Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registration substance did not exhibit any genotoxic activity in three in-vitro test systems.

Link to relevant study records

Referenceopen allclose all

Endpoint:
genetic toxicity in vitro, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 February 2016 TO 18 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Genamin DMG 75
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
“In vitro Mammalian Chromosome Aberration Test” adopted on 26 September 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In vitro Chromosomal aberration test
Specific details on test material used for the study:
Test Item Name: Genamin DMG 75
Chemical Name ( IUPAC): N, N-Dimethyl-D-glucamine
CAS No.: 76326-99-3
Physical Appearance
(with colour) :Slightly yellowish liquid
Batch No.: RAK-KRS-00022
Purity (Declared by sponsor and/ or as per Certificate of Analysis) : 71.2%; (Active components ca. 75% in ca. 25% aqueous solution)
Batch Produced by: Global Amines Germany GmbH
84504 Burgkirchen, Germany
Date of Manufacture : April 2015
Date of Analysis : 24.09.2015
Date of Expiry/Valid up to : 01.04.2018
Storage Conditions: Ambient (21 to 29°C)
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Details on mammalian cell type (if applicable):
CHO-K1 cell line procured from ATCC (American Type Culture Collection).
Additional strain / cell type characteristics:
other: Chromosome number of CHO-K1 is 2n=22.
Metabolic activation:
with and without
Metabolic activation system:
male Wistar Rats
Test concentrations with justification for top dose:
Based on the precipitation and pH test, 2 mg/mL was chosen as the highest dose for the initial cytotoxicity test. 2 mg/mL was chosen as the highest concentration for the chromosomal aberration test as the initial study was non toxic at 2 mg/mL
Vehicle / solvent:
Distilled Water- Vehicle(s) used: water]
- Justification for choice of vehicle: based on the results of solubility test
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Distilled Water
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
CHO-K1 cell line procured from ATCC, Hams F12 medium containing 10% FBS
and 1 % penicillin-streptomycin incubated at 37°C with 5% CO2.
Rationale for test conditions:
As per guidelines
Evaluation criteria:
The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the metaphases with aberrations.
Gaps were recorded separately.
Statistics:
Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 for differences among vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p < 0.05) and the statistical significance was designated by the superscripts though out the report as stated below:
* Statistically significant (p < 0.05) change than the vehicle control group.
Key result
Species / strain:
other: CHOK-1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change
- Water solubility: 2mg/mL
- Precipitation: no precipitation at 2mg/mL

Remarks on result:
other: non-clastogenic

 

TABLE 1.           SUMMARY OF PERCENTAGE RICC FOR INITIAL CYTOTOXICITY TEST

Refer Appendix 2

Set No.

Treatment

Dose (mg/mL)

Initial Cell Count

(1×105 cells/flask)

Final Cell Count

(1×105cells/flask)

Final – initial cell count

(1×105cells/flask)

RICC

Reduction in RICC (%)

Set 1 (+S9)                  (3 to 6 hours)

Vehicle control

-

2.78

8.63

5.85

100

0

Test item

[Genamin DMG 75]

0.5

2.78

7.95

5.18

88.46

11.54

1

2.78

7.65

4.88

83.33

16.67

2

2.78

7.58

4.80

82.05

17.95

 

Set 2 (-S9)                     (3 to 6 hours)

Vehicle control

-

2.78

7.95

5.18

100.00

0

Test item

[Genamin DMG 75]

0.5

2.78

7.65

4.88

94.20

5.80

1

2.78

7.65

4.88

94.20

5.80

2

2.78

7.50

4.73

91.30

8.70

 

Set 3 (-S9)                   (18 to 20 hours)

Vehicle control

-

2.78

7.88

5.10

100.00

0

Test item

[Genamin DMG 75]

0.5

2.78

7.43

4.65

91.18

8.82

1

2.78

7.05

4.28

83.82

16.18

2

2.78

7.05

4.28

83.82

16.18

RICC:Relative increase in cell count, +S9: with metabolic activation, -S9: without metabolic activation.  

 

TABLE 2.     SUMMARY OFCHROMOSOMAL ABERRATIONSANDRICC

                                                                                              Refer Appendix3

Set No.

Treatment

Dose (mg/mL)

Initial Cell Count

(1×105cells/flask)

Final Cell Count

(1×105cells/flask)

Final – initial cell count

(1×105cells/flask)

RICC

Reduction in RICC (%)

Mean of Total Aberrations with Gaps

Mean of Total Aberrations without Gaps

Mean of Total Aberrated cells without Gaps

Percentage Mean of Aberrated Cells without Gaps

Set 1 (+S9) (3 to 6 hours)

Vehicle control

-

2.63

7.44

4.81

100

0

1.0

1.0

1.0

0.7

Test item

[Genamin DMG 75]

0.5

2.63

6.94

4.31

89.61

10.39

1.5

1.5

1.0

0.7

1

2.63

6.75

4.13

85.71

14.29

2.5

2.0

1.0

0.7

2

2.63

6.63

4.00

83.12

16.88

1.0

1.0

1.0

0.7

Positive Control

(Cyclophosphamide)

10 µg/mL

2.63

6.19

3.56

74.03

25.97

25.5

24.0

12.5

8.3*

RICC: Relative increase in cell count; *: Statistically significant;+S9: with metabolic activation.

 

 

 

 

  

 

TABLE 2 (Contd..,). SUMMARY OFCHROMOSOMAL ABERRATIONSAND RICC

                                                                                              Refer Appendix 3

Set No.

Treatment

Dose (mg/mL)

 

 

Initial Cell Count

(1×105cells/flask)

 

 

Final Cell Count

(1×105cells/flask)

Final – initial cell count

(1×105cells/flask)

RICC

Reduction in RICC (%)

Mean of Total Aberrations with Gaps

Mean of Total Aberrations without Gaps

Mean of Total Aberrated cells without Gaps

Percentage Mean of Aberrated Cells without Gaps

Set 2 (-S9) (3 to 6 hours)

Vehicle control

-

2.63

7.13

4.50

100.00

0.00

2.0

2.0

1.0

0.7

Test item

[Genamin DMG 75]

0.5

2.63

7.00

4.38

97.22

2.78

1.0

1.0

1.0

0.7

1

2.63

6.75

4.13

91.67

8.33

1.0

1.0

1.0

0.7

2

2.63

6.69

4.06

90.28

9.72

1.5

1.5

1.0

0.7

Positive Control

(Mitomycin-C)

0.05 µg/mL

2.63

6.44

3.81

84.72

15.28

21.5

19.5

13.5

9.0*

RICC: Relative increase in cell count; *: Statistically significant;-S9: without metabolic activation.

 

 

  

TABLE 2 (Contd..,). SUMMARY OF CHROMOSOMAL ABERRATIONS AND RICC

                                                                                              Refer Appendix 3

Set No.

Treatment

Dose (mg/mL)

Initial Cell Count

(1×105cells/flask)

Final Cell Count

(1×105cells/flask)

Final – initial cell count

(1×105cells/flask)

RICC

Reduction in RICC (%)

Mean of Total Aberrations with Gaps

Mean of Total Aberrations without Gaps

Mean of Total Aberrated cells without Gaps

Percentage Mean of Aberrated Cells without Gaps

Set 3 (-S9) (18 to 20 hours)

Vehicle control

-

2.63

7.19

4.56

100.00

0.00

1.0

1.0

1.0

0.7

Test item

[Genamin DMG 75]

0.5

2.63

7.06

4.44

97.26

2.74

1.0

1.0

1.0

0.7

1

2.63

6.69

4.06

89.04

10.96

1.0

1.0

1.0

0.7

2

2.63

6.50

3.88

84.93

15.07

2.0

2.0

1.0

0.7

Positive Control

(Mitomycin-C)

0.05 µg/mL

2.63

6.00

3.38

73.97

26.03

19.5

18.0

11.5

7.7*

RICC: Relative increase in cell count; *: Statistically significant;-S9: without metabolic activation.

 

 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

Conclusions:
The gentoxicity of the registration substance was investigated according to the Guideline OECD 473. The registration substance did not exhibit any mutagenic acitivity.
Executive summary:

The gentoxicity of the registration substance was investigated according to the Guideline OECD 476. The registration substance was incubated with Chinese hamster ovary cells and the cells were investigated for the increases of chromosome aberration. No increase of chromosome aberration was found. The registration substance did not exhibit any clastogenic activity.

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:
2015-12-10 to 2016-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
His, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
A correction factor of 1.40 was applied to consider the purity of the test item.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A. dest.
Untreated negative controls:
yes
Remarks:
A. dest., BSL Lot No. 151006, 151125 and 151230
Negative solvent / vehicle controls:
yes
Remarks:
A. dest., BSL Lot No. 151006, 151125 and 151230
Positive controls:
yes
Positive control substance:
other:
Remarks:
Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for E.coli. Positive control with metabolic activation: 2-aminoanthracene for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
-For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.



NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E.coli the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Genamin DMG 75 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.
Conclusions:
The genotoxicity of the registration substance was investigated according to the Guideline OECD 471. The registration substance did not exhibit any mutagenic acitivity.
Executive summary:

The genotoxicity of the registration substance was investigated according to the Guideline OECD 471. The registration substance did not exhibit any mutagenic acitivity.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 February 2016 to 22 April 2016
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)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: IN VITRO MAMMALIAN CELL GENE MUTATION TEST
Specific details on test material used for the study:
Test Item Name: Genamin DMG 75
Chemical Name ( IUPAC): N, N-Dimethyl-D-glucamine
CAS No.: 76326-99-3
Physical Appearance (with colour): Slightly yellowish liquid
Batch No.: RAK-KRS-00022
Purity (Declared by sponsor and/ or as per Certificate of Analysis): 71.2%; (Active components ca. 75% in ca. 25% aqueous solution)
Batch Produced by: Global Amines Germany GmbH
84504 Burgkirchen, Germany
Date of Manufacture: April 2015
Date of Analysis: 24.09.2015
Date of Expiry/valid up to: 01.04.2018
Storage Condition: Ambient (21 to 29°C)

Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
AA8
Details on mammalian cell type (if applicable):
CHO AA8 cells, procured from ATCC
Metabolic activation:
with and without
Metabolic activation system:
Male Wistar Rats
Test concentrations with justification for top dose:
Based on the precipitation and pH test, 2 mg/mL was chosen as the highest dose for the initial cytotoxicity test.
2 mg/mL was chosen as the highest concentration for the gene mutation test as initial study was non toxic at 2 mg/mL.
Vehicle / solvent:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
Distilled Water
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
CHO AA8 cells, procured from ATCC and cells were maintained in alpha MEM with 10% FBS and 1% Pen-Strep, at 37ºC with 5% CO2.
Rationale for test conditions:
As per Guideline
Evaluation criteria:
Mutant Frequency
Statistics:
Data of mutant frequencies was analyzed for differences among vehicle control, treatment and positive control groups using SPSS Software version 22 at a 95% level (p<0.05) of significance.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No pH change at 2 mg/mL
- Water solubility: 2mg/mL
- Precipitation: Not precipitated at 2 mg/mL


 

TABLE 1.           SUMMARY OF PERCENTAGE RICC FOR INITIAL CYTOTOXICITY TEST

Refer Appendix 2

Set No.

Treatment

Dose (mg/mL)

Initial Cell Count

(1×105 cells/flask)

Final Cell Count

(1×105cells/flask)

Final – initial cell count

(1×105cells/flask)

RICC

Reduction in RICC (%)

Set 1 (+S9)                  (3 to 6 hours)

Vehicle control

-

2.78

8.63

5.85

100

0

Test item

[Genamin DMG 75]

0.5

2.78

7.95

5.18

88.46

11.54

1

2.78

7.65

4.88

83.33

16.67

2

2.78

7.58

4.80

82.05

17.95

 

Set 2 (-S9)                     (3 to 6 hours)

Vehicle control

-

2.78

7.95

5.18

100.00

0

Test item

[Genamin DMG 75]

0.5

2.78

7.65

4.88

94.20

5.80

1

2.78

7.65

4.88

94.20

5.80

2

2.78

7.50

4.73

91.30

8.70

 

Set 3 (-S9)                   (18 to 20 hours)

Vehicle control

-

2.78

7.88

5.10

100.00

0

Test item

[Genamin DMG 75]

0.5

2.78

7.43

4.65

91.18

8.82

1

2.78

7.05

4.28

83.82

16.18

2

2.78

7.05

4.28

83.82

16.18

RICC:Relative increase in cell count, +S9: with metabolic activation, -S9: without metabolic activation.  

TABLE 2.           SUMMARY OF PARALLEL CYTOTOXICITY TEST- GENE MUTATION TEST

                                                                                              Refer Appendix2

Set No.

Treatment

Dose (mg/mL)

Average colony count

Adjusted Cloning Efficiency (ACE)

Relative Survival (%)

Set 1 +S9

Vehicle control

-

 

173.50

1.13

-

Genamin

DMG 75

 

0.25

 

172.00

1.08

95.41

 

0.5

 

169.17

1.05

92.51

 

1

 

167.17

1.03

91.10

 

2

 

168.00

1.02

90.22

Benzo(a)pyrene

 

0.003

 

150.67

0.82

72.62

 

Set 2 -S9

Vehicle control

-

175.67

1.10

-

Genamin

DMG 75

 

0.25

 

173.33

1.07

97.61

 

0.5

 

172.17

1.05

95.58

 

1

 

171.17

1.03

93.99

 

2

 

169.83

1.02

92.56

4 Nitroquinoline 1-oxide

0.001

151.00

0.81

73.16

 +S9: with metabolic activation; -S9: without metabolic activation.  

 


 

TABLE 3.           SUMMARY OF GENE MUTATION TEST

Refer Appendix 3

Set No.

Treatment

Dose (mg/mL)

Average colony count

Cloning Efficiency

Average Mutant Colonies/ 106cells

Mutant Frequency/ 106cells

Set 1 +S9

Vehicle control

-

173.17

0.87

13.5

15.59

Genamin

DMG 75

 

0.25

 

170.83

0.85

13.5

15.81

 

0.5

 

170.00

0.85

12.5

14.69

 

1

 

170.67

0.85

12.5

14.65

 

2

 

169.67

0.85

13.0

15.33

Benzo(a)pyrene

0.003

150.50

0.75

119.5

158.85*

 

Set 2 -S9

Vehicle control

-

174.83

0.87

14.5

16.58

Genamin

DMG 75

 

0.25

 

171.50

0.86

12.5

14.57

 

0.5

 

169.33

0.85

14.0

16.54

 

1

 

167.83

0.84

11.5

13.70

 

2

 

168.33

0.84

12.0

14.26

4 Nitroquinoline 1-oxide

0.001

147.17

0.74

119.5

162.41*

  +S9: with metabolic activation; -S9: without metabolic activation; *: Statistically significant  

 

 

 

 

 

 

 

 

 

 

 

 

Conclusions:
The gentoxicity of the registration substance was investigated according to the Guideline OECD 476. The reigstration substance did not exhibit any mutagenic acitivity.

Executive summary:

The gentoxicity of the registration substance in mammalian cells was investigated according to the Guideline OECD 476. The registration substance did not exhibit any mutagenic acitivity.

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

Additional information

Justification for classification or non-classification

Three in-vitro test data are available for the assessment of the genotoxicity of the registration substance. In all three tests the registration substance was found to be not mutagenic and/or not clastogenic. No classification is justified.