Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

Bacterial reverse mutation assay:

BASF (1998) performed an Ames test (plate incorporation and pre-incubation test) withS. typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 andE. colistrain WP2 uvr A with and without metabolic activation.

Following test concentrations were applied in triplicate: 0, 20, 100, 500, 2500, 5000 µg/plate (standard plate test and preincubation test). Solvent and positive controls were run in triplicate and were considered to be valid.According to the results of the study, the test substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay with and without metabolic activation. This K1 study is selected as key study. In addition, a supporting K2 Ames study is available from the Central Labor Accident Prevention Institute, Nippon Bioassay Research Center (1988), in which an ambiguous result for mutagenicity was observed.

Chromosome aberration test:

Genetic Laboratory, JBC (1994) performed an in vitro chromosome aberration test in CHL/IU male Chinese hamster lung fibroblasts with and without metabolic activation. Following doses were tested in duplicate:

Continuous treatment for 24h or 48h : 63, 125, 250, 500, 1000 µg/ml (without or with S9)

Pulse treatment for 6h, and 18h recovery: 313, 625, 1250, 2500, 5000 µg/ml (with or without S9)

According to the report, the positive result in the first pulse test was a false positive result. The dose-dependent increase could not be repeated in a second and third pulse test, although in the third test positive responses were observed. The report concludes that the test substance is mutagenic. However, we conclude, based on the results in this test, that the test substance is negative in the presence of S9, and ambiguous in the absence of S9. In addition, an in vivo micronucleus test was performed: the test substance was concluded to be negative in the in vivo mouse micronucleus assay.

CHO/HGPRT Assay:

BioReliance (2012) performed an in vitro mammalian cell gene mutation assay in Chinese hamster Ovary (CHO) cells with and without metabolic activation. Following concentrations were tested in duplicate: 250, 500, 1500, 3000, 4000, 5000 µg/mL (5 hours of exposure). A vehicle control (sterile distilled water) and positive control (ethylmethanesulphonate without metabolic activation; benzo(a)pyrene with metabolic activation) were scored as well. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, the test substance was concluded to be negative with and without metabolic activation. Positive and negative controls were considered valid. No cytotoxicity was observed.

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
Remarks:
Guideline study, GLP study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only 2-aminoanthracene as positive control with metabolic activation)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: H 50541 H x B (Substance number: 95/452)
- Analytical purity: 98.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (N2 conditions)
- Stability under test conditions: guaranteed throughout study period via reanalysis

OTHER SPECIFICS:
- Name of test substance (as cited in study report): 2,2' -Dimorpholinodiethylether
- Physical state: liquid
Target gene:
His- and Trp-Operon
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 1254-induced rat liver S9-mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2,500, 5,000 µg/plate (standard plate test and preincubation test)
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: with S9: 2-aminoanthracene (all tester strains); without S9: N-methyl-N'-nitro-N-nitrosoguanidine (TA 1535, TA 100), 4-nitro-o-phenylendiamine (TA 98), 9-aminoacridine (TA 1537), N-ethyl-N'-nitro-N-nitrosoguanidine (E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION (preincubation test)
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

DURATION (standard plate test)
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 plates/dose/experiment.


DETERMINATION OF CYTOTOXICITY
- Method: reduction of the number of revertants compared to negative control; reduction of titer; reduction of backgroud growth

Evaluation criteria:
The test subtance is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9-mix or after adding a metabolizing system.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation up to 5,000 µg/plate (highest dose tested)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Standard plate test:

 Dose (µg/plate)

 TA1535   

 TA100   

 TA1537   

 TA98   

    E. coli WP2 uvrA

 

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

-S9

 +S9

-S9 

+S9 

 0

20±5

19±2

128±10

123±4

10±1

10±1

31±4

47±5

32±1

42±2

20 

19±2

18±2

115±20

125±12

10±2

11±4

32±3

46±4

33±5

41±3

100

19±5

17±3

111±7

121±14

10±1

7±1

31±5

39±8

30±2

37±6

 500

19±3

14±1

128±8

107±14

9±3

8±1

27±5

33±10

30±1

39±2

 2500

19±3

16±4

123±7

121±15

10±1

6±0

31±4

38±5

32±4

42±2

 5000

18±3

15±2

140±3

97±17

8±1

8±1

28±6

28±8

30±3

43±2

 2 -AA

-

149±7

-

1467±

133

 -

149±24

1241±

29

 -

236±

17

 MNNG

783±

37

-

1027±

49

-

-

 -

-

 -

-

-

 AAC

-

-

-

-

804±13

-

-

-

-

-

 NOPD

 -

-

-

-

-

-

1308±

135

-

 ENNG

 -

-

-

-

-

-

-

-

521±

26

-

Mean ± SD

 

Preincubation-test:

 Dose (µg/plate)

 TA1535   

 TA100   

 TA1537   

 TA98   

 E. coli WP2

uvrA   

 

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

-S9

 +S9

 -S9

+S9 

 0

18±1

19±2

133±4

130±17

11±2

11±3

29±1

40±3

32±4

38±3

20

18±2

16±2

116±9

108±9

11±1

11±2

23±2

42±1

31±6

32±5

 100

21±1

17±0

124±6

121±10

8±1

11±1

24±2

36±5

28±4

33±5

 500

17±2

14±1

111±0

125±13

8±2

11±2

24±2

38±1

30±6

25±4

 2500

19±1

16±3

117±15

132±7

8±2

9±1

25±1

40±3

28±3

28±4

 5000

16±2

15±2

102±16

138±14

7±1

7±2

20±3

38±7

26±2

30±51

2-AA

-

116±13

-

699±

2

-

119±9

-

727±

12

-

177±11

 MNNG

978±

135

-

1177± 126

-

-

-

-

-

-

-

 AAC

-

-

-

-

402±62

-

-

-

-

-

NOPD

-

-

-

-

-

-

1148±

103

-

-

-

 ENNG

-

 -

 -

 -

-

 -

-

569±49

 -

Mean ± SD

X: reduced background growth

2-AA: 2-aminoanthracene

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

ENNG; N-ethyl-N-nitro-N-nitrosoguanidine

NOPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

 

According to the results of the study, the test substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental conditions chosen here. The positive controls gave the expected results.

 

Conclusions:
According to the results of the study, the test substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental conditions chosen here. The positive controls gave the expected results.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-06-07 to 1993-07-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed similar to OECD TG 473. No historical control data is present. Results and conclusions in this report are contradictory.
Qualifier:
equivalent or similar 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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 6950-77-89
- Expiration date of the lot/batch: not indicated
- Purity test date: not indicated
- Analytical purity: 99.12%
- Impurities (identity and concentrations): 0.78% hydroxyethoxyethylmorpholine

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark
- Stability under test conditions : stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: stable in physiological saline and DMSO

OTHER SPECIFICS:
- Name of test material (as cited in study report): Bis(2-morpholinoethyl)ether
- Substance type: liquid
- Physical state: liquid
- Other: other name Texacat DMDEE; molecular weight 244; melting point -28°C; boiling point 309°C

Species / strain / cell type:
mammalian cell line, other: CHL/IU male Chinese hamster lung fibroblasts
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's MEM medium supplemented with 10% calf serum (heat inactivated at 56°C for 30 min)
Additional strain / cell type characteristics:
other: modal chromosome number, 25
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-benzoflavone induced SD rat liver S9
Test concentrations with justification for top dose:
Continuous treatment for 24h or 48h : 63, 125, 250, 500, 1000 µg/ml (without or with S9)
Pulse treatment for 6h, and 18h recovery: 313, 625, 1250, 2500, 5000 µg/ml (with or without S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiological saline
- Justification for choice of solvent/vehicle: not indicated
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9 Migrated to IUCLID6: 0.05 µg/ml in phys. saline
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 Migrated to IUCLID6: 20 µg/ml in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration: 24 or 48h (continuous treatment); 6h (pulse treatment)
- Expression time (cells in growth medium): 18h (pulse treatment)
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): The cells were fixed by fresh methanol/acetic acid (3:1). The fixative was exchanged at least three times. No information is provided on fixation time.

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg/ml)
STAIN (for cytogenetic assays): 1.3% Giemsa solution

NUMBER OF REPLICATIONS: 2 plates per dose level; continuous treatment test not repeated, pulse treatment test repeated twice

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no
Evaluation criteria:
Negative if less than 4.9% aberrant cells
Suspicious if 5-10% aberrant cells
Positive (+, ++, +++) if resp. 10-20%, 20-50% or >50% aberrant cells.
Statistics:
No statistics performed
Species / strain:
mammalian cell line, other: CHL/IU male Chinese hamster lung fibroblasts
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
false positive according to study director
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mammalian cell line, other: CHL/IU male Chinese hamster lung fibroblasts
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: no

RANGE-FINDING/SCREENING STUDIES: performed to determine the effect of the substance on the mitotic index. Continuous treatment for 24h or 48h (without S9) and pulse treatement for 6h (with and without S9).

COMPARISON WITH HISTORICAL CONTROL DATA: no info on historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Continuous treatment 24h or 48h without S9: no significant increase in the number of aberrant cells. It is concluded that the substance is not clastogenic in the absence of S9

Pulse treatment 6h and 18h recovery without S9: dose-related increase in the number of aberrant cells (2.5% at 1250 µg/ml, 5% at 2500 µg/ml and 7% at 5000 µg/ml). The pulse treatment in the absence of S9 is invalid as the positive control did not significantly increase the proportion of cells with structural aberrations.

Pulse treatment 6h and 18h recovery with S9: no significant increase in the number of aberrant cells. It is concluded that the substance is not clastogenic in the presence of S9.

Pulse treatment 6h and 18h recovery without S9 (repeat 1): no significant increase in the number of aberrant cells. The pulse treatment studies in the absence of metabolic activation are invalid as the positive control did not significantly increase the proportion of cells with structural aberrations.

Pulse treatment 6h and 18h recovery without S9 (repeat 2): non-dose-related increase in the number of aberrant cells (6.5% at 1250 µg/ml, 5.0% at 2500 µg/ml and 4.5% at 5000 µg/ml). The pulse treatment studies in the absence of metabolic activation are invalid as the positive control did not significantly increase the proportion of cells with structural aberrations.

In conclusion: the substance is not clastogenic based on continuous exposures in the absence of S9 and pulse exposures in the presence of S9.

Conclusions:
According to the report, the positive result in the first pulse test was a false positive result. The dose-dependent increase could not be repeated in a second and third pulse test, although in the third test positive responses were observed.
The report concludes that the test substance is mutagenic. It should be mentioned that there were some issues with the positive controls in the test. However, it was concluded that the substance is not clastogenic based on continuous exposures in the absence of S9 and pulse exposures in the presence of S9.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-10-27 to 2011-12-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP study performed according to OECD Guideline 476.
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1D403
- Analytical purity: 100%
- Composition of test material, percentage of components: 0.04 wt% water, 8.4 meq/g total amine

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature, in the dark

OTHER SPECIFICS:
- Name of test material (as cited in study report): JEFFCAT DMDEE
- Substance type: colorless to light yellow liquid
- Physical state: liquid
Target gene:
HGPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: F12FBS5+Hx
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: Cells used in the mutation assay were within four subpassages from cleansing in order to assure karyotypic stability.
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Toxicity Assay: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/mL
Mutagenesis Assay: 250, 500, 1500, 3000, 4000, 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: used at stock concentration of 20 µL/mL, for a final concentration of 0.2 µL/mL as positive control for non-activated test system
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: used at a stock concentration of 400 µg/mL, for a final concentration of 4 µg/mL, as positive control for the S9-activated test system
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 7 to 9 days
- Selection time (if incubation with a selection agent): 7 to 10 days

SELECTION AGENT (mutation assays): After the expression period, cells are grown in medium with and without 6-thioguanine.

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 2x1E06 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
- The test article was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least two consecutive concentrations showing mutant frequencies > 40 mutants per 1E06 clonable cells.
- If a single point above 40 mutants per 1E06 clonable cells was observed at the highest concentration, the test article was considered suspect
- If no culture exhibited a mutant frequency of > 40 mutants per 1E06 clonable cells, the test article was considered negative.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Relative cloning efficiency (mutagenesis assay) was 81% and 105% at the highest concentration tested in the non-activated and S9-activated systems, respectively.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: The osmolality of the solvent control was 287 mmol/kg and the osmolality of the top concentration, 5000 µg/mL, was 301 mmol/kg.
- Water solubility: Sterile distilled water was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in water at a concentration of 50 mg/mL, the maximum concentration tested.
- Precipitation: No test article precipitate was observed at any concentration in the treatment medium.

RANGE-FINDING/SCREENING STUDIES: Based on the results of the toxicity test, the concentrations chosen for the mutagenesis assay ranged from 500 to 5000 µg/mL for the non-activated cultures and 250 to 5000 µg/mL S9-activated cultures.

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

None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 1E06 clonable cells.

Conclusions:
The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, the test substance was concluded to be negative with and without metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo:

In vivo micronucleus test:

Huntsman (2012) performed an in vivo micronucleus test in male and female ICR mice via single oral administration of 500, 1000 or 2000 mg/kg DMDEE (nominal concentration). Concurrent vehicle (purified water) was used as a control.

Cyclophosphamide (50 mg/kg) was used as a positive control substance. Under the conditions of the study conduct, a single oral administration of DMDEE 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 bone marrow of male and female ICR mice. Therefore, DMDEE was concluded to be negative in the mouse micronucleus assay.


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
Study period:
2011-10-11 to 2011-12-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP study performed according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test).
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1D403
- Purity: 100% (provided by sponsor)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15 to 30°C), in the dark without desiccant

OTHER SPECIFICS:
- Name of test material (as cited in study report): JEFFCAT DMDEE
- Substance type: light yellow liquid
- Physical state: liquid
- Other: assignment of code AD36HK; stability not determined
- Total amine content: 8.4 meq/g
- Water content: 0.04 wt%
- Viscosity: 29 cSt

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 6 weeks
- Weight at study initiation: dose range finding study: male: 31.8 - 33.4 g, female: 24.3 - 25.4 g; definitive micronucleus study: male mice: 30.0 - 36.1 g, female mice: 24.5 - 30.3 g
- Assigned to test groups randomly: yes, under following basis: In each study, mice were assigned to the appropriate number of treatment groups using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight varation of the animals did not exceed ± 20% from their group mean. Following randomization, animals were identified by sequentially numbered ear tags assigned to each animal during 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 treatment group. Raw data records and specimens were also identified by the unique test animal number.
- Fasting period before study: no
- Housing: Animals were housed in an AAALAC-accredited facility with a controlled environment of
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days (dose-range finder) or 6 days (definitive study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: All dose formulations were administered at a dose volume of 20 mL/kg by a single oral administration using appropriate size disposable polypropylene syringes with gastric intubation tubes (needles).
The test substance dose formulations were prepared fresh for each phase of the study prior to dose administration. The formulation (100 mg/mL) for the DRF study and all formulations (25, 50 and 100 mg/mL) for definitive study were prepared as follows:
An appropriate amount of the test substance was weighed for each concentration separately.
An appropriate volume of the vehicle, ~80% of the final volume was added to the respective containers.
Each formulation was vortexed for 1 minute.
Remaining volume of the vehicle was added to achieve the final volume.
All formulations appeared as clear solutions.
Duration of treatment / exposure:
Single oral administration
Frequency of treatment:
Single oral administration
Post exposure period:
Dose range finding study: mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration on Study Day 0 and on Study Days 1 and 3. Following the last observation on Study Day 3, animals were euthanized by exposure to CO2 verified by toe pinch reflex and discarded without further examination.
Definitive micronucleus study: In-Life evaluations: mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity, which were recorded. Bone marrow collection 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.
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Based on the information provided by the Sponsor, the oral LD50 dose was > 2000 mg/kg. In order to confirm this information, in the dose-range finding study, 5 male and 5 female mice were orally exposed 2000 mg/kg, the highest regulatory recommended dose.
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Based on the information provided by the Sponsor, the oral LD50 dose was > 2000 mg/kg. In order to confirm this information, in the dose-range finding study, 5 male and 5 female mice were orally exposed 2000 mg/kg, the highest regulatory recommended dose.
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Based on the information provided by the Sponsor, the oral LD50 dose was > 2000 mg/kg. In order to confirm this information, in the dose-range finding study, 5 male and 5 female mice were orally exposed 2000 mg/kg, the highest regulatory recommended dose.
No. of animals per sex per dose:
500 and 1000 mg/kg: 5
vehicle and 2000 mg/kg: 10
positive control: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 50 mg/kg
Tissues and cell types examined:
Tissue: bone marrow of the femurs
Cell types examined:
- Polychromatic erythrocytes (PCEs): 2000 PCEs per animal were screened (scored) for the presence of micronuclei resulting in evaluation of a total of 10000 PCEs per each treatment group.
- Normochromatic erythrocytes (NCEs): The number of NCEs and micronucleated NCEs (MNCEs) in the field of 1000 total erythrocytes (ECs) is determined for each animal in order to determine the proportion of polychromatic erythrocytes to total erythrocytes (PCEs/ECs ratio).
- Micronuclei (M): Micronuclei are round, fluorescent green-stained nuclear (chromosome) fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei may occur in PCEs (MPCEs) or NCEs (MNCEs).
- The proportion of polychromatic erythrocytes to total erythrocytes were also determined per total of 1000 erythrocytes (PCEs/ECs ratio) for each animal.
Details of tissue and slide preparation:
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 labelled 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 re-suspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Slides were labelled with the experiment and animal numbers using a lead pencil. One set of slides was stained with a nucleic acid-specific stain, acridine orange, and was used in microscopic evaluation.
Evaluation criteria:
In this study, the test substance was jugded negative if no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groups relative to the concurrent negative (vehicle) groups was observed.
The test substance would have been considered to induce a positive response if the incidence of micronucleated polychromatic erythrocytes at one or more doses in statistically elevated relative to the vehicle control (p < or = 0.05, binomial distribution, Kastenbaum-Bowman tables).
However, the results of the statistical analysis would not be the only criteria in determination of the test substance positivity. A dose-dependent increase or biological relevance of the data would have been taken in consideration. In addition, if criteria for either a positive or negative genotox response were not met, the results would have been judged as equivocal.
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10000 PCEs for each treatment group was determined.
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time. In order 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 mouse and treatment group (PCEs/ECs ratio). The proportion of polychromatic erythrocytes to total erythrocytes in test substance animals should not be less than 20% of the control value.

Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
Piloerection and/or lethargy are among the clinical effects observed during the course of the study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the dose-range finding study, no mortality was observed. Piloerection and/or lethargy are among the clinical signs observed during the course of the study. No appreciable changes in the group mean body weights were seen.

RESULTS OF DEFINITIVE STUDY
In the definitive study, following results were generated:
- No mortality was observed in any of the treatment groups. All mice in the control substance (vehicle or positive) groups and all mice in the 500 and 1000 mg/kg treatment groups appeared normal during the study project. Piloerection was seen at 2000 mg/kg uring the course of the study.
- In the bone marrow, no appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test substance groups relative to the respective vehicle control groups were observed, suggesting that the substance did not inhibit erythropoiesis.
- No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (p > 0.05, binomial distribution, Kastenbaum-Bowman Tables).
- CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p< or = 0.05, binomial distribution, Kastenbaum-Bowman Tables) in both male and female mice. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

Criteria for a valid test:

All criteria for a valid test were met because of the following:

- The incidence of micronucleated polychromatic erythrocytes in the vehicle control groups did not exceed the historical vehicle control range.

- The incidence of micronucleated polychromatic erythrocytes in the male and female positive control groups was significantly increased relative to the respective vehicle control groups (p < or = 0.05, binomial distribution, Kastenbaum-Bowman Tables).

However, the results of the statistical analysis would not be the only criteria in determination of the test substance positivity. A dose-dependent increase or biological relevance of the data would have been taken in consideration. In addition, if criteria for either a positive or negative genotoxic response were not met, the results would have been judged as equivocal.

Conclusions:
Under the conditions of the study conduct, a single oral administration of the test substance 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 bone marrow of male and female ICR mice. Therefore, the substance was concluded to be negative in the mouse micronucleus assay.
This test was performed to fulfil other regulatory requirements outside the EU.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:
Bacterial reverse mutation assay: performed according to a method equivalent to OECD Guideline 471 and EU method B13/14 in S. typhimurium TA 1535, TA 1537, TA 98 and TA 98 and TA 100 and E. coli WP2 uvrA (BASF, 1998). According to the results of the study, the test substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay with and without metabolic activation. This study is selected as key study. In addition, a supporting K2 Ames study is available from the Central Labor Accident Prevention Institute, Nippon Bioassay Research Center (1988), in which an ambiguous result for mutagenicity was observed.
Chromosome aberration test: performed in CHL/IU male Chinese hamster lung fibroblasts according to a method equivalent to OECD Guideline 471. Based on the results of this test, it is concluded that the test substance is negative in the presence of S9, and ambiguous in the absence of S9.
Mammalian cell gene mutation test: performed according to OECD Guideline 476 in Chinese hamster Ovary (CHO) cells. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, the test substance was concluded to be negative with and without metabolic activation.

Genetic toxicity in vivo:
Micronucleus test: performed according to a method according to OECD Guideline 474 in mouse ICR male/female mice (Huntsman, 2012).


Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation (EC) 1272/2008, DMDEE should not be classified for mutagenicity.