Registration Dossier

Administrative data

Key value for chemical safety assessment

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:
23 May 2005 to 14 September 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to GLP and in accordance with international guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: Testing Method Concerning Designated Chemical Substances (November 21, 2003, Pharmaceutical and Food affairs No. 1121002, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor, and Welfare, November 13, 2003
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Test concentrations with justification for top dose:
See Table 1, 2, and 3

Untreated negative controls:
yes
Remarks:
purified water
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Remarks:
purified water
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
Untreated negative controls:
yes
Remarks:
purified water
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine hydrochloride (9-AA)
Untreated negative controls:
yes
Remarks:
purified water
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 90 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Main test was conducted in duplicate; in each of the main tests, concentrations were run in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see Results Table 4, 5, and 6
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see Results Table 4, 5, and 6
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see Results Table 4, 5, and 6
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see Results Table 4, 5, and 6
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: see Results Table 4, 5, and 6

Table 4.

With (+) or without (-) S9 mix Dose Level (µg/plate)* Number of revertants (number of colonies/plate)  
Base-pair change type   Frameshift type  
TA 100 (mean)  TA1535 (mean)  WP2uvrA/pKM101 (mean)  TA98 (mean)  TA 1537 (mean) 
(SD) (SD) (SD) (SD) (SD)
S9 mix (-) Negative Control 116   14   79   18   10  
121 (122) 8 (11) 84 (85) 16 (17) 15 (12)
130 (7) 11 (3) 91 (6) 17 (1) 10 (3)
39.1 136   8   Cytotoxicity  24   10  
103 (124) 10 (9) 15 (17) 10 (11)
132 (18) 10 (1) 13 (6) 14 (2)
78.1 112   9   22   13  
137 (128) 8 (9) 17 (19) 13 (14)
136 (14) 9 (1) 18 (3) 15 (1)
156 138   7   74   18   12  
164 (148) 10 (10) 77 (80) 24 (22) 13 (14)
142 (14) 14 (4) 90 (9) 24 (3) 16 (2)
313 127   14   83   24   15  
121 (129) 13 (14) 95 (87) 25 (22) 13 (13)
140 (10) 16 (2) 83 (7) 18 (4) 12 (2)
625 132   10   80   25   15  
115 (130) 10 (10) 77 (84) 24 (24) 16 (14)
142 (14) 10 (0) 94 (9) 24 (1) 12 (2)
1250 92*   7*   89   11*   5*  
58* (77) 10* (7) 61 (76) 13* (12) 4* (4)
82* (17) 5* (3) 77 (14) 11* (1) 3* (1)
2500 Cytotoxicity   Cytotoxicity 0    Cytotoxicity  Cytotoxicity
0 (0)
0 0
5000 0  
0 (0)
0 (0)

Table 5.

S9 mix (+) Negative Control 128   9   119   25   14  
135 (131) 8 (11) 108 (111) 33 (26) 16 (16)
143 (7) 17 (5) 105 (7) 26 (7) 17 (2)
9.77  Cytotoxicity 12    Cytotoxicity Cytotoxicity  Cytotoxicity
14 (12)
10 (2)
19.5 15  
11 (12)
9 (3)
39.1 132   13   99   21   18  
138 (131) 9 (12) 125 (114) 34 (26) 16 (16)
124 (7) 15 (3) 118 (13) 24 (7) 14 (2)
78.1 138   8   115   24   16  
132 (130) 9 (11) 121 (114) 29 (27) 16 (16)
119 (10) 17 (5) 105 (8) 27 (3) 16 (0)
156 120   9   129   27   15  
127 (123) 10 (11) 123 (118) 24 (27) 14 (17)
121 (4) 14 (3) 103 (14) 29 (3) 21 (4)
313 135   11   127   24   17  
120 (131) 9 (13) 105 (116) 30 (29) 18 (17)
138 (10) 18 (5) 115 (11) 34 (5) 16 (1)
625 126   13   145   26   15  
119 (125) 12 (13) 114 (128) 31 (27) 16 (15)
129 (5) 15 (2) 126 (16) 24 (4) 13 (2)
1250 108   13   120   14   9  
93 (115) 6 (8) 166 (136) 12 (12) 10 (9)
143 (26) 4 (5) 123 (26) 11 (2) 7 (2)

Table 6.

Positive control S9 mix (+) Name 2-AA 2-AA 2-AA 2-AA 2-AA
Dose (ug/plate) 1 2 2 0.5 2
Number of colonies/plate 1416   236   998   432   194  
1473 (1502) 201 (241) 938 (966) 430 (435) 197 (189)
1573 (61) 287 (43) 963 (30) 443 (7) 177 (11)
Positive control S9 mix (-) Name AF-2 NaN3 AF-2 AF-2 9-AA
Dose (ug/plate) 0.01 0.5 0.005 0.1 80
Number of colonies/plate 744   590   1189   931   434  
640 (723) 601 (603) 1102 (1108) 959 (934) 341 (371)
785 (75) 618 (14) 1032 (79) 913 (23) 337 (55)


Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that 1,3-Cyclohexanedimethanamine had not shown any mutagenic activity under the test conditions used. The test was conducted up to the point of microbial toxicity for each strain tested, with and without metabolic activation.
Executive summary:

A bacterial reverse mutation assay (Ames test) was conducted to determine the potential for the test substance 1,3-Cyclohexanedimethanamine to cause gene mutation. The test was conucted according to OECD test guideline 471 and official Japanese guidelines, and in compliance with GLP. The test involved exposing strains of Salmonella typhimurium (TA100, TA1535, TA1537, and TA98) and one strain of Escherichia coli (WP2uvrA/pKM101) to a series of solutions of the test substance in distilled water, by the preincubation method. A preliminary test was conducted to determine the concentrations to be investigated in the main tests, followed by the main test which was performed in duplicate. In the main test, microbial toxicity (cytotoxicity) was seen for each Salmonella strain at 1250 µg/plate and at 2500 µg/plate for E. coli in the absence of S9 mix (metabolic activation). Microbial toxicity was observed for all strains tested at a concentration of 1250 µg/plate in the presence of S9 mix. None of the strains tested, either in the presence or the absence of S9 mix / metabolic activation showed a significant increase in the number of revertant colonies relevant to the concurrent negative controls (a two-fold increase was considered significant). The concurrent positive control plates showed an increase in the number of revertant colonies relative to the negative controls, and so the test was shown to be valid. It was concluded that 1,3-Cyclohexanedimethanamine had not shown any mutagenic activity under the test conditions used.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 May 2005 - 15 September 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP and international test guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Testing Method Concerning Designated Chemical Substances (November 21, 2003, Pharmaceutical and Food affairs No. 1121002, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor, and Welfare
Qualifier:
according to
Guideline:
other: November 13, 2003, Manufacturing Industries Bureau, No. 2, Ministry of Economy, Trade, and Industry, No. 031121002, Environmental Policy Bureau, Ministry of the Environment
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: female Chinese hamsters CHL/IU lung cells
Details on mammalian cell type (if applicable):
- Type and identity of media: DMSO
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
cell growth inhibition test: 0.125, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 5, 7.5, 10, 12.5 mg/mL; cell growth inhibition test-2 and chromosomal aberration test [main test]: 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 mg/mL; chromosomal aberration test [confirmation test]: 4, 4.5 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:physiol. saline
- Justification for choice of solvent/vehicle: In a previous solvent selection test, the test substance was found to dissolve in saline at up to 50 mg/mL with no increases in temperature, foaming, or discolouration.
Untreated negative controls:
yes
Remarks:
physiological saline
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
used in the -S9 mix assay
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
used in the +S9 mix assay
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 hours (with 18 hour recovery period)
- Selection time (if incubation with a selection agent): 2 hours

SELECTION AGENT (mutation assays): colcemid

NUMBER OF REPLICATIONS: 2 plates per concentration

NUMBER OF CELLS EVALUATED: 100 Cells per plate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
Species / strain:
other: Chinese hampster CHL/ lung cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Based on incidence of structural chromosome aberrations. Chromosome aberration was considered secondary to severe cytotoxicity, however.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: Chinese hampster CHL/lung cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance from the test medium was not recognised at the beginning or the end of any of the tests.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Cell growth inhibition test

Precipitation of the test substance was not recognized in the medium at the beginning or the end of the treatment in any test substance treatment groups under any treatment conditions.

The concentration giving50% inhibition of cell growth (IC50) were 297mg/mLin the –S9 mix assay and 353mg/mLin the +S9 mix assay.

On the other hand, cell growth was not inhibited by more than 50%in the 24-hour assay.

 

   Cell growth inhibition test-2

Precipitation of the test substance was not recognized in the medium at the beginning or the end of the treatment in any test substance treatment groups.

The IC50was 320mg/mL in the 24-hour assay.

 

Chromosomal aberration test

 Main test

Precipitation of the test substance was not recognized in the medium at the beginning or the end of the treatment in any test substance treatment groups under any treatment conditions.

No remarkable decrease in the relative mitotic index was seen as compared to the cell growth index in any treatment conditions.

The incidences of cells with structural chromosome aberrations were 8.5%, 16.8%, and 16.5% at 400, 450, and 500mg/mL, respectively, in the–S9 mix assay and 5.5% at 500 mg/mL in the +S9 mix assay.

D20value was 0.56 mg/mL in the–S9 mix assay.

The incidences of cells with numerical aberrations were less than 5% in all test substance treatment groups under all treatment conditions.

In the negative control group, both the incidences of cells with structural and numerical aberrations were less than 5% under all treatment conditions. In the positive control group, the incidence of cells with structural aberrations was more than 10% under all treatment conditions.

 

Confirmation test

Precipitation of the test substance was not recognized in the medium at the beginning or the end of the treatment in any test substance treatment groups.

A remarkable decrease in the relative mitotic indexwas not seen compared with the result of cell growth index.

The incidences of cells with structural and numerical aberrations were less than 5% in all test substance treatment groups.

In the negative control group, both the incidences of cells with structural and numerical aberrations were less than 5%. In the positive control group, the incidence of cells with structural aberrations was more than 10%.

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
positive with metabolic activation

In conclusion, 1,3-Cyclohexanedimethanamine was considered to have the potential to induce chromosomal aberration in CHL/IU cells under the conditions employed in this study (short-term treatment assay in the absence of S9 mix) .


Executive summary:

An in vitro chromosomal aberration study of 1,3-Cyclohexanedimethanamine was conducted with CHL/IU cells derived from the lungs of female Chinese hamsters as the indicator cells.

 

Based on the results of a preliminary test, a cell growth inhibition test was conducted as follows in short-term treatment assays in the absence of S9 mix (–S9 mix assay) and in the presence of S9 mix (+S9 mix assay), and in a continuous treatment assay for 24 hours (24-hour assay).

−S9 mix: 125, 250, 500, 750, 1000, 1250, 1500 mg/mL

+S9 mix:125, 250, 500, 750, 1000, 1250, 1500 mg/mL

24 hours: 12.5, 25, 50, 75, 100, 125, 150, 200mg/mL

As a result, 50% cell growth inhibition concentrations (IC50) were 297 mg/mL in the –S9 mix assay and 353 mg/mL in the +S9 mix assay. On the other hand, since 50% cell growth inhibition was not observed in the 24-hour assay, the cell growth inhibition test-2 was conducted as follows.

24 hours: 100, 150, 200, 250, 300, 350, 400, 450, 500 mg/mL

As a result, IC50was 320mg/mL in the 24-hour assay.

 

Based on the results of IC50, the chromosomal aberration test was conducted as follows.

−S9 mix: 200, 250, 300, 350, 400, 450, 500 mg/mL

+S9 mix:250, 300, 350, 400, 450, 500 mg/mL

As a result, the incidences of structural chromosome aberrations were 8.5%, 16.8%, and 16.5% at 400, 450, and 500 mg/mL, respectively, in the S9 mix assay. On the other hand, the incidence of structural chromosome aberrations was 5.5% (inconclusive: 5% or more, but less than 10%) at 500 mg/mL in the +S9 mix assay. Therefore, a confirmation test was conducted at 400, 450 and 500 mg/mL in the +S9 mix assay.

As a result, the incidences of structural aberrations were less than 5% in all test substance treatment groups with no reproducibility.

 

In conclusion, 1,3-Cyclohexanedimethanamine was considered to have the potential to induce chromosomal aberrations in CHL/IU cells under the conditions (short-term treatment assay in the absence of S9 mix) employed in this study.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although the test methodology followed was consistent with the Ames test used in a number of international test guidelines, no formal recognised Test Guideline was cited in the study report, and no claim of GLP compliance was made. As the study report lacks detail regarding the test methodology, the study data cannot be considered reliable.
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
not specified
Remarks:
No claim regarding GLP compliance was included in the test report.
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix

Substance

Concentration of test material (ug/plate)

With (+) / Without (-) S9

Reverse Mutation number of colonies per plate

 

 

 

Basic replacement type

Frame shift type

 

 

 

TA100

TA1535

WP2UVRA

TA98

TA1537

TA1538

Solvent Contract (Control??)

 

 

86

17

8

17

18

26

 

 

107

15

9

30

21

17

Concentration of test material

5

-

 

 

 

 

18

10

 

 

 

 

15

25

10

-

74

6

19

22

14

20

110

11

16

17

7

14

50

-

42

8

14

20

9

24

79

14

19

24

12

17

100

-

54

9

13

14

9

7

74

14

11

23

14

16

500

-

46

11

9

0

11

13

100

11

15

18

11

22

1000

-

39

5

10

14

*

15

66

6

17

2

*

12

5000

-

30

6

*

*

*

*

47

19

*

*

*

*

Solvent Contract (Control??)

 

+

77

11

11

14

13

26

75

15

12

37

34

44

Concentration of test material

10

+

44

16

8

27

3

16

 

71

19

9

26

13

30

50

+

43

8

11

37

5

23

 

69

11

19

32

12

34

100

+

41

18

8

41

7

15

 

58

16

10

29

12

30

500

+

55

10

10

37

11

18

 

56

12

8

18

6

42

1000

+

76

9

8

52

4

26

 

63

22

7

36

13

46

5000

0

47

12

11

*

6

*

 

57

6

10

*

10

*

S9 Mix is not needed

Name

ENNG

ENNG

ENNG

2-NF

9-AA

2-NF

Concentration (micrograms per plate)

2

5

5

2

100

5

Number of colonies per plate

430

1198

1051

455

320

828

398

878

1001

367

311

876

S9 Mix is needed

Name

2-AA

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration (micrograms per plate)

5

20

40

5

20

5

Number of colonies per plate

370

182

198

621

503

584

463

96

237

1580

388

514

Conclusions:
Interpretation of results (migrated information):
negative

Consideration of test results by Test Operation Controller, mutagenicity was not found in all of 6 strains under any conditions.
Executive summary:
A bacterial mutation assay was conducted according to EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria). Mutagenicity from exposure to 1,3 -BAC was not found under any of the test conditions.
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:
23 October 2009 - 21 January 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to official test guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
other: JMHW Genotoxicity Testing Guideline, PAB Notification No. 1604 (1 November 1999)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The study report includes a statement of GLP compliance signed by the study director, a certificate of quality assurance, and a certificate of GLP Compliance issued by the UK GLP Monitoring Authority.
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Crl:CD1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: UK
- Age at study initiation: Micronucleus test (main test) approximately 43 days old.
- Weight at study initiation: 29.1 g to 33.0 g
- Assigned to test groups randomly:Yes
- Diet (e.g. ad libitum): Free access to pelleted rat and mouse maintenance diet.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C.
- Humidity (%): 40 to 70% relative humidity.
- Photoperiod (hrs dark / hrs light): 12 hours light per day.

IN-LIFE DATES: From: 28 October 2009 To: Approx. 11 December 2009 (assumes final sacrifice of animals 48 hours after start of Micronucleus test).
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water; pH was adjusted (from pH 12 to a pH which could be tolerated by the rodents, pH<9) using Hydrochloric Acid and / or Sodium Hydroxide
- Justification for choice of solvent/vehicle: The test material is highly soluble in water.
- Concentration of test material in vehicle: 0 (control), 43.75, 87.5, 175 mg/mL.
- Amount of vehicle (if gavage or dermal): 10 mL/kg/day
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were sonicated or heated to 37°C where required to form a solution.
Duration of treatment / exposure:
Two treatments, approximately 24 hours apart. Sacrifice was performed approximately 24 hours after the second treatment.
Frequency of treatment:
Doses were administered approximately 24 hours apart.
Post exposure period:
24 Hours
Remarks:
Doses / Concentrations:
0 mg/kg/day (Vehicle control)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
437.5 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
875 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1750 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
6 Males (test only conducted in males, as no difference in toxicity was found between sexes in a preliminary test).
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Justification for choice of positive control(s): Mitomycin C is a recommended positive control material suggested in the OECD test guideline.
- Route of administration: Oral gavage
- Doses / concentrations: 5 Males dosed at 12 mg/kg/day (0.6 mg/mL, 20 mL/kg dose). Positive control group was dosed once only, 24 hours prior to termination.
Tissues and cell types examined:
Bone marrow from both femurs of each rat was collected. Polychromatic erythrocytes were examined.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.
The resulting cell suspensions were centrifuged at 1000 rpm (150 × g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976).


METHOD OF ANALYSIS:
Fixed for a minimum of 10 minutes in methanol and allowed to air-dry, rinsed in purified water, stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes. Cells were then washed in purified water for 5 minutes, and rinsed in cold tap water for 2 minutes. Slides were stored at room temperature protected from light until required. Immediately prior to scoring, slides are wet mounted with coverslips using purified water.
Evaluation criteria:
Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.
Statistics:
For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3, then on Groups 1 and 2. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
Sex:
male
Genotoxicity:
ambiguous
Remarks:
statistically significant decrease in the proportion of polychromatic erythrocytes was observed at 1750 mg/kg/day only
Toxicity:
yes
Remarks:
The decrease was considered indicative of toxicity.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
No statistically signifcant increase in the induction of micronucleated polychromatic erythrocytes in male Crl:CD1TM mice compared to control.
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
A preliminary toxicity (range-finding) test was conducted to determine the maximum tolerated dose of the test material. 2 males and 2 females in each group were dosed at 500, 1000, 2000, 1500, and 1750 mg/kg/day, then observed for 48 hours after the first dose. Clinical signs and mortalities were recorded. At the end of the observation period, surviving animals were killed and discarded.
Excessive clinical signs were seen at 2000 mg/kg/day (the standard limit for this test), and so 1750 mg/kg/day was chosen as the highest concentration for the main (micronucleus) test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):The test substance did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes in male animals.
- Ratio of PCE/NCE (for Micronucleus assay):The test substance did cause a statistically significant decrease (p<0.05) in the proportion of polychromatic erythrocytes at 1750 mg/kg/day in male animals only. This decrease is considered as being indicative of toxicity. All individual and group mean values were within the laboratory historical control range.
Conclusions:
Interpretation of results (migrated information): negative
The test substance is not considered to be genotoxic as no clear statistically significant increases in micronuclei (MPCE) were observed in any treatment groups relative to the control. 
Executive summary:

An In-Vivo Mammalian Erythrocyte Micronucleus Test was conducted to assess the mutagenic potential of the test substance 1,3-Bis(aminomethyl)cyclohexane. The study was conducted according to a number of international test guidelines including OECD test guideline 474 and EC test guideline B12. The study was conducted in compliance with GLP.

A range finding study test was performed to determine the maximum tolerated dose, and then the main study was conducted at levels 437.5, 875, and 1750 mg/kg/day. No difference in response was seen between males and females in the range finding test, and so the dose groups consisted of six male mice only. A vehicle control group and positive control group were tested contemporaneously.

No statistically significant increase was seen in the induction of micronucleated polychromatic erythrocytes. A statistically significant decrease was seen in the proportion of polychromatic erythrocytes at 1750 mg/kg/day only, but this observation was believed to be indicative of toxicity.

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

Additional information

A bacterial reverse mutation assay (Ames test; Kashima Labs, 2006) was conducted to determine the potential for the test substance 1,3-Cyclohexanedimethanamine to cause gene mutation. The test was conucted according to OECD test guideline 471 and official Japanese guidelines, and in compliance with GLP. The test involved exposing strains of Salmonella typhimurium (TA100, TA1535, TA1537, and TA98) and one strain of Escherichia coli (WP2uvrA/pKM101) to a series of solutions of the test substance in distilled water, by the preincubation method. A preliminary test was conducted to determine the concentrations to be investigated in the main tests, followed by the main test which was performed in duplicate. In the main test, microbial toxicity (cytotoxicity) was seen for each Salmonella strain at 1250 µg/plate and at 2500 µg/plate for E. coli in the absence of S9 mix (metabolic activation). microbial toxicity was observed for all strains testd at a concentration of1250 µg/plate in the presence of S9 mix. None of the strains tested, either in the presence or the absence of S9 mix / metabolic activation showed a significant increase in the number of revertant colonies relevant to the concurrent negative controls (a two-fold increase was considered significant). The concurrent positive control plates showed an increase in the number of revertant colonies relative to the negative controls, and so the test was shown to be valid. It was concluded that 1,3-Cyclohexanedimethanamine had not shown any mutagenic activity under the test conditions used.  

A bacterial mutation assay was conducted according to EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria). Mutagenicity from exposure to 1,3 -BAC was not found under any of the test conditions.  In a chromosomal aberration study (Kashima labs, 2006), with mammalian cells, 1,3-Cyclohexanedimethanamine was considered to have the potential to induce chromosomal aberrations in CHL/IU cells under the conditions (short-term treatment assay in the absence of S9 mix) employed.  

An In-Vivo Mammalian Erythrocyte Micronucleus Test (HLS, 2010) was conducted to assess the mutagenic potential of the test substance 1,3-Bis(aminomethyl)cyclohexane. The study was conducted according to a number of international test guidelines including OECD test guideline 474 and EC test guideline B12. The study was conducted in compliance with GLP. A range finding study test was performed to determine the maximum tolerated dose, and then the main study was conducted at levels 437.5, 875, and 1750 mg/kg/day. No difference in response was seen between males and females in the range finding test, and so the dose groups consisted of six male mice only. A vehicle control group and positive control group were tested contemporaneously. No statistically significant increase was seen in the induction of micronucleated polychromatic erythrocytes. A statistically significant decrease was seen in the proportion of polychromatic erythrocytes at 1750 mg/kg/day only, but this observation was believed to be indicative of toxicity.  

Short description of key information:  

- Ames test - no evidence of mutagenic activity in four strains of Salmonella typhimurium or one strain of Escherichia coli, with or without metabolic activation.

- Chromosome aberration test in mammalian cells (Chinese Hamster Lung cells)- potential to cause chromosome aberration in a short-term exposure test without metabolic activation.

- In-vivo mammalian micronucleus assay - no significant increase in micronucleated polychromatic erythrocytes. Significant decrease in proportion of polychromatic erythrocytes seen at highest dose (1750 mg/kg/day) only, believed to be indicative of toxicity.  

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Two in-vitro studies (Ames test and Chromosomal Aberration test, both conducted by Kashima Labs in 2006) and one in-vivo study (Mouse micronucleus test, HLS 2010) have been conducted. Of these tests, the Ames and Mouse mirconucleus tests were both negative for mutagenic activity, whereas the Chromosomal Aberration test concluded that 1,3 -BAC did present a potential for mutagenicity activity. As the mouse micronucleus test was conducted in-vivo, the results from this test take precedence over the results from the in-vitro tests for the purposes of classification, and as such the overall weight of evidence suggests that 1,3 -BAC does not require classification as a hazard for Germ cell mutagenicity.

Classification does not apply for mutagenic endpoints under the CLP Regulation (EC regulation 1272/2008).