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Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-03-05 to 2009-04-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision repair system. Tester strains TA98 and TA100 also contain the pKM101 plasmid (carrying the R factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch repair process.

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 fr meshift 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).

The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et al., 1990). Tester strain WP2 uvrA has a deletion in the uvrA gene resulting in a deficient DNA excision repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base pair substitution mutations (Green and Muriel, 1976).
Test concentrations with justification for top dose:
The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells.
Details on test system and experimental conditions:
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 received from Dr. Bruce
Ames’ designated distributor, Discovery Partners International, San Diego, California. E. coli
tester strains were received from the National Collection of Industrial and Marine Bacteria,
Aberdeen, Scotland.

Overnight cultures were prepared by inoculating from the appropriate master plate or from the
appropriate frozen permanent stock into a vessel containing ~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 resting shaker/incubator at room
temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm
at 37±2°C approximately 12 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 0.3x10e9 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 lot of S9 was prepared by and
purchased from MolTox (Boone, NC). 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 at
least two promutagens 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
BioReliance 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 using dimethyl sulfoxide (DMSO) to determine the highest
soluble or workable stock concentration, up to 500 mg/mL.
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 was 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 was equal to or
greater than 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 will be evaluated as negative, if it is
neither positive nor equivocal.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
Historical Negative and Positive Control Values
2005 – 2007

revertants per plate
Strain Control Activation
None Rat Liver
Mean SD Min Max Mean SD Min Max
TA98 Neg 17 6 5 56 25 8 6 60
Pos 189 112 30 1812 526 343 73 2669
TA100 Neg 133 30 52 250 143 32 67 263
Pos 548 148 112 4349 698 343 232 2652
TA1535 Neg 20 7 4 53 16 5 4 45
Pos 420 146 54 1019 104 59 18 985
TA1537 Neg 7 3 1 20 8 3 1 29
Pos 740 356 14 2514 88 87 13 1297
WP2 uvrA Neg 21 9 5 69 23 10 4 65
Pos 214 137 28 1086 268 174 29 1178
SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

Bacterial Mutation Assay

Summary of Results - Initial Toxicity-Mutation Assay

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Test Article Id

:

DADPM/DEG-PO

 

Study Number

:

AC23JM.503.BTL

 

Experiment No

:

B1

 

 Average Revertants Per Plate ± Standard Deviation

 

Activation Condition

:

None

 

Dose (µg per plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

 

Vehicle

16

±

4

215

±

23

18

±

4

6

±

3

29

±

1

 

1.5

14

±

4

207

±

5

19

±

4

6

±

1

27

±

2

 

5.0

14

±

3

196

±

24

17

±

1

3

±

1

33

±

6

 

15

9

±

3

185

±

27

16

±

0

8

±

0

31

±

6

 

50

15

±

4

154

±

23

17

±

1

9

±

1

31

±

0

 

150

9

±

0

146

±

1

19

±

2

6

±

2

31

±

4

 

500

11

±

1

167

±

1

15

±

0

5

±

1

27

±

1

 

1500

19

±

1

153

±

3

12

±

1

9

±

1

22

±

1

 

5000

10

±

1

145

±

6

19

±

1

9

±

2

33

±

5

 

Positive

278

±

53

651

±

33

489

±

28

1977

±

23

405

±

28

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Activation Condition

:

Rat Liver S9

 

Dose (µg per plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

 

Vehicle

26

±

1

168

±

23

14

±

1

8

±

0

32

±

8

 

1.5

29

±

1

160

±

7

17

±

5

11

±

0

31

±

6

 

5.0

30

±

1

174

±

6

18

±

2

5

±

1

34

±

6

 

15

29

±

3

180

±

5

14

±

0

5

±

1

28

±

8

 

50

35

±

3

179

±

3

16

±

1

7

±

4

33

±

1

 

150

28

±

1

163

±

18

10

±

4

10

±

4

27

±

4

 

500

41

±

11

164

±

5

17

±

5

8

±

1

37

±

2

 

1500

29

±

4

188

±

9

16

±

4

6

±

3

36

±

3

 

5000

31

±

1

163

±

4

13

±

4

8

±

0

37

±

6

 

Positive

345

±

56

1064

±

67

92

±

3

86

±

1

262

±

25

 

Vehicle = Vehicle Control

 

Positive = Positive Control (50 µL plating aliquot)

Plating aliquot = 50 µL

Bacterial Mutation Assay

Summary of Results - Confirmatory Mutagenicity Assay

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Test Article Id

:

DADPM/DEG-PO

 

Study Number

:

AC23JM.503.BTL

 

Experiment No

:

B2

 

 

 

 

 

 

 

 

 

 Average Revertants Per Plate ± Standard Deviation

 

Activation Condition

:

None

 

Dose (µg per plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

 

Vehicle

11

±

3

122

±

9

15

±

3

8

±

3

26

±

7

 

50

17

±

5

107

±

13

17

±

2

5

±

3

34

±

3

 

150

15

±

6

144

±

16

17

±

8

7

±

3

34

±

6

 

500

16

±

4

125

±

4

17

±

3

6

±

1

35

±

4

 

1500

12

±

6

140

±

8

17

±

2

4

±

3

25

±

3

 

5000

14

±

6

129

±

20

19

±

5

4

±

2

27

±

6

 

Positive

206

±

24

593

±

81

453

±

12

1674

±

221

352

±

53

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Activation Condition

:

Rat Liver S9

 

Dose (µg per plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

 

Vehicle

20

±

8

143

±

9

14

±

5

4

±

2

29

±

2

 

50

19

±

8

119

±

24

12

±

3

6

±

3

29

±

8

 

150

24

±

2

115

±

3

15

±

1

4

±

3

45

±

6

 

500

27

±

5

117

±

16

14

±

4

5

±

1

35

±

4

 

1500

35

±

6

132

±

13

10

±

2

4

±

3

32

±

6

 

5000

33

±

6

115

±

11

11

±

4

5

±

4

33

±

11

 

Positive

341

±

49

705

±

51

91

±

14

52

±

12

271

±

45

 

Vehicle = Vehicle Control

 

Positive = Positive Control (50 µL plating aliquot)

Plating aliquot = 50 µL

Conclusions:
Interpretation of results (migrated information):
negative under the conditions of this study, test article DADPM/DEG-PO did not cause a positive mutagenic response in either the presence or absence of Aroclor induced rat liver S9.

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, test article DADPM/DEG-PO did not cause a positive mutagenic response in either the presence or absence of Aroclor induced rat liver S9.
Executive summary:

The purpose of this study was to evaluate the mutagenic potential of the test article by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9.

The test article, DADPM/DEG-PO, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation 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 vortexing for 10 minutes and sonication at 18°C for 2 minutes, the test article formed a soluble and clear solution at approximately 500 mg/mL, the maximum concentration tested.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. In the initial toxicity-mutation assay, no positive mutagenic response was observed. Precipitate was observed at 5000 µg per plate. No appreciable toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 µg per plate.

In the confirmatory mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate. Precipitate was observed at 5000 µg per plate with a few test conditions. No appreciable toxicity was observed. 

Under the conditions of this study, test article Daltolac XR 159 (DADPM/DEG-PO) was concluded to be negative in the Bacterial Reverse Mutation Assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 March 2021 to 07 April 2021
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine phosphoribosyl transferase (HPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese hamster ovary (CHO-K1)
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
50, 100, 150, 200, 250 and 312.5 ug/mL

The test material was dosed at concentrations up to 5000 µg/mL. Precipitate was observed by eye at the end of treatment at concentrations of 312.5 µg/mL and above and this was, therefore, the highest concentration plated for determination of relative survival (RS) in both the absence and presence of S9 mix. Exposure to the test material for 3 hours at concentrations from 39.06 to 312.5 µg/mL in both the absence and presence of S9 mix resulted in RS values from 102 to 1% and 102 to 26% respectively. Concentrations for the main test were based upon these data.
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Details on test system and experimental conditions:
Preliminary Toxicity Test Procedure
A cell suspension was prepared at 3 x 105 cells/mL. Aliquots of 20 mL of this suspension were dispensed into 150 cm2 flasks, one flask per concentration of test item and two for the vehicle controls both in the absence and in the presence of S9 mix. Eight concentrations of the test item were used. Two additional flasks were prepared to determine average cell density across all flasks at the beginning of the treatment period to ensure a minimum of 10 x 106 cells being present during treatment; this value was subsequently also used to calculate the adjusted cloning efficiency. The cells were incubated for approximately 24 hours at between 34 and 39C, in a humidified atmosphere of 5% CO2 in air, prior to exposure to the test item on Day 1. Prior to treatment, the medium was replaced with 16 mL of fresh medium and aliquots of 4 mL of H0 or S9 mix as appropriate followed by 200 L of test item (at 100 times the desired final concentration) or vehicle. The flasks were returned to the incubator for a further three hours.
At the end of the exposure period the cells were harvested. Samples were taken from each culture, the cells counted and the cell density calculated. Three flasks per culture were seeded with 200 cells each (survival flasks). The plates were incubated at between 34 and 39C, in a humidified atmosphere of 5% CO2 in air, for seven days, after which time colonies growing in the plate were fixed and stained in a methanol:Giemsa solution (4:1 v/v). Colonies were counted and the Day 1 relative survival was calculated. These data were used to determine the concentrations of the test item used in the main tests.

Main Test Procedure
The procedure for the main tests was the same as that for the preliminary tests, with the following exceptions: positive control cultures were included for all tests; duplicate cultures were prepared for all cultures (quadruplicate cultures for vehicle controls); and following preparation of survival flasks, 2 x 106 cells from each culture were seeded into 150 cm2 flasks containing 30 mL H10 and incubated for seven days to allow expression of the mutant phenotype. The cultures were sub-cultured during the expression period and after a total of seven days, were harvested. For each culture, three flasks were seeded with 200 cells each, to determine cloning efficiency and five flasks with 5 x 105 cells each in selective medium to determine cloning efficiency. The flasks were returned to the incubator for approximately seven days at between 34 and 39C in a humidified atmosphere of 5% CO2 in air. At the end of this incubation period, colonies growing in the flasks were fixed and stained in a methanol:Giemsa solution (4:1 v/v) and counted.
Evaluation criteria:
Acceptance criteria for test item:
The highest concentration tested was one that allowed the maximum exposure up to 5000 g/mL for freely soluble compounds, or the limit of toxicity (i.e. relative survival (RS) reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analyzable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RS.
Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 1 and 20 x 10-6.
The mean cloning efficiency was between 65 and 120%.
Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.
The concurrent vehicle control must be considered acceptable for addition to the laboratories historical vehicle control data base (ideally within the 95% confidence limits). Where concurrent vehicle control data fall outside the 95% confidence limit they may be acceptable for inclusion in the historical control distribution as long as these data are not extreme outliers and there is evidence that the test system is ‘under control’ and there is evidence of no technical or human failure.
Acceptance criteria for positive controls:
Positive controls showed a statistically significant increase in mean total MF above the mean concurrent vehicle control MF and within, or close to, the range of the historical control data.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1             Summary of results



















































































































Test Item



Concentration


(µg/mL)



3-hour Treatment ‑S9 mix



95% confidence limits of HCD



3-hour Treatment +S9 mix



95% confidence limits of HCD



Mean RS (%)



Mean MFa



Mean RS (%)



Mean MFa



DMSO



0



100



6.25



3.6 – 13.7



100



5.83



2.3 – 16.0



DADPM/DEG/PO



50



105



6.04



 



95



6.88



 



DADPM/DEG/PO



100



92



5.05



 



88



5.65



 



DADPM/DEG/PO



150



21



4.93



 



74



5.39



 



DADPM/DEG/PO



200



9b



6.54



 



52



5.36



 



DADPM/DEG/PO



250



c



 



 



45



5.27



 



DADPM/DEG/PO



312.5



c



 



 



20b



3.82



 



Ethyl methanesulphonate



250



93



69.25***



30.4 – 125.7



NT



NT



 



3-methylcholanthrene



5



NT



NT



 



68



57.68***



33.2 – 121.4



a. Mutant frequencies expressed per 106 viable cells


b. Precipitate observed at the end of treatment


c. Precipitate observed at the end of treatment, cultures discarded


 


RS:  Relative Survival


MF:  Mutant Frequency


NT:  Not tested


 


*** p<0.001; all other cultures p≥0.05.  Treated groups were compared to the vehicle control using one-tailed Dunnett’s tests for an increase and the positive control was compared to the vehicle control using a one-tailed t‑test for an increase


 


Conclusions:
Cultures were exposed to the test material at concentrations from 50 to 312.5 µg/mL. Precipitate was observed by eye at the end of treatment at a concentration of 312.5 µg/mL. Exposure to the test material resulted in mean RS values from 95 to 20%. All cultures were plated out for determination of cloning efficiency and mutant frequency. No significant increases in mean mutant frequency were observed after exposure to the test material at any concentration analysed. None of the treated groups induced mean mutant frequencies above the laboratory historical control data 95% confidence limits and the test for non-linearity was applied across all treatment groups and was not statistically significant. A test for linear trend was applied across all treatment groups and was statistically significant (p= 0.010) resulting from a negative trend and therefore was not biologically relevant.
3MC, the positive control, induced a significant increase in mean mutant frequency.
Executive summary:

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, under the experimental conditions described.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-02-28 to 2013-04-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System:
ICR (CD-1) mice were obtained from Harlan, Frederick, MD. At the time of dose administration for each phase of the study, the mice were 6 weeks old. Mouse body weights recorded at randomization were within the following ranges:
Study Phase: 27.4 – 30.9 grams (Males) and 23.7 – 26.8 grams (females)
Definitive Micronucleus Study: 28.9 – 33.3 grams (Males)

Animal Receipt and Acclimation:
Virus antibody-free (VAF) mice were obtained from a supplier that monitored mice for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were acclimated for 5 or 6 days after receipt. At BioReliance, mice were observed each day for signs of illness and other conditions of poor health. All mice were judged to be healthy prior to utilization in the study.

Animal Welfare Provisions and Animal Care:
All procedures involving mice performed at BioReliance follow the specifications recommended in The Guide for the Care and Use of Laboratory Animals adopted by BioReliance.
Animals were housed in an AAALAC-accredited facility with a controlled environment of 50 ± 20% relative humidity and 72 ± 3°F temperature with a 12-hour light/dark cycle. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour.

Mice of the same sex were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.
Heat-treated Sani-Chip hardwood chips were used for bedding to absorb liquids (P.J. Murphy Forest Products, Montville, NJ). Bedding was analyzed by the Manufacturer for any contaminants.
Animals were allowed free access to tap water, which meets U.S. EPA drinking water standards [water source is Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum.

Randomization:
In each assay, mice were assigned to the appropriate number of treatment groups. Animals were assigned to these groups using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals did not exceed ±20% from their group mean. Following randomization, animals were identified by sequentially numbered ear tags assigned to each animal during the randomization process. The cage card contained the following information: the animal number, sex, study number, test substance identification, treatment group number, dose level and route of administration. Cage cards were color coded as a function of the treatment group. Raw data records and specimens were also identified by the unique test animal number.

Synopsis:
The study was conducted in two phases: a dose range-finding assay that evaluated the toxicity of the test substance and a definitive micronucleus assay that evaluated the genotoxic potential of the test substance. Based on the absence of mortality in the dose range-finding assay, male mice were exposed to DADPM/DEG-PO at 500, 1000 and 2000 mg/kg in the definitive micronucleus assay. At 24 and 48 hours post dose, bone marrow was collected and microscopically evaluated for the incidence of mnPCEs (Table 1).
Route of administration:
oral: gavage
Vehicle:
Mineral oil
Details on exposure:
Dose Administration Procedure:
All dose formulations were administered at a dose volume of 10 mL/kg by single oral administration using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). All mice in the experimental and control groups were weighed immediately before dose administration and the administered volume was based on individual body weight. Mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity.

Definitive Micronucleus Assay
Mineral oil was also used as the negative (vehicle) control and Cyclophosphamide monohydrate (CP), at a dose of 50 mg/kg, was used as the positive control substance in the definitive micronucleus assay. The test or control substances were administered by single oral gavage at a dose volume of 10 mL/kg body weight. Based on the absence of mortality in the dose range-finding assay, 5 male mice in each group were exposed to DADPM/DEG-PO at the OECD guideline recommended high dose of 2000 mg/kg and two lower dose levels, 500 and 1000 mg/kg (Table 1). Animals were observed for clinical signs of toxicity following each dose administration and at least once daily thereafter.
Duration of treatment / exposure:
Dose Range-Finding Assay:
The dose range-finding assay was conducted exposing three mice/sex to DADPM/DEG-PO at a dose of 2000 mg/kg. Mice were observed for clinical signs of toxicity, following each dose administration and at least once daily thereafter. Following the two-day observation period, all surviving animals were euthanized by carbon dioxide inhalation verified by toe pinch reflex, and discarded without further examination.
Frequency of treatment:
A single oral gavage dose.
Post exposure period:
24 and 48 hours
Remarks:
Doses / Concentrations:
0
Basis:
other:
Positive control(s):
Cyclophosphamide monohydrate (CP) at a dose of 50 mg/kg
Tissues and cell types examined:
At the scheduled time point, animals were euthanized and the femoral bone marrow was collected.
Details of tissue and slide preparation:
At the scheduled bone marrow collection time, five mice per sex per treatment were euthanized by CO2 asphyxiation verified by toe pinch reflex. Immediately following euthanasia, the femurs were 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 labeled centrifuge tube containing approximately 1 mL of fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100xg for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended 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 air dried and fixed in methanol. One set of slides was stained with a nucleic acid-specific stain, acridine orange, and was used in microscopic evaluation. The second set of slides was stored at BioReliance at room temperature as a backup.
Evaluation criteria:
The bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Bone marrow was evaluated by fluorescent microscopy. The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost-like, dark green NCEs, respectively). Slides initially were scanned using medium magnification to locate suitable areas where the cells were well spread and stained. Next, cells were scored using a high power oil immersion lens as follows.
The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus, occasional cells with more than one micronucleus are counted as one micronucleated PCE (mnPCE), not two (or more) micronuclei.
At least 2000 PCEs/animal were scored for the presence of micronuclei (mnPCEs) whenever possible. In addition, at least 1000 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity.
Statistics:
The frequency of mnPCEs and the proportion of PCEs to total erythrocytes were determined for each animal and treatment group. Statistical significance (p . 0.05) was determined using binomial distribution tables (Kastenbaum and Bowman, 1970).
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose Range-Finding Assay:
No mortality was observed. Piloerection or diarrhea was noted in male mice, and diarrhea was noted in female mice. No appreciable changes in the mean group body weight were observed during the study period.

Definitive Micronucleus Study:
No mortality was observed in any of the treatment groups. Diarrhea was noted in animals in the vehicle control and the 500, 1000 mg/kg treatment groups. Diarrhea or piloerection was noted in animals in the 2000 mg/kg treatment group. All mice in the positive control group appeared normal during the study period.

The incidence of micronucleated polychromatic erythrocytes per 10,000 polychromatic erythrocytes scored (2000 PCEs/mouse) and the proportion of polychromatic erythrocytes per total erythrocytes are summarized and presented for each treatment group by sacrifice time in Table 2.

• No reductions were observed in the PCEs/EC ratio with the test item at any dose level relative to the vehicle control group.
• No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance-treated groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman tables).
• CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p = 0.05, Kastenbaum-Bowman tables).
• The incidence of mnPCEs in the vehicle control group at 24 hours post dose was outside the historical control data (0-7 mnPCEs/10,000 PCEs) for vehicle control. However, the incidence of mnPCEs for individual animal was within the range for all animals. Therefore, the Study Director concluded that this out of specification results did not adversely impact the integrity of the data or the validity of the study conclusion.

Table 2: Summary of results

Toxic/Cytotoxic Effects: No appreciable reductions in the PCEs/EC ratio were observed at any dose level relative to the respective vehicle control groups.

Genotoxic Effects: No statistically significant increase in the incidence of mnPCEs was observed at either 24 or 48 hours post dose.

Evidence of Exposure: Piloerection or diarrhea was noted in some animals dosed at 500, 1000 and 2000 mg/kg.

 Sampling time  Controls/Test Substance  Dose (mg/kg)  Sex/No. of Animals/Group     PCE#/1000 Erythrocyte (Mean+/-SD)  Number mnPCE/PCE scored   
 24 hours post-dose  Mineral oil  0  Male  5  0.8 +/- 0.27  8 / 10,000 
 24 hours post-dose  DADPM/DEG-PO  500  Male  5  1.0 +/- 0.35  10  / 10,000
 24 hours post-dose  DADPM/DEG-PO  1000  Male  5  0.8 +/- 0.27  8  / 10,000
 24 hours post-dose  DADPM/DEG-PO  2000  Male  5  1.1 +/- 0.55  11  / 10,000
 24 hours post-dose  Cyclophosphamide  50  Male  5  14.5 +/- 2.57  *145 / 10, 000
               
 48 hours post-dose  Mineral oil  0  Male  5 0.7 +/- 0.27  7 / 10,000 
   DADPM/DEG-PO  2000  Male  5  1.1 +/- 0.89  11  /10,000

* Statistically significant increase relative to the vehicle control, p = 0.05 (Kastenbaum-Bowman tables binomial distribution)                     

# PCE = Polychromatic erythrocytes; b mnPCE = Micronucleated polychromatic erythrocytes 

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the study conducted as described above, a single oral administration of DADPM/DEG-PO at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male ICR mice. Therefore, DADPM/DEG-PO was concluded to be negative in the mouse micronucleus assay.
Executive summary:

The test substance, DADPM/DEG-PO, was evaluated for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (mnPCEs) cells in ICH (CD-1) mouse bone marrow.


The study was conducted in two phases: a dose range-finding assay to evaluate the toxicity of the test substance and a definitive micronucleus assay to evaluate the genotoxic potential of the test substance.


DADPM/DEG-PO was formulated using mineral oil as the vehicle and it was also used as the negative (vehicle) control in the definitive micronucleus assay. Cyclophosphamide monohydrate (CP), at a dose of 50 mg/kg, was used as the positive control substance in the definitive micronucleus assay. In both assays, test or control substances were administered by single oral gavage at a dose volume of 10 mL/kg body weight. Animals were observed for clinical signs of toxicity following each dose administration and at least once daily thereafter.


In the dose range-finding assay, three mice/sex were exposed to DADPM/DEG-PO at the maximum OECD guideline recommended dose level of 2000 mg/kg. No mortality was observed. Piloerection or diarrhea was noted in male mice, and diarrhea was noted in female mice. No appreciable changes in the mean group body weight were observed during the study period.


Due to the absence of mortality at 2000 mg/kg in the dose range-finding assay, the definitive micronucleus assay was conducted exposing the animals to DADPM/DEG-PO at dose levels of 500, 1000 and 2000 mg/kg. The vehicle and positive control substances were tested concurrently. Since no substantial differences in the clinical signs of toxicity between the sexes were observed in the dose range-finding assay, only male mice were used in the definitive micronucleus assay.


At the scheduled time point, animals were euthanized and the femoral bone marrow was collected. Bone marrow smears (slides) were prepared and stained with acridine orange, a nucleic acid specific stain and were examined microscopically for the presence of micronuclei (mnPCEs). A statistical analysis of the data was performed using the Kastenbaum-Bowman tables (binomial distribution, p = 0.05). In addition, the ratio of PCEs to total erythrocytes (PCEs/EC ratio) was also evaluated as an indication of bone marrow toxicity.


The following observations were made in the definitive micronucleus assay:


• No mortality was observed in any of the treatment groups. Diarrhea was noted in animals in the vehicle control and the 500, 1000 mg/kg treatment groups. Diarrhea or piloerection was noted in animals in the 2000 mg/kg treatment group. All mice in the positive control group appeared normal during the study period.


 


• No reductions were observed in the PCEs/EC ratio with DADPM/DEG-PO at any dose level relative to the vehicle control group.


• No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance-treated groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman tables).


• CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p = 0.05, Kastenbaum-Bowman tables).


• The incidence of mnPCEs in the vehicle control group at 24 hours post dose was outside the historical control data (0-7 mnPCEs/10,000 PCEs) for vehicle control. However, the incidence of mnPCEs for individual animal was within the range for all animals. Therefore, the Study Director concluded that this out of specification results did not adversely impact the integrity of the data or the validity of the study conclusion.


Based upon this, all criteria for a valid test were met as specified in the protocol.


Under the conditions of the study as described in this report, a single oral administration of DADPM/DEG-PO at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male ICR (CD-1) mice. Therefore, DADPM/DEG-PO was concluded to be negative in the mouse micronucleus assay.


This in vivo micronucleus test was requested by the National Institute of Environmental Research (NIER) of Korea for their registration of DADPM/DEG-PO.

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

Additional information

DADPM/DEG/PO was evaluated in a bacterial reverse mutation assay (OECD 471 guideline study). The test material did not induce an increase in the frequency of revertant colonies in either the absence or presence of S9 -mix in either of two separate experiments (BioReliance, 2009).


 


DADPM/DEG/PO did not induce mutagenic effects in the in vitro gene mutation assay (HPRT test, according to OECD 476 and under GLP) with Chinese hamster ovary cells in the presence and absence of a metabolic activation system (Covance, 2021).


 


DADPM/DEG/PO was also evaluated in an in vivo mouse micronucleus assay (OECD 474). The test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male ICR (CD-1) mice. Therefore, DADPM/DEG-PO was concluded to be negative in the mouse micronucleus assay (BioReliance 2013). The in vivo micronucleus test was requested by the National Institute of Environmental Research (NIER) of Korea for their registration of DADPM/DEG/PO



Short description of key information:
DADPM/DEG/PO was tested in an in vitro bacterial reverse mutation assay, an in vitro HPRT and in vivo mouse micronucleus test. All three studies showed negative results. 

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The findings from the in-vitro and in vivo genotoxicity testing do not warrant classification.