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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,3-Bis(aminomethyl)cyclohexane
- Physical state: liquid
- Stability under test conditions: confirmed stable (IR spectrophotometry)
- Storage condition of test material: Refrigeration (actual temperature: 2.8°C to 8.4°C, permissible range: 1°C to 10°C), shielded from light, nitrogen-sealed

Method

Species / strainopen allclose all
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

Controlsopen allclose all
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

Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables

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)


Applicant's summary and conclusion

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.