ± 0.000

4.0

0.0

125 BPA-DA

-

20

6.3

200

0.000 ± 0.000

3.5

0.0

250 BPA-DA

-

20

6.2

200

0.010 ± 0.100

2.5

0.1

500 BPA-DA

-

20

4.0

200

0.005 ± 0.071

4.5

0.5

Positive control (MMC)

0.1

-

20

7.1

100a

0.320 ± 0.764

2.0

20.0**

Treatment:  Cells from all treatment regimens were harvested 20 hours after the initiation of the treatments.

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

Percent Aberrant Cells:  **, p is less than or equal to 0.01; using the Fisher's exact test.

a Numerical aberrations are out of 100 cells scored.

Conclusions:
Under the conditions of the assay, the test material was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.
Executive summary:

The study was conducted in Chinese hamster Ovary (CHO) cells in accordance with OECD 473 under GLP conditions (BioReliance, 2004).

CHO cells were exposed to the test material in DMSO at concentrations up to 1500 µg/mL without metabolic activation and up to 750 µg/mL with metabolic activation. The positive and solvent controls fulfilled the requirements for a valid test.

Under the conditions of the assay, the test material was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
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:
0, 50, 75, 100, 125 and 150 µg/mL with activation
0, 25, 50, 75, 100 and 125 µg/mL without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
Description of test procedure: The preliminary toxicity assay was used to establish the optimal dose levels for the mutagenesis assay. L5178Y cells were exposed to the solvent alone and nine concentrations of test article ranging from 0.05 to 500 µg/mL in both the absence and presence of S9-activation with a 4-hour exposure and without activation with a 24-hour exposure. Cell population density was determined 24 and 48 hours after the initial exposure to the test article. The cultures were adjusted to 3x10^5 cells/mL after 24 hours only. Toxicity was measured as suspension growth of the treated cultures relative to the growth of the solvent control cultures after 48 hours.

The mutagenesis assay was carried out by combining 6x10^6 L5178Y/TK+/- cells, medium or S9 activation mixture and 100 uL dosing solution of test or control article in solvent or solvent alone and incubated for 4 (with and without activation) or 24 (without activation) hours. The positive controls were treated with MMS (at 15 and 20 ug/mL for the 4-hour exposure or 2.5 and 5.0 µg/mL for the 24-hour exposure) and 7,12 DMBA (5.0 and 7.5 µg/mL).

Expression of the mutant phenotype: For expression of the mutant phenotype, the cultures were counted and adjusted to 3x10^5 cells/mL at approximately 24 and 48 hours after treatment. Cultures with less than 3x10^5 cells/mL were not adjusted. For expression of the TK+/- cells, two flasks per culture were cloned for TFT (trifluorothymidine, the selective agent) or VC (viable count). The cells were diluted in cloning medium to concentrations of 3x10^6 cells/100/mL for the TFT flask and 600 cells/100mL for the VC flask. Cells were plated and incubated for 10-14 days.

Scoring procedures: The VC plates were counted for the total number of colonies per plate and the total relative growth determined. The TFT-resistant colonies were counted for each culture with >= 20% total relative growth (including at least one concentration with >= 10% but <= 20% total growth). The diameters of the TFT-resistant colonies for the positive and solvent controls and, in the case of a positive response, the test article-treated cultures were determined over a range of approximately 0.2 to 1.1 mm.
Evaluation criteria:
Evaluation of results: The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency (number of mutants per 10^6 surviving cells) was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies in the three corresponding VC plates and multiplying by the dilution factor (2x10^-4) then multiplying by 10^6. In evaluation of the data, increases in mutant frequencies that occurred only at highly toxic concentrations (i.e., less than 10% total growth) were not considered biologically relevant. The following criteria are presented as a guide to interpretation of the data:
(1) A result was considered positive if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10% or greater total growth exhibited mutant frequencies of ≥ 100 mutants per 10^6 clonable cells over the background level,
(2) A result was considered equivocal if the mutant frequency in treated cultures was between 55 and 99 mutants per 10^6 clonable cells over the background level,
(3) A result was considered negative if the mutant frequency in treated cultures was fewer than 55 mutants per 10^6 clonable cells over the background level.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The maximum dose tested in the preliminary toxicity assay was 500 ug/mL. Visible precipitate was present at ≥ 150 µg/mL in treatment medium. No visible precipitate was present at concentrations of ≥ 50 µg/mL in treatment medium. The osmolality of the solvent control was 442 mmol/kg and the osmolality of the highest soluble dose, 50 µg/mL, was 437 mmol/kg. Suspension growth relative to the solvent controls was 0% at 150 µg/mL without activation with 4- and 24-hour exposures and 7% at 500 µg/mL with S9 activation and 4-hour exposure. Based on the results of this preliminary test the concentrations selected for the mutagenesis assay were 0, 50, 75, 100, 125 and 150 µg/mL with activation and 0, 25, 50, 75, 100 and 125 µg/mL without activation.

Cytotoxicity conc.:
With metabolic activation: 500 µg/mL
Without metabolic activation: ≥ 150 ug/mL

Results for cultures treated for four hours (initial assay): Visible precipitate was present at 150 µg/mL. In the non-activated system, suspension growth ranged from 14 to 107%. In the activated system, suspension growth ranged from 103 to 115%. One cloned culture treated with 125 µg/mL without activation exhibited a mutant frequency between 55 and 99 mutants per 10^6 clonable cells greater than the solvent control. The total growths ranged from 14 to 112% for the non-activated cultures at concentrations of 25 to 125 µg/mL and 108 to 146% for the S9-activated cultures at concentrations of 50 to 150 µg/mL. The results of the initial 4-hour exposure assay were negative in the absence and presence of S9 activation. Because no unique metabolic requirements were known about the test article, only an extended treatment assay was performed in the absence of S9 for a 24-hour exposure period.

Results for cultures treated for 24 hours (extended treatment assay): Visible precipitate was present at 150 µg/mL. Cultures treated with concentrations of 25, 50, 75, 100 and 125 µg/mL were cloned and produced a range in suspension growth of 89 to 115%.

One cloned culture (treated with 125 µg/mL) exhibited a mutant frequency of between 55 and 99 mutants per 10^6 clonable cells over that of the solvent control. A dose-response trend was not observed. The total growths ranged from 98% to 133% at concentrations of 25 to 125 µg/mL.

The TFT-resistant colonies for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS positive control yielded the expected increase in small colonies, verifying the adequacy of the methods used to detect small colony mutants.

Cloning Data for L5178Y/TK+/- Mouse Lymphoma Cells Treated with BPA-DA in the Absence of Exogenous Metabolic Activation Initial Assay (4-hour exposure)

Dose Level (µg/mL)

Replicate

TFT Colonies

VC Colonies

Mutant Freq.a

Induced Mutant Freq.b

%

Total Growthc

Counts

Mean

Counts

Mean

0 (solvent)

1

94

90

92

92 ± 2

176

140

171

162 ± 16

113

--

--

0 (solvent)

2

34

22

+

28 ± 5

168

130

127

142 ± 19

40

--

--

Mean Solvent Mutant Frequency = 76

25

A

84

67

17

56 ± 28

165

133

178

159 ± 19

71

-6

112

25

B

30

37

33

33 ± 3

151

169

164

161 ± 8

41

-35

109

50

A

112

103

50

88 ± 27

176

175

189

180 ± 6

98

22

110

50

B

38

68

52

53 ± 12

185

151

186

174 ± 16

61

-16

106

75

A

83

58

21

54 ± 25

155

150

162

156 ± 5

69

-7

18

75

B

65

70

28

54 ± 19

152

129

131

137 ± 10

79

3

22

100

A

77

67

73

72 ± 4

165

153

150

156 ± 6

93

16

18

100

B

79

76

68

74 ± 5

151

171

165

162 ± 8

92

15

15

125

A

63

79

78

73 ± 7

158

162

164

161 ± 2

91

14

19

125

B

94

76

86

85 ± 7

105

122

122

116 ± 8

147

70

14

Positive Control - Methyl Methanesulfonate (ug/mL)

15

--

104

83

+

94 ± 9

77

103

93

91 ± 11

205

129

39

20

--

212

155

129

165 ± 35

71

48

66

62 ± 10

536

460

22

Solvent = DMSO

+ = Culture lost

a Mutant frequency (per 106 surviving cells) = (Average # TFT colonies / average # VC colonies) x 200

b Induced mutant frequency (per 106surviving cells) = mutant frequency - average mutant frequency of solvent controls

c % Total growth = (% suspension growth x % cloning growth) / 100

Cloning Data for L5178Y/TK+/- Mouse Lymphoma Cells Treated with BPA-DA in the Presence of Exogenous Metabolic Activation Initial Assay (4-hour exposure)

Dose Level (µg/mL)

Replicate

TFT Colonies

VC Colonies

Mutant Freq.a

Induced Mutant Freq.b

%

Total Growthc

Counts

Mean

Counts

Mean

0 (solvent)

1

59

68

35

54 ± 14

212

101

65

126 ± 63

86

--

--

0 (solvent)

2

80

17

89

62 ± 32

80

212

232

175 ± 67

71

--

--

Mean Solvent Mutant Frequency = 78

50

A

73

63

55

64 ± 7

169

152

199

173 ± 19

73

-5

130

50

B

50

13

64

42 ± 22

181

194

191

189 ± 6

45

-33

141

75

A

71

60

81

71 ± 9

146

168

162

159 ± 9

89

11

121

75

B

72

86

84

81 ± 6

195

159

166

173 ± 16

93

15

130

100

A

96

85

93

91 ± 5

215

198

158

190 ± 24

96

18

146

100

B

94

78

81

84 ± 7

195

190

156

180 ± 17

94

15

138

125

A

101

78

70

83 ± 13

177

196

202

192 ± 11

87

8

133

125

B

101

87

90

93 ± 6

162

177

96

145 ± 35

128

49

108

150*

A

89

94

79

87 ± 6

177

194

179

183 ± 8

95

17

125

150*

B

85

80

57

74 ± 12

178

157

182

172 ± 11

86

8

122

Positive Control - 7,12 Dimethylbenz(a)anthracene (ug/mL)

2.5

--

198

51

60

103 ± 67

54

50

80

61 ± 13

336

258

35

5

--

238

269

275

261 ± 16

99

59

50

69 ± 21

752

674

14

Solvent = DMSO

* Precipitating concentration

a Mutant frequency (per 106 surviving cells) = (Average # TFT colonies / average # VC colonies) x 200

b Induced mutant frequency (per 106surviving cells) = mutant frequency - average mutant frequency of solvent controls

c % Total growth = (% suspension growth x % cloning growth) / 100

Cloning Data for L5178Y/TK+/- Mouse Lymphoma Cells Treated with BPA-DA in the Absence of Exogenous Metabolic Activation Extended Treatment Assay (24-hour exposure)

Dose Level (µg/mL)

Replicate

TFT Colonies

VC Colonies

Mutant Freq.a

Induced Mutant Freq.b

%

Total Growthc

Counts

Mean

Counts

Mean

0 (solvent)

1

60

32

57

50 ± 13

158

160

143

154 ± 8

65

--

--

0 (solvent)

2

24

30

37

30 ± 5

120

131

127

126 ± 5

48

--

--

Mean Solvent Mutant Frequency = 56

25

A

72

32

39

48 ± 17

171

140

160

157 ± 13

61

4

100

25

B

39

40

40

40 ± 0

158

146

137

147 ± 9

54

-2

98

50

A

47

54

24

42 ± 13

175

165

162

167  ± 6

50

-7

113

50

B

117

86

40

81 ± 32

131

160

155

149 ± 13

109

53

108

75

A

40

45

33

39 ± 5

183

130

143

152 ± 23

52

-5

114

75

B

32

24

32

29 ± 4

136

164

198

166 ± 25

35

-21

133

100

A

104

109

46

86 ± 29

190

145

142

159 ± 22

109

52

111

100

B

66

78

78

74 ± 6

137

163

197

166 ± 25

89

33

127

125

A

87

41

94

74 ± 24

176

169

117

154 ± 26

96

40

113

125

B

72

74

86

77 ± 6

122

119

168

136 ± 22

113

57

113

Positive Control - Methyl Methanesulfonate (ug/mL)

5

--

163

136

132

144 ± 14

66

76

86

76 ± 8

378

322

40

7.5

--

214

156

140

170 ± 32

70

72

85

76 ± 7

449

393

32

Solvent = DMSO

a Mutant frequency (per 106 surviving cells) = (Average # TFT colonies / average # VC colonies) x 200

b Induced mutant frequency (per 106surviving cells) = mutant frequency - average mutant frequency of solvent controls

c % Total growth = (% suspension growth x % cloning growth) / 100

Conclusions:
Under the conditions of this study, the mutagenicity of the test material was concluded to be negative without activation with 4- and 24-hour exposures and negative with S9 activation with a 4-hour exposure.
Executive summary:

The study was conducted in mouse lymphoma L5178Y cells in accordance with OECD 476 under GLP conditions (BioReliance, 2004).

Mouse lymphoma L5178Y cells were exposed to the test material in DMSO at concentrations up to 150 µg/mL with metabolic activation (4-hour exposure) and up to 125 µg/mL without metabolic activation (4- and 24-hour exposure). All criteria for a valid study were met as described in the protocol.

Under the conditions of this study, the mutagenicity of the test material was concluded to be negative with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
other: US EPA Health Effects Test Guidelines, HG-Gene Muta - S. typhimurium, EPA Report No. 560/6-84-002, October 1984
Deviations:
yes
Remarks:
Confirmatory assay not performed
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate prepared from Aroclor 1254 induced Sprague-Dawley male rats.
Test concentrations with justification for top dose:
0, 0.3, 1.0, 3.0, 10 and 30 mg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-Aminoanthracene and 4-nitro-o-phenylenediamine
Remarks:
All strains with metabolic activation, 2.5 µg/plate
Details on test system and experimental conditions:
The Salmonella typhimurium strains were supplied by Dr. Bruce N. Ames, University of California, Berkeley, USA. A preliminary cytotoxicity test was conducted with 10 dose levels of the test substance ranging from 0.01 to 50 mg/plate to determine the doses for the mutagenicity assay. No cytotoxicity was evident; however, the highest dose (50 mg/plate) produced a dense precipitate which precluded the ability to count colonies. For the mutagenicity assay, 100 uL of the appropriate solvent, control or test chemical solution was added to a sterile tube containing 2 mL of top agar and a 100 uL aliquot of the appropriate bacterial culture. Either 0.5 mL of S9 mix or 0.5 mL of phosphate-buffered saline was added for tests with or without metabolic activation, respectively. The top agar mixture was then poured onto a Vogel-Bonner Medium E agar plate. Each dose was tested in triplicate with all five bacterial strains. The plates were transferred to a darkened 37°C incubator after hardening and incubated for 48-72 hours. Bacterial colonies were counted manually or by an automatic counter. An examination of the background lawn was also recorded.
Solvent and Positive controls: The test substance was suspended in dimethylsulfoxide (DMSO), which also was used as the solvent control. For the nonactivation assay, the following positive control substances were used: 4-nitro-o-phenylenediamine (0.01 mg/plate for strains TA98 and TA1538); sodium azide (0.01 mg/plate for strains TA100 and TA1535); and 9-aminoacridine (0.06 mg/plate for strain 1537). 2-Aminoanthracene (2.5 ug/plate) was used as the positive control substance for the activation assay in all strains.
Evaluation criteria:
The spontaneous reversion for the solvent controls should be within the laboratories historical range. The positive controls should demonstrate that the test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose and there is evidence of a dose related increase in the number of revertant colonies.
Key result
Species / strain:
other: S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
With metabolic activation: 30 mg/plate in TA1537 and TA1538 only; Without metabolic activation: >= 10 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The number of revertants/plate produced by treatment of the bacteria with the test substance at all concentrations and in all tester strains was reported to be less than or approximately equal to the number of revertants in the vehicle-treated negative control group, with and without metabolic activation. The test material was therefore concluded to not be mutagenic in this assay.

Mean Number of Revertants Per Plate

Activation:  None

Dose (mg/plate)

TA98

TA100

TA1535

TA1537

TA1538

Solvent (DMSO)

18

94

19

7

7

0.3

20

72

13

6

9

1.0

17

70

11

2 (t)

3 (t)

3.0

21

77

10

4

6

10

17

55 (s)

6 (s/t)

5

3 (s/t)

30

19

49 (s)

12 (t)

4 (t)

6 (s)

Positive Control

928

1847

1740

208

1130

t = Toxic: Clearing of background lawn, or average number of colonies less than ½ the solvent control value.

s = Sparse growth of background lawn; counts not included in calculation of mean.

Mean Number of Revertants Per Plate

Activation:  Rat Liver S9

Dose (mg/plate)

TA98

TA100

TA1535

TA1537

TA1538

Solvent (DMSO)

30

95

9

6

21

0.3

21

84

11

4

16

1.0

30

82

9

6

15

3.0

25

77

10

5

17

10

19

76

9

1 (s/t)

10 (t)

30

20

77

7

3

10 (t)

Positive Control

2372

1910

83

125

1164

t = Toxic: Clearing of background lawn, or average number of colonies less than ½ the solvent control value.

s = Sparse growth of background lawn; counts not included in calculation of mean.

Conclusions:
Under the conditions of this study, the test material did not exhibit mutagenic activity in any of the assays conducted in this evaluation and was considered not mutagenic.
Executive summary:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and TA1538 were exposed to the test material in DMSO at concentrations up to 30 mg/plate both with and without metabolic activation in accordance with the US EPA Health Effects Test Guidelines under GLP conditions (Bushy Run Research Center, 1988).

Under the conditions of this study, the test material did not exhibit mutagenic activity in any of the assays conducted in this evaluation and was considered not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Ames et al., Mutation Research 31:347, 1975
Description of test procedure: The plate test consisted of direct revertant colony counts obtained from a set of selective agar plates seeded with populations of mutant cells suspended in a semisolid overlay. Approximately 10^8 cells were treated with the test substance in the presence and absence of a metabolic activation system (Aroclor 1254-treated rat liver supernatant). One plate was used per concentration. The plates were incubated for 48 hours at 37 degrees C, and scored for the number of colonies growing on each plate.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538
Species / strain / cell type:
Saccharomyces cerevisiae
Remarks:
Strain D4
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate prepared from Aroclor 1254 induced male Sprague-Dawley rats.
Test concentrations with justification for top dose:
0, 0.1, 1.0, 10, 100 and 500 µg/plate
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: Methylnitrosoguanidine
Remarks:
Without metabolic activation: Strains TA1535, TA100 and D4
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation: Strains TA1538 and TA98
Positive controls:
yes
Positive control substance:
other: quinacrine mustard
Remarks:
Without metabolic activation: Strain TA1537
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
With metabolic activation: Strains TA1535 and TA100
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
With metabolic activation: TA1538 and TA98
Positive controls:
yes
Positive control substance:
other: 8-aminoquinoline
Remarks:
With metabolic activation: TA1537
Positive controls:
yes
Positive control substance:
not specified
Remarks:
With metabolic activation: Strain D4
Details on test system and experimental conditions:
The plate test consisted of direct revertant colony counts obtained from a set of selective agar plates seeded with populations of mutant cells suspended in a semisolid overlay. Approximately 10^8 cells were treated with the test substance in the presence and absence of a metabolic activation system (Aroclor 1254-treated rat liver supernatant). One plate was used per concentration. The plates were incubated for 48 hours at 37 °C, and scored for the number of colonies growing on each plate.
Evaluation criteria:
The solvent control values must be within the normal historical control range and the presence of a dose response is required for establishing mutagenicity. For strains TA1535, TA1537 and TA1538, if the solvent control value is within the normal range, a test substance producing a positive response over three concentrations with the lowest increase equal to twice the solvent control is considered mutagenic. For strains TA98, TA100 and D4, a test substance producing a positive response over three concentrations with the lowest increase equal to twice the solvent control (TA100) or two to three times the solvent control (TA98 and D4) is considered mutagenic. In addition, a positive response must be repeated in a separate assay.
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Saccharomyces cerevisiae
Remarks:
Strain D4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
The number of revertants/plate produced by treatment of the bacteria with the test substance at all concentrations and in all tester strains was reported to be less than or approximately equal to the number of revertants in the solvent-treated negative control group, with and without metabolic activation.

Revertants Per Plate

Activation:  None

Dose (µg/plate)

TA1535

TA1537

TA1538

TA98

TA100

D4

Solvent (DMSO)

10

14

13

28

169

32

0.1

16

15

11

26

140

38

1.0

19

10

16

31

189

37

10

17

12

16

30

202

41

100

17

15

13

29

206

42

500

12

17

15

28

233

28

Positive Control

653

619

>1000

>1000

>1000

593

Revertants Per Plate

Activation:  Rat Liver S9

Dose (µg/plate)

TA1535

TA1537

TA1538

TA98

TA100

D4

Solvent (DMSO)

17

13

24

35

267

23

0.1

10

18

16

31

196

21

1.0

17

12

24

34

238

10

10

17

14

29

26

217

23

100

11

17

20

38

254

28

500

9

10

13

32

245

25

Positive Control

131

213

574

891

831

48

Conclusions:
Under the conditions of this study the test material did not exhibit mutagenic activity in any of the assays conducted in this evaluation and was considered not mutagenic according to the evaluation criteria.
Executive summary:

S. typhimurium TA1535, TA1537, TA1538, TA98 and TA 100 and Saccharomyces cerevisiae (Strain D4) were exposed to the test material in DMSO at concentrations up to 500 µg/plate both with and without metabolic activation in accordance with the method of Ames et al. (Litton Bionetics, Inc., 1977).

Under the conditions of this study the test material did not exhibit mutagenic activity in any of the assays conducted in this evaluation and was considered not mutagenic according to the evaluation criteria.

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
no guideline available
Principles of method if other than guideline:
Ames et al., Mutation Research 31:347, 1975

Description of test procedure: The plate test consisted of direct revertant colony counts obtained from a set of selective agar plates seeded with populations of mutant cells suspended in a semisolid overlay. Approximately 0.1 to 0.2 mL of the test organisms were treated with the test substance in the presence and absence of a metabolic activation system (Aroclor 1254-treated rat liver supernatant). One plate was used per concentration. The plates were incubated for approximately 48 hours at 37 °C, and scored for the number of colonies growing on each plate.

Criteria for evaluating results: The solvent control values must be within the normal historical control range and the presence of a dose response is required for establishing mutagenicity. For strains TA1535, TA1537 and TA1538, a test substance producing a positive response equal to three times the solvent control value is considered mutagenic. For strains TA98 and TA100, a test substance producing a positive response equal to twice the solvent control value is considered mutagenic. In addition, a positive response must be repeated in a separate assay.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate prepared from Aroclor 1254 induced Sprague-Dawley male rats.
Test concentrations with justification for top dose:
0, 1, 10, 100, 500, 1000, 2500, 5000 and 10,000 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Anthramine-With metabolic activation: All strains
Details on test system and experimental conditions:
Approximately 0.1 to 0.2 mL of the test organisms were treated with the test substance in the presence and absence of a metabolic activation system (Aroclor 1254-treated rat liver supernatant). One plate was used per concentration. The plates were incubated for approximately 48 hours at 37 °C, and scored for the number of colonies growing on each plate.
Key result
Species / strain:
S. typhimurium, other: TA1537, TA1538, TA1535 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
With and without metabolic activation at 5000-10000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
With and without metabolic activation at 10000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The number of revertants/plate produced by treatment of the bacteria with the test substance at all concentrations and in all tester strains, was reported to be less than or approximately equal to the number of revertants in the solvent-treated negative control group, with and without metabolic activation. 

Revertants Per Plate

Activation:  None

Dose (µg/plate)

TA1535

TA1537

TA1538

TA98

TA100

Solvent (DMSO)

18

8

10

52

118

Solvent (DMSO)

19

14

17

54

132

1.0

18

12

18

45

99

10

11

6

23

56

128

100

13

7

13

44

123

500

16

8

19

46

111

1000

11

5

17

57

116

2500

14

4

11

56

84

5000

3

0

0

35

43

10,000

1

0

0

4

8

Positive Control

584

190

820

876

1143

Positive Control

645

299

925

913

1249

Revertants Per Plate

Activation:  Rat Liver S9

Dose (µg/plate)

TA1535

TA1537

TA1538

TA98

TA100

Solvent (DMSO)

15

9

21

68

137

Solvent (DMSO)

17

20

23

73

138

1.0

18

10

28

47

114

10

10

10

30

69

148

100

9

12

18

67

147

500

17

5

20

57

124

1000

18

12

20

65

134

2500

10

9

16

53

104

5000

9

2

4

54

86

10,000

3

0

0

18

42

Positive Control

443

227

1555

1510

1608

Positive Control

450

308

1756

1554

1661

Conclusions:
Under the conditions of this study the test material did not exhibit mutagenic activity in any of the assays conducted in this evaluation and was considered not mutagenic according to the evaluation criteria.
Executive summary:

S. typhimurium TA1535, TA1537, TA1538, TA98 and TA 100 were exposed to the test material in DMSO at concentrations up to 10 000 µg/plate both with and without metabolic activation in accordance with the method of Ames et al. under GLP conditions (Litton Bionetics, Inc., 1981).

Under the conditions of this study the test material did not exhibit mutagenic activity in any of the assays and was considered not mutagenic.

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

Genetic toxicity in vivo

Description of key information

IN VIVO

An in vivo micronucleus assay conducted in ICR mice in accordance with OECD 474 concluded that BPA-DA is non-mutagenic.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Remarks:
OECD GLP, US FDA GLP (21 CFR 58); US EPA GLP (40 CFR 160 and 40 CFR 792), UK GLP and Japanese GLP Standard
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
The ICR mice were obtained from Harlan Sprague Dawley, Inc. and were approximately 6-8 weeks of age at study initiation, weighing 25.6-33.4 g (males) and 19.8-27.6g (females). Up to five mice of the same sex were group-housed in polycarbonate cages. The controlled environment parameters were 72 ± 3°F, 50 ± 20% relative humidity and a 12-hour light/dark cycle. The mice had free access to certified rodent chow and tap water.
Route of administration:
intraperitoneal
Vehicle:
Corn oil was determined to be the vehicle of choice based on a solubility determination of BPA-DA and compatibility of the vehicle with the test system. BPA-DA was a workable suspension in corn oil at 200 mg/mL, the maximum concentration tested in the solubility test.
Details on exposure:
In the pilot toxicity assay, one group of five male and five female mice were exposed to BPA-DA at a dose of 2000 mg/kg body weight and four groups of two male mice each to 1, 10, 100, or 1000 mg/kg. BPA-DA dosing formulations were administered at a volume of 20 mL/kg by a single IP injection. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration and 1 and 3 days after dose administration.
For the toxicity assay and supplement, all mice were weighed immediately prior to dose administration and the dose volume was based on individual body weight. In the toxicity study, animals were exposed to 1200, 1400, 1600 or 1800 mg/kg BPA-DA. Since mortality was observed at all these doses, two lower doses, 700 and 1000 mg/kg, were tested in the supplemental toxicity study. BPA-DA dosing formulations were administered at a volume of 20 mL/kg by a single IP injection. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration and 1 and 3 days after dose administration.

For the definitive micronucleus assay, ICR mice were assigned to seven groups, each containing 5 males and 5 females. Animals in five of these groups were treated either with the controls (negative or positive) or with BPA-DA at a dose of 200, 400 or 800 mg/kg and were euthanized 24 hours after treatment. Animals in the other two groups were treated either with the negative control or BPA-DA at a dose of 800 mg/kg and were euthanized 48 hours after treatment. BPA-DA vehicle mixture, the vehicle alone or the positive control was administered by a single IP injection at a dose volume of 20 mL/kg. IP injection was selected to maximize delivery of BPA-DA to the test system. All mice in the experimental and control groups were weighed immediately before dose administration, and the dose volume was based on individual body weight. Mice were observed after dose administration for clinical signs of toxicity.
Frequency of treatment:
Single treatment
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Preliminary Assay: 5/sex at 2000 mg BPA-DA/kg; 2 males/group at 1, 10, 100, or 1000 mg BPA-DA/kg
Toxicity and Supplement Assay: 5/sex/group
Definitive Assay: 5/sex/group
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes, Cyclophosphamide monohydrate (CP, CAS number 6055-19-2)
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
At the scheduled sacrifice times, five mice per sex per dose were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes, and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May Gruenwald Giemsa and permanently mounted.
Bone marrow cells [polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)], were analysed for the presence of micronuclei. Polychromatic erythrocytes are young, immature red blood cells that stain bluish while normochromatic erythrocytes or normocytes are mature red blood cells that stain pink. Micronuclei are round, darkly-staining nuclear fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei can occur in both PCEs (MPCEs) and NCEs (MNCEs).
To control for bias, slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification (10 x 40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10 x 100), 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
To quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and dose group.
As a guide to interpretation of the data, BPA-DA was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p ≤ 0.05, Kastenbaum Bowman Tables) at any sampling time. However, values that were statistically significant but did not exceed the range of historical negative or vehicle controls were judged as not biologically significant. BPA-DA was judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and dose group. Statistical significance was determined using the Kastenbaum Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
males greater than 400 mg/kg and females greater than 800 mg/kg
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the pilot study, mortality was observed in 4/5 male mice and 4/5 female mice at 2000 mg/kg. Clinical signs following dose administration included: piloerection in males at doses ≥10 mg/kg and in females at 2000 mg/kg. Lethargy was seen in males at 1000 mg/kg and in males and females at 2000 mg/kg and hunched position in males and females at 2000 mg/kg.
In the toxicity study, mortality was observed in 5/5 male and 5/5 female mice at 1800 mg/kg, 5/5 males and 2/5 females at 1600 mg/kg, 4/5 males and 3/5 females at 1400 mg/kg, in 4/5 males and 3/5 females at 1200 mg/kg and in 1/5 males and 1/5 females at 1000 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in males and females at all doses and hunched position in males at 1200 mg/kg and in males and females at all doses ≥ 1400 mg/kg. Based upon these results, the high dose for the micronucleus test was set at 800 mg/kg, which was estimated to be the maximum tolerated dose.
In the definitive micronucleus study, no mortality was observed in any male or female. Clinical signs following dose administration included: piloerection in males and females at all doses and lethargy in males at 400 mg/kg and in males and females at 800 mg/kg. All other animals treated with control articles appeared normal during the course of the study.
Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after treatment were examined microscopically for presence of micronuclei (MPCEs or MNCEs). Reductions of 15%, 37% and 56% in the ratio of polychromatic erythrocytes to total erythrocytes were observed at 800 mg/kg in female group 24 hours postdose and in male and female group 48 hours postdose, respectively. These reductions demonstrate that there was bioavailability of BPA-DA to the bone marrow target tissue. The number of micronucleated polychromatic erythrocytes per 10,000 polychromatic erythrocytes in BPA-DA-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose or bone marrow collection time (p > 0.05, Kastenbaum-Bowman Tables).

In this study, all criteria for a valid test were met as specified in the protocol. CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p < 0.05, Kastenbaum-Bowman Tables). The negative and positive controls were consistent with the historical control data, indicating that there was no problem with the test system or the quality of the test.

Summary of Bone Marrow Micronucleus Analysis Following a Single Dose of Bisphenol A Dianhydride (BPA-DA; CAS# 38103-06-9) in ICR Mice

Treatment (20 mL/kg)

Sex

Time (hr)

Number of Mice

PCE/Total             Erythrocytes        (Mean ± SD)

Change from Control (%)

Micronucleated Polychromatic Erythrocytes

Number per 1000 PCEs (Mean ± SD)

Number per PCEs Scored

Corn oil*

M

24

5

0.485 ± 0.06

---

0.8 ± 0.27

8 / 10000

F

24

5

0.464 ± 0.09

---

0.4 ± 0.42

4 / 10000

BPA-DA

200 mg/kg

M

24

5

0.464 ± 0.07

-4

0.4 ± 0.42

4 / 10000

F

24

5

0.460 ± 0.05

-1

0.4 ± 0.22

4 / 10000

400 mg/kg

M

24

5

0.457 ± 0.08

-6

0.5 ± 0.35

5 / 10000

F

24

5

0.456 ± 0.07

-2

0.6 ± 0.42

6 / 10000

800 mg/kg

M

24

5

0.471 ± 0.05

-3

0.3 ± 0.45

3 / 10000

F

24

5

0.396 ± 0.08

-15

0.6 ± 0.22

6 / 10000

CP*

50 mg/kg

M

24

5

0.341 ± 0.04

-30

20.1 ± 3.34

*201 / 10000

F

24

5

0.324 ± 0.01

-30

19.4 ± 3.15

*194 / 10000

Corn oil*

M

48

5

0.480 ± 0.03

---

0.7 ± 0.27

7 / 10000

F

48

5

0.519 ± 0.02

---

0.5 ± 0.00

5 / 10000

BPA-DA

800 mg/kg

M

48

5

0.301 ± 0.06

-37

0.7 ± 0.27

7 / 10000

F

48

5

0.228 ± 0.09

-56

0.9 ± 0.55

9 / 10000

*Statistically significant, p 0.05 (Kastenbaum-Bowman Tables)

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 24 Hours Following a Single Dose of Bisphenol A Dianhydride (BPA-DA; CAS# 38103-06-9) in ICR Mice

Treatment (20 mL/kg)

Sex

Animal Number

PCE/Total Erythrocytes

Micronucleated PCE (Number/PCE scored)

Corn oil*

M

1

0.467

1 / 2000

2

0.435

2 / 2000

3

0.420

2 / 2000

4

0.564

1 / 2000

5

0.540

2 / 2000

F

6

0.340

0 / 2000

7

0.571

1 / 2000

8

0.519

2 / 2000

9

0.407

1 / 2000

10

0.485

0 / 2000

BPA-DA

200 mg/kg

M

11

0.464

1 / 2000

12

0.411

2 / 2000

13

0.510

0 / 2000

14

0.377

1 / 2000

15

0.556

0 / 2000

F

16

0.416

1 / 2000

17

0.474

1 / 2000

18

0.528

0 / 2000

19

0.461

1 / 2000

20

0.421

1 / 2000

400 mg/kg

M

21

0.461

1 / 2000

22

0.322

1 / 2000

23

0.467

1 / 2000

24

0.482

0 / 2000

25

0.552

2 / 2000

F

26

0.461

2 / 2000

27

0.414

1 / 2000

28

0.411

1 / 2000

29

0.568

2 / 2000

30

0.425

0 / 2000

800 mg/kg

M

31

0.417

0 / 2000

32

0.519

0 / 2000

33

0.531

2 / 2000

34

0.472

0 / 2000

35

0.417

1 / 2000

F

36

0.409

1 / 2000

37

0.470

1 / 2000

38

0.317

1 / 2000

39

0.310

2 / 2000

40

0.474

1 / 2000

CP*

50 mg/kg

M

71

0.314

35 / 2000

72

0.383

33 / 2000

73

0.319

42 / 2000

74

0.376

41 / 2000

75

0.314

50 / 2000

F

76

0.310

44 / 2000

77

0.338

36 / 2000

78

0.332

43 / 2000

79

0.323

29 / 2000

80

0.316

42 / 2000

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 48 Hours Following a Single Dose of  Bisphenol A Dianhydride (BPA-DA; CAS# 38103-06-9) in ICR Mice

Treatment (20 mL/kg)

Sex

Animal Number

PCE/Total Erythrocytes

Micronucleated PCE (Number/PCE scored)

Corn oil*

M

81

0.478

2 / 2000

82

0.458

2 / 2000

83

0.473

1 / 2000

84

0.524

1 / 2000

85

0.465

1 / 2000

F

86

0.520

1 / 2000

87

0.536

1 / 2000

88

0.535

1 / 2000

89

0.481

1 / 2000

90

0.523

1 / 2000

BPA-DA

800 mg/kg

M

91

0.251

1 / 2000

92

0.312

1 / 2000

93

0.343

2 / 2000

94

0.367

2 / 2000

95

0.231

1 / 2000

F

96

0.309

2 / 2000

97

0.112

3 / 2000

98

0.200

2 / 2000

99

0.194

2 / 2000

100

0.326

0 / 2000

Conclusions:
Under the conditions of the assay described in this report, a single intraperitoneal administration of the test material at doses up to 800 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test material was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

The study was conducted in ICR mice in accordance with OECD 474 under GLP conditions (BioReliance, 2003).

Male and female ICR mice were dosed via the intraperitoneal route with test material concentrations of 200, 400 or 800 mg/kg body weight in corn oil.

No mortality was observed. Clinical signs following dose administration included: piloerection in males and females at all doses and lethargy in males at 400 mg/kg and in males and females at 800 mg/kg. All other animals treated with control articles appeared normal during the course of the study.

Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after treatment were examined microscopically for presence of micronuclei (MPCEs or MNCEs). Reductions of 15, 37 and 56 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed at 800 mg/kg in female group 24 hours post-dose and in male and female group 48 hours post-dose, respectively. These reductions demonstrate that there was bioavailability of the test material to the bone marrow target tissue.

Under the conditions of the assay, the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, it was concluded to be negative in the micronucleus test.

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

Additional information

In vitro

Bacterial reverse mutation assay

Four studies are available to address this endpoint, one key and three supporting. The key study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). The supporting studies were all awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The key study was conducted in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A in accordance with OECD 471 under GLP conditions (BioReliance, 2002).

Bacteria were exposed to the test material at concentrations up to 5000 µg/plate in DMSO both with and without S9 metabolic activation.

Under the conditions of this study, BPA-DA did not cause a positive response in either the presence or absence of Aroclor induced rat liver S9 and is therefore non-mutagenic.

 

In the first supporting study, S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and TA1538 were exposed to the test material in DMSO at concentrations up to 30 mg/plate both with and without metabolic activation in accordance with the US EPA Health Effects Test Guidelines under GLP conditions (Bushy Run Research Center, 1988).

The test substance did not exhibit mutagenic activity in any of the assays and was considered not mutagenic under these test conditions.

 

In the second supporting study, S. typhimurium TA1535, TA1537, TA1538, TA98 and TA 100 were exposed to the test material in DMSO at concentrations up to 10 000 µg/plate both with and without metabolic activation in accordance with the method of Ames et al. under GLP conditions (Litton Bionetics, Inc., 1981).

The test substance did not exhibit mutagenic activity in any of the assays and was considered not mutagenic under these test conditions.

 

In the third supporting study, S. typhimurium TA1535, TA1537, TA1538, TA98 and TA 100 and Saccharomyces cerevisiae (Strain D4) were exposed to the test material in DMSO at concentrations up to 500 µg/plate both with and without metabolic activation in accordance with the method of Ames et al. (Litton Bionetics, Inc., 1977).

The test substance did not exhibit mutagenic activity in any of the assays and was considered not mutagenic under these test conditions.

 

Chromosome aberration test

The key study was conducted in Chinese hamster Ovary (CHO) cells in accordance with OECD 473 under GLP conditions (BioReliance, 2004). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

CHO cells were exposed to the test material in DMSO at concentrations up to 1500 µg/mL without metabolic activation and up to 750 µg/mL with metabolic activation.

Under the conditions of the assay, BPA-DA was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

 

Mammalian cell gene mutation assay

The key study was conducted in mouse lymphoma L5178Y cells in accordance with OECD 476 under GLP conditions (BioReliance, 2004). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Mouse lymphoma L5178Y cells were exposed to the test material in DMSO at concentrations up to 150 µg/mL with metabolic activation (4-hour exposure) and up to 125 µg/mL without metabolic activation (4- and 24-hour exposure).

Under the conditions of this study, the mutagenicity of BPA-DA was concluded to be negative with and without metabolic activation.

 

In vivo

Micronucleus assay

The key study was conducted in ICR mice in accordance with OECD 474 under GLP conditions (BioReliance, 2003). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Male and female ICR mice were dosed via the intraperitoneal route with test material concentrations of 200, 400 or 800 mg/kg body weight in corn oil.

No mortality was observed. Clinical signs following dose administration included: piloerection in males and females at all doses and lethargy in males at 400 mg/kg and in males and females at 800 mg/kg. All other animals treated with control articles appeared normal during the course of the study.

Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after treatment were examined microscopically for presence of micronuclei (MPCEs or MNCEs). Reductions of 15, 37 and 56 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed at 800 mg/kg in female group 24 hours post-dose and in male and female group 48 hours post-dose, respectively. These reductions demonstrate that there was bioavailability of BPA-DA to the bone marrow target tissue.

Under the conditions of the assay, BPA-DA did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, it was concluded to be negative in the micronucleus test.

Justification for selection of genetic toxicity endpoint

No single study is selected as key on the basis that the different studies address different aspects of genetic toxicity and the data are therefore all considered to be key.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to mutagenicity.