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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
5 October 1984 - 22 February 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, the study is comparable to OECD 476. The purity is not indicated and analytical certificate is not available.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Hexamethylene Diamine
- Physical state: clear liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Lot/batch No.: no data
- Stability under test conditions: no data
- Storage condition of test material: in the plastic container received from the sponsor, maintained in frozen aliquots in a Revco Ultra-low Freezer.

Method

Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
serum free medium F12 (for wash)
F12FCM5 (for plate)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
At dose levels of 25, 50, 100, 175 and 250 µg/mL of treatment volume without metabolic activation preparation.
At doses levels of 50, 100, 300, 450 and 600 µg/mL of treatment volume with a 5% concentration of metabolic activation preparation.
See below Table 7.6.1/1
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water supplied by Pharmakon Research International.
- Justification for choice of solvent/vehicle: no data
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate (EMS) and Dimethylnitrosamine (DMN)
Remarks:
Positive controls: EMS was used at 200 µg/mL without S9 activation and DMN was used at 100 µg/mL with S9 activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- in suspension for preliminary cytotoxicity and mutagenicity screen
- in agar (plate incorporation) for the main test


DURATION
- Preincubation period: No
- Exposure duration: 5 h
- Selection time (if incubation with a selection agent): 7 days


SELECTION AGENT (mutation assays): hypoxanthine


NUMBER OF REPLICATIONS:
Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
- The test substance was tested to the level of at least 10% survival or to a level deemed acceptable by the sponsor.
- At least three of the five test substance concentrations should produce toxicity ranging from 10 to 100% survival in the cytotoxicity assay.
- ideally, at least one concentration should induce toxicity of less than 50% survival in the assay.
- The plating efficiency of the solvent control was at least 50%.
- The spontaneous mutation frequency of the solvent control was within 2 standard deviations of the historical mean frequency for Pharmakon Research International, Inc.
Statistics:
Dose-response analyses were performed on transformed mutation frequency data by the one-way analysis of variance method outlined by Snee and Irr (1981). Dose-response was considered significant if p < 0.01.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No additional data
Remarks on result:
other: strain/cell type: CHO-K1-BH4
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Preliminary cytotoxicity Screen

 

Relative Cell Survival (%)

S.9 concentrations

Test article

µg/mL

0%

1%

2%

5%

10%

Untreated

-

100a

77.6

103.6

99.5

102.0

HMD

3

105.0

144.1

130.2

96.1

100.3

HMD

9

109.9

116.2

128.8

123.6

107.9

HMD

30

117.6

112.1

99.7

101.6

96.1

HMD

91

98.0

108.3

117.6

109.1

103.5

HMD

304

53.2

115.3

109.8

97.6

89.0

HMD

912

0

0

0

0

0

HMD

3039

0

0

0

0

0

HMD

9116

0

0

0

0

0

aAbsolute cloning efficiency = 63.5 %

Table 7.6.1/3: Preliminary mutagenicity Screen

Compound

µg/mL

S.9

(±)

Relativea Initial Survival (%)

Total No. of Mutants

(5 plates)

Cloning Efficiency (%)

Mutant Frequency (Mutants/106clonables cells)

Mean Mutation Frequency

 

1% S.9 Concentration

Untreated

-

-

-

-

99.6

100.4

13

8

108.3

97.5

12.0

8.2

 

10.1

-

-

+ 1%

+ 1%

102.9

98.9

4

9

96.0

86.8

4.2

10.4

 

7.3

HMD

100

100

+ 1%

+ 1%

85.2

86.8

6

2

63.8

96.0

7.2

2.1

 

4.6

400

400

+ 1%

+ 1%

2.4

3.1

4

11

91.5

83.0

4.4

13.3

 

8.9

600

600

+ 1%

+ 1%

-

-

-

-

-

-

-

-

 

-

 

2% S.9 Concentration

Untreated

-

-

-

-

99.6

100.4

13

8

108.3

9705

12.0

8.2

 

10.1

-

-

+ 2%

+ 2%

88.5

102.9

12

5

92.8

95.2

12.9

5.3

 

9.1

HMD

100

100

+ 2%

+ 2%

91.2

94.9

6

10

87.7

96.8

6.8

10.3

 

8.6

400

400

+ 2%

+ 2%

55.4

53.2

4

3

96.3

85.8

4.2

3.5

 

3.9

600

600

+ 2%

+ 2%

35.7

25.8

5

2

111.5

87.7

4.5

2.3

 

3.4

 

5% S.9 Concentration

Untreated

-

-

-

-

99.6

100.4

13

8

108.3

97.5

12.0

8.2

 

10.1

-

-

+ 5%

+ 5%

97.7

91.0

9

5

99.5

92.2

9.0

5.4

 

7.2

HMD

100

100

+ 5%

+ 5%

85.5

86.0

5

10

86.7

105.7

5.8

9.5

 

7.7

400

400

+ 5%

+ 5%

66.1

52.6

13

4

99.5

98.0

13.1

4.1

 

8.6

600

600

+ 5%

+ 5%

23.8

27.6

9

3

98.5

80.5

9.1

3.7

 

6.5

 

10% S.9 Concentration

Untreated

-

-

 

99.6

100.4

13

8

108.3

97.5

12.0

8.2

 

10.1

-

-

+ 10%

+ 10%

95.4

83.9

12

11

105.5

100.7

11.4

10.9

 

11.1

HMD

100

100

+ 10%

+ 10%

86.2

77.9

10

10

90.2

85.7

11.1

11.7

 

11.4

400

400

+ 10%

+ 10%

68.9

62.6

7

8

81.8

92.5

8.6

8.6

 

8.6

600

600

+ 10%

+ 10%

37.4

38.3

7

16

89.3

87.2

7.8

18.3

 

13.0

DMN

 

100

100

+ 10%

+ 10%

28.9

28.1

146

126

61.5

64.2

237.4

196.3

 

216.9

200

200

-

-

72.4

53.2

161

158

83.2

71.2

193.5

221.9

 

207.6

 

0% S.9 Concentration

Untreated

-

-

-

-

98.4

101.6

5

8

92.5

97.0

5.4

8.2

 

6.8

HMD

50

50

-

-

103.8

96.2

2

3

76.2

85.3

2.6

3.5

 

3.1

100

100

-

-

60.7

64.8

8

2

75.3

89.5

10.6

2.2

 

6.4

200

200

-

-

51.5

39.5

4

3

76.8

95.3

5.2

3.1

 

4.1

250

250

-

-

28.4

22.7

11

22

87.0

78.5

12.6

28.0

 

20.3

EMS

200

200

-

-

54.1

52.3

156

172

70.2

69.8

222.2

246.4

 

234.3

aAbsolute cloning efficiency: -    Mean for 1-2-5-10% = 79.9

                                          -    Mean for 0% = 68.4

Table 7.6.1/4: Mutagenicity Data

Compound

µg/mL

S.9

(±)

Relativea Initial Survival (%)

Total No. of Mutants

(5 plates)

Cloning Efficiency (%)

Mutant Frequency (Mutants/106clonables cells)

Mean Mutation Frequency

 

1% S.9 Concentration

HMD

 

25

25

25

-

-

-

109.4

105.6

99.5

4

5

7

90.2

94.3

86.0

4.4

5.3

8.1

 

 

6.0

50

50

50

-

-

-

83.4

98.6

74.3

1

0

16

86.3

93.7

84.7

1.2

0b

18.9

 

 

6.7

100

100

100

-

-

-

66.0

62.4

47.4

4

3

2

84.7

86.0

79.2

4.7

3.5

2.5

 

 

3.6

175

175

175

-

-

-

58.3

54.9

38.7

1

4

0

88.7

93.3

86.7

1.1

4.3

0b

 

 

1.8

250

250

250

-

-

-

35.5

15.5

39.6

6

4

3

93.5

83.8

74.8

6.4

4.8

4.0

 

 

5.0

 

 

HMD

50

50

50

+ 5%

+ 5%

+ 5%

81.1

82.1

98.9

9

3

14

84.8

87.7

105.8

10.6

3.4

13.2

 

 

9.0

100

100

100

+ 5%

+ 5%

+ 5%

85.3

83.1

78.4

8

6

2

91.5

92.7

87.0

8.7

6.5

2.3

 

 

5.9

300

300

300

+ 5%

+ 5%

+ 5%

76.7

69.3

73.5

3

5

7

100.5

108.3

94.5

3.0

4.6

7.4

 

 

5.0

450

450

450

+ 5%

+ 5%

+ 5%

55.4

47.1

40.3

2

3

5

91.8

76.7

88.7

2.0

3.8

6.0

 

 

4.0

600

600

600

+ 5%

+ 5%

+ 5%

36.1

40.6

30.5

1

15

12

91.8

76.7

86.7

1.1

19.6

13.5

 

 

11.4

aAbsolute cloning efficiency: Mean = 93.5

bNot scorable mutants.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay

Under the test conditions, Hexamethylene diamine was found not to induce mutations in this in vitro CHO/HGPRT mammalian cell forward gene mutation assay.
Executive summary:

The ability of Hexamethylene Diamine to induce mutations at the hypoxanthine-guanine phosphoribosyl transférase (hgprt) locus in Chinese hamster Owary (CHO) cells (clone K1 designated CHO-K1-BH4) was tested following directives of the OECD guideline 476 and in compliance with GLP.

A Preliminary Cytotoxicity Screen was performed to evaluate HMD for cytotoxicity in CHO at 8 dose levels with and without metabolic activation. Hexamethylene Diamine was then evaluated in a Preliminary Mutagenicity Screen at several dose levels, with and without metabolic activation. Based on the results of the Preliminary Mutagenicity, the test article mutagenicity was tested with and without a 5% concentration of metabolic activation preparation.

Hexamethylene Diamine was evaluated in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay at dose levels of 25, 50, 100, 175 and 250 µg/mL of treatment volume without metabolic activation preparation and at dose levels of 50, 100, 300, 450 and 600 µg/mL of treatment volume with a 5% concentration of metabolic activation preparation.

The positive control articles, EMS and DMN, indicate that the integrity of the test system was valid.

No statistically significant increases of mutants in HMD treated cultures were observed when compared to the negative controls. Results for Hexametylene Diamine were negative in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay at the dose levels and S9 concentrations tested.

Under the test conditions, HMD didn't induce mutagenicity in mammalian cells in the absence and the presence of activation system.