Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro was evaluated by a weight of evidence approach.

The test substance was found to be negative in all 8 studies (Ames, Ames, CA, TK, HGPRT, UDS, MN, SCE). Therefore, the test substance is considered to be not mutagenic in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The compound was tested for mutagenicity in a collaborative study (3 laboratories) as described by Maron and Ames (Mutat. Res. 133: 173-215, 1983) and as specified for TA102 by Levin et al. (Proc. Natl. Acad. Sci. U.S.A. 79: 7445-7449, 1982).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Caprolactam
- Analytical purity: highest purity
Target gene:
Salmonella typhimurium: histidine operon
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with
Metabolic activation system:
Aroclor 1254 induced liver S9 mix
Test concentrations with justification for top dose:
up to 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
As described by Maron and Ames (Mutat. Res. 133: 173-215, 1983) and as specified for TA102 by Levin et al (Proc. Natl. Acad. Sci. U.S.A. 79: 7445-7449, 1982).
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
The test substance was found to be negative in TA102.
Executive summary:

The test substance was tested for mutagenicity in a collaborative study (3 laboratories) and was found to be negative in TA102.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The chemical was tested with the TK+/- mouse lymphoma mutagen assay system as described by Clive et al. Mutation Res. 59, 61-108, 1979.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
- Name of test material (as cited in study report): Caprolactam
- Analytical purity: no data
Target gene:
TK+-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
500, 1000, 2000, 3000, 4000, 5000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate (EMS) or methylmethanesulfonate (MMS)
Remarks:
without S9
Details on test system and experimental conditions:
- As described by Clive et al. Mutation Res. 59, 61-108, 1979.
- NUMBER OF REPLICATIONS: 3
Evaluation criteria:
Two or more experiments were considered for an evaluation of the mutagenic activity.
- Test is positive when significant response for at least 1 of the 3 highest dose sets and a significant trend (p ≤ 0.05).
- Test is questionable when significant response for 1 of the 3 highest dose sets but no significant trend, or significant trend but no significant dose set.
- Test is inconclusive when significant response for a dose set other than 1 of the 3 highest but no significant trend, or no significant responses or trend, but the relative total growth is greater than 30% and higher toxicity can be attained.
- Test has no response when no significant responses or trend, and the relative total growth is greater than 30% under conditions where a 1.5-fold increase in dose causes precipitation or where the 5 mg/ml (or 5 μl/ml) concentration limit is attained.
- Test is negative when no significant responses or trend, and either the relative total growth is less than 30% or excessive toxicity occurs for a 1.5-fold higher dose.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1: Without metabolic activation

Treatment (µg/ml)

Total Mutants Colonies

Cloning efficiency

Relative total growth (%)

Mutant frequency (10 E-6 units)

Trial 1

Trial 2

Trial 1

Trial 2

Trial 1

Trial 2

Trial 1

Trial 2

0

47.0

61.0

86.2

49.8

100.0

100.0

18.2

40.8

0

70.0

79.0

80.0

77.2

100.0

100.0

29.2

34.1

0

114.0

62.0

95.8

97.8

100.0

100.0

39.6

21.1

0

100.0

81.0

c

88.5

c

100.0

c

30.6

500

79.0

50.0

121.6

96.8

126.5

94.8

24.8

22.0

500

92.0

82.0

106.7

87.9

119.7

136.0

32.9

39.7

500

102.0

73.0

106.5

106.6

114.6

112.8

36.5

29.1

1000

92.0

66.0

97.4

84.1

92.4

129.9

36.1

33.4

1000

70.0

42.0

76.2

129.6

93.5

117.0

35.1

13.8

1000

84.0

74.0

92.6

86.2

112.1

102.8

34.6

36.5

2000

77.0

52.0

92.0

106.9

89.2

67.1

31.9

20.7

2000

104.0

75.0

87.8

104.1

90.9

141.5

45.2

30.7

2000

124.0

62.0

124.1

74.9

94.2

81.0

38.1

35.2

3000

118.0

43.0

101.6

82.4

85.9

81.9

44.4

22.2

3000

91.0

53.0

87.6

79.8

76.9

63.0

39.6

28.3

3000

99.0

71.0

114.5

76.6

86.0

70.7

33.0

39.4

4000

104.0

109.0

122.6

113.7

102.0

92.7

32.4

40.8

4000

105.0

101.0

126.2

85.8

120.6

74.9

31.8

50.1

4000

82.0

90.0

124.9

110.3

93.9

82.7

25.1

34.7

5000

76.0

73.0

97.2

75.6

83.7

90.0

29.9

41.1

5000

112.0

82.0

116.3

89.6

91.5

71.8

36.8

38.9

5000

89.0

88.0

98.3

97.7

109.9

74.6

34.6

38.3

PC

718.0

380.0

81.8

16.5

77.5

6.8

292.5

767.7

PC

817.0

515.0

75.5

20.7

78.7

9.0

360.7

830.6

PC

c

611.0

90.7

55.7

71.6

44.0

c

365.9

PC

-

570.0

-

55.8

-

46.8

-

340.3

c: contaminated

PC: positive control. In Trial 1, the PC was0.25 µg/ml. In Trial 2, the PC was MMS 10 and 5 nl/ml.

Table 2: S9 Activated

Treatment (µg/ml)

Total Mutants Colonies

Cloning efficiency

Relative total growth (%)

Mutant frequency (10 E-6 units)

Trial 1

Trial 2

Trial 1

Trial 2

Trial 1

Trial 2

Trial 1

Trial 2

0

34.0

79.0

55.0

95.2

100.0

100.0

20.6

27.7

0

c

139.0

72.8

83.8

100.0

100.0

c

55.3

0

35.0

154.0

68.7

106.2

100.0

100.0

17.0

48.3

0

29.0

151.0

72.3

109.2

100.0

100.0

13.4

46.1

500

38.0

118.0

78.6

110.0

69.2

69.1

24.0

36.2

500

34.0

122.0

93.0

81.3

89.6

71.0

18.1

50.7

500

31.0

127.0

82.3

77.8

83.0

76.6

18.7

55.2

1000

44.0

111.0

92.0

67.3

79.0

61.3

23.7

55.8

1000

39.0

106.0

93.3

75.4

109.7

63.8

20.7

47.5

1000

33.0

100.0

102.4

67.3

84.8

63.4

160.

34.2

2000

63.0

96.0

101.2

75.4

53.6

63.4

30.9

38.1

2000

-

151.0

-

98.9

-

57.4

-

63.7

2000

-

91.0

-

85.2

-

70.4

-

39.1

3000

53.0

161.0

76.6

80.1

72.2

57.4

34.3

50.9

3000

48.0

155.0

108.6

78.6

76.2

56.7

21.9

61.0

3000

-

151.0

-

106.8

-

74.2

-

49.6

4000

c

147.0

79.4

85.9

43.4

62.4

c

58.2

4000

c

175.0

125.0

102.9

75.8

59.1

c

55.2

4000

c

163.0

97.7

85.4

93.6

91.4

c

55.7

5000

20.0

148.0

78.4

107.2

75.7

94.6

12.6

42.9

5000

36.0

199.0

c

104.6

c

102.4

c

62.5

5000

34.0

150.0

c

116.6

c

79.5

c

43.2

PC

353.0

691.0

89.0

107.7

60.8

56.0

132.2

275.3

PC

-

679.0

-

117.3

-

48.5

-

284.7

PC

-

682.0

-

57.2

-

17.4

-

397.7

c: contaminated

PC: positive control, Methylcholanthrene (MCA) 2.5 µg/ml

Conclusions:
The test substance is negative in the mouse lymphoma assay both in the absence and presence of exogenous metabolic activation.
Executive summary:

The chemical was tested with the TK+/- mouse lymphoma mutagen assay system. The test substance was not or only weakly toxic for concentrations up to the testing limit of 5000 μg/ml. 5 trials performed with or without S9 activation gave no responses that could be interpreted as evidence for mutagenesis. The 5th trial (S9 activated) was rejected due to high or low viable colony count. The largest observed increase in mutant frequency, 1.7-fold for the 3000 μg/ml treatment in Trial 1 (S9 activated), was not statistically significant, and the low mutant colony counts obtained at the higher, weakly toxic doses did not provide evidence for mutagenesis. Thus, under the conditions of this study, the test substance is negative in the mouse lymphoma assay both in the absence and presence of exogenous metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): Caprolactam
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
Main test (without S9): 50, 160, 500, 1600 and 5000 µg/ml
Main test (with S9): 16, 50, 160, 500, 1600 and 5000 µg/ml
Repeat test (with and without S9): 2000, 3000, 4000 and 5000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
Details on test system and experimental conditions:
Testing was performed as reported by Galloway, S.M. et al. (1985). Environ Mutagen 7: 1-51
Evaluation criteria:
A dose-related increase was regarded as positive response. Chromatid/chromosome gaps were excluded when computing dose-response. A repeat of each test was required, and the results of two tests were then used to assign a negative, questionable, or positive conclusion.
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

Table 1: Main test

Treatment (µg/ml)

Abs. /100 cells (% cells w/abs.)

Without S9

With S9

0

3 (3)

0 (0)

16

1 (1)

50

2 (2)

1 (1)

160

3 (3)

0 (0)

500

0 (0)

0 (0)

1600

2 (2)

0 (0)

5000

2 (2)

1 (1)

Positive control

54 (38)

106 (61)

 

Table 2: Repeat test

Treatment (µg/ml)

Abs. /100 cells (% cells w/abs.)

Without S9

With S9

0

0 (0)

2(2)

2000

4 (4)

0 (0)

3000

4 (4)

0 (0)

4000

1 (1)

1 (1)

5000

1 (1)

3 (3)

Positive control

54 (38)

49 (32)

 

Conclusions:
The test substance was found to be negative.
Executive summary:

An in vitro mammalian chromosome aberration study similar to OECD TG 473 was performed. The treatment of CHO cells with up to 5 mg/ml of the test substance in the presence and absence of S9 did not increase the frequency of chromosomal aberrations. Under the condition of this assay, the test substance was found to be negative in the in-vitro Mammalian Chromosome Aberration Test both in the absence and presence of exogenous metabolic activation. 

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Cells were seeded and incubated for 24h. Following 1h treatment, cultures were washed 3 times and incubated for a further 24h. 500 cells were scored from each 2 cultures, yielding 1000 cells per treatment.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
566 -11300 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: methyl methanesulfonate, dimethyl nitrosamine
Evaluation criteria:
A dose-related increase in micronucleus frequency greater than twice the historical solvent control value for this study was considered a positive response.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 11300 µg/ml
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity was determined by observing inhibition of cell growth in 24-well cluster dishes. 12 concentrations of the chemical were used, with 2 replicate wells per dish. To select a range of concentrations to be used in subsequent experiments, a cytotoxicity threshold concentration was determined which was found to be 2.26 µg/ml (-S9) and 5.66 µg/ml (+S9). This value represents the lower limit of cytotoxicity as determined by visible inhibition of cell growth.
Conclusions:
No increase in micronucleus frequency was observed both in presence and absence of S9 mix. Under the conditions of this study, the test substance is negative in this in vitro CHO/ micronucleus assay.
Executive summary:

Cells were seeded and incubated for 24h. Following 1h treatment, cultures were washed 3 times and incubated for a further 24h. 500 cells were scored from each 2 cultures, yielding 1000 cells per treatment.

No increase in micronucleus frequency was observed both in presence and absence of S9 mix. Under the conditions of this study, the test substance is negative in this in vitro CHO/ micronucleus assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The chemical was tested for mutagenic activity at the HGPRT locus as described by Fox 1982 (Mut. Res. 100, 235-238), and McMillan and Fox 1979 (Mut. Res. 60, 97-107).
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and ß-naphthoflavone induced rat liver S-9 mix
Test concentrations with justification for top dose:
300, 1000, 2000, 3000 and 4000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Details on test system and experimental conditions:
As described by Fox 1982 (Mut. Res. 100, 235-238), and McMillan and Fox 1979 (Mut. Res. 60, 97-107).
Evaluation criteria:
not reported
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
- Precipitation: no precipitation was observed even at the maximum concentrations tested


RANGE-FINDING/SCREENING STUDIES: Preliminary cytotoxicity study was carried out with V79 cells using Microtitre assay at concentration range from 10 mg/ml to 5 µg/ml in the presence or absence of serum, and in the presence or absence of an exogenous metabolising system (S9 mix). The maximum non-toxic dose was found to be 2.5 mg/ml in the absence of serum, both with and without S9 mix. Based on initial cytotoxicity studies, a dose range was chosen for use in assays for inhibition of colony-forming ability and found to be 0.3 mg/ml (highest non-toxic dose) and >4mg/ml (concentration for 50% kill).

  Table 1: Mutation frequency at expression times I

Treatment (µg/ml)

Mutation frequency at expression times of -

5 days

8 days

12 days

-S9

+S9

-S9

+S9

-S9

+S9

300

NT

3.1 x 10E-6

NT

6.8 x 10E-6

NT

NT

1000

1.7 x 10E-6

5.9 x 10E-6

< 5.0 x 10E-6

3.2 x 10E-6

NT

NT

2000

2.1 x 10E-6

NT

< 6.2 x 10E-6

NT

NT

NT

3000

NT

4.5 x 10E-6

NT

1.2 x 10E-5

NT

NT

4000

8.3 x 10E-6

NT

1.3 x 10E-5

NT

NT

NT

Vehicle

1.9 x 10E-5

3.1 x 10E-6

< 2.8 x 10E-6

6.3 x 10E-6

NT

NT

PC

3.0 x 10E-6

7.6 x 10E-6

1.0 x 10E-6

9.3 x 10E-6

NT

NT

 

  Table 1: Mutation frequency at expression times II

Treatment (µg/ml)

Mutation frequency at expression times of -

5 days

8 days

12 days

-S9

+S9

-S9

+S9

-S9

+S9

2000

NT

< 4.9 x 10E-6

< 1.8 x 10E-6

< 3.4 x 10E-6

2.6 x 10E-6

< 3.5 x 10E-6

3000

NT

8.0 x 10E-6

2.7 x 10E-6

3.6 x 10E-6

5.4 x 10E-6

< 3.4 x 10E-6

Vehicle

NT

5.6 x 10E-6

2.2 x 10E-6

< 2.7 x 10E-6

8.8 x 10E-6

< 3.6 x 10E-6

2000*a

NT

7.7 x 10E-6

< 3.7 x 10E-6

< 2.7 x 10E-6

< 6.2 x 10E-6

3.6 x 10E-6

3000*a

NT

4.5 x 10E-6

2.9 x 10E-6

3.0 x 10E-6

< 6.5 x 10E-6

3.7 x 10E-6

Vehicle*a

NT

< 6.6 x 10E-6

< 3.6 x 10E-6

3.5 x 10E-6

1.9 x 10E-6

3.0 x 10E-6

NT: not tested

PC: Positive control

*a: serum (FCA) was added

Conclusions:
The test substance was found to be negative in the HGPRT/V79 assay both in the absence and presence of exogenous metabolic activation. 
Executive summary:

The chemical was tested for mutagenic activity at the HGPRT locus. No significant increase in the mutatin frequency was observed at all dose levels up to 12-day expression times, in the presence and absence of both serum and activation. Under the condition of this assay, the test substance was found to be negative in the HGPRT/V79 assay both in the absence and presence of exogenous metabolic activation. 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Caprolactam
- Analytical purity: >99%
Target gene:
Salmonella typhimurium: histidine operon
Species / strain / cell type:
other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced liver S9 mix
Test concentrations with justification for top dose:
5000, 10770, 23210 and 50000 µg/plate
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: dinitrotoluene (500 μg, TA1535 and TA100), quinacrine mustard (10 μg, TA1537) and nitrofluorene (100 μg, TA1538 and TA98)
Remarks:
without S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: anthramine (100 μg, TA1535 and TA 100), aminoquinoline (100 μg, TA1537) and acetamidofluorene (500 μg, TA1538 and TA98)
Remarks:
with S9
Details on test system and experimental conditions:
According to Ames et al. 1975; Mut.Res. 31, 347
Evaluation criteria:
not reported
Species / strain:
other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1: Mean Reverants per plate (average of 2 plates)

Dose (µg/plate)

        TA 98

       TA 100

      TA 1535

      TA 1537

      TA1538

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

13

17

54

55

54

52

6

7

8

11

5000

16

20

56

67

52

63

6

8

8

16

10770

15

18

54

66

60

56

4

8

10

16

23210

11

22

57

52

66

61

3

6

11

12

50000

8

16

50

54

56

51

4

8

9

12

Positive control

558

1178

237

242

78

0

122

379

1209

1156

 

Conclusions:
The test substance was not mutagenic in any of the five strains of S. typhimurium.
Executive summary:

An Ames test similar to OECD TG 471 was performed. Under test conditions chosen, the test substance was not mutagenic in any of the five strains of S. typhimurium used in this testand doses up to 50 mg/plate. The authors also reported that studies using lower concentrations of the test substance gave similar results. Under the condition of this assay, the test substance was not mutagenic in the microbial assay either in the presence or absence of metabolic activation system.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Specific details on test material used for the study:
- Name of test material (as cited in study report): Caprolactam
- Analytical purity: no data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
Main test (with S9): 50, 160, 500, 1600 and 5000 µg/ml
Main test (without S9): 16, 50, 160, 500, 1600 and 5000 µg/ml
Repeat test (with and without S9): 2000, 3000, 4000 and 5000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
withour S9
Details on test system and experimental conditions:
Testing was performed as reported by Galloway, S.M. et al. (1985). Environ Mutagen 7: 1-51
Evaluation criteria:
As described in Galloway, S.M. et al. (1985). Environ Mutagen 7: 1-51.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/ml in main test
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: 10 or 11 dose levels in descending half-log series were used for the first SCE tests beginning with 5 mg/ml (highest dose). Cells were harvested by shake-off. The 3-5 highest doses yielding sufficient mitotic cells were scored. Results from these initial SCE tests were used to determine dose ranges for all subsequent tests.

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

Table 1: Main test

Treatment (µg/ml)

SCE/cell ± S.E.

Without S9

With S9

0

7.6±0.4

8.4±0.4

16

8.3±0.4

-

50

8.6±0.4

8.1±0.4

160

8.5±0.4

8.7±0.4

500

8.4±0.4

8.4±0.4

1600

8.3±0.4

8.5±0.5

5000

toxic

8.4±0.3

Positive control

51.1±1.5

24.6±0.7

 

Table 2: Repeat test

Treatment (µg/ml)

SCE/cell ± S.E.

Without S9

With S9

0

8.2±0.4

8.1±0.4

2000

8.5±0.4

8.5±0.4

3000

9.7±0.5

8.7±0.5

4000

9.3±0.4

8.3±0.5

5000

8.3±0.4

8.6±0.3

Positive control

47.3±1.4

38.6±1.0

 

Conclusions:
The test substance was found to be negative in the in-vitro Sister Chromatid Exchange Assay in Mammalian Cells both in the absence and presence of exogenous metabolic activation. 
Executive summary:

The treatment of CHO cells with up to 5 mg/ml test substance in the presence and absence of S9 did not increase the frequency of SCE. Under the condition of this assay, the test substance was found to be negative in the in-vitro Sister Chromatid Exchange Assay in Mammalian Cells both in the absence and presence of exogenous metabolic activation. 

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Unscheduled DNA synthesis in hepatocytes was performed as described by Probst et al. Environm. Mutagen 3, 11-32 (1981).
GLP compliance:
not specified
Type of assay:
other: UDS
Species / strain / cell type:
hepatocytes: primary cultures of adult rat
Metabolic activation:
without
Test concentrations with justification for top dose:
0.5, 1, 5, 10, 50, 100, 500, 1000, 5000 and 10000 nmoles/ml, which is equivalent to 0.056, 0.113, 0.56, 1.13, 5.65, 11.3, 56.5, 113, 565 and 1130 µg/ml (Probst, 1985)
0.01-1000 µg/ml (Williams, 1985)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and 2-acetylaminofluorene (AAF)
Details on test system and experimental conditions:
As described by Probst et al. Environm. Mutagen 3, 11-32 (1981).
Evaluation criteria:
A compound was judged to have induced a positive response for UDS when at least 2 successive concentrations produced nuclear grain counts which exceeded those of the control by 3 standard deviations of the control value.
Species / strain:
hepatocytes: primary cultures of adult rat
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1: Net nuclear silver grains

Concentration (µg/ml)

Net nuclear silver grains (Mean±SD)*a

Study No. 1

Study No. 2

1130

NT

-0.92±1.05

565

NT

-0.43±1.59

113

-0.63±1.89

-1.87±1.37

56.5

-1.24±1.17

-2.75±2.33

11.3

-2.00±2.15

-1.15±1.47

5.65

-1.56±1.11

-0.26±1.70

1.13

-1.01±1.50

-0.97±1.94

0.56

-0.74±2.58

-0.43±1.83

0.113

-2.08±1.93

-0.56±1.46

0.056

-0.58±1.46

-0.24±1.35

*a: Represents counts of nuclei from 20 morphologically unaltered cells from each treatment

NT: not tested.

Conclusions:
The test suibstance was found to be negative in that assay.
Executive summary:

Primary cultures of adult rat hepatocytes were incubated for 20 h with 8 concentrations of the test substance. Unscheduled DNA synthesis was measured by autoradiography, and the study was replicated using 2 independent hepatocyte preparations. A positive autoradiographic response for UDS was noted in cultures treated with either the ultimate carcinogen MNNG or the procarcinogen 2AAF. At concentrations up to 1130 µg/ml of the test substance, no cytotoxicity was evident and the incidence of net nuclear silver grains was not different from the DMSO-treated control and, therefore, there was no evidence for the induction of UDS.

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

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo was evaluated by a weight of evidence approach.

The tests for chromosomal aberration, induction of DNA strand breaks or unscheduled DNA synthesis as well as the micronucleus assay were negative for the test substance.

Therefore, the test substance is considered to be not mutagenic in vivo.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Principles of method if other than guideline:
The present experiment was conducted according to the test procedure described in Annex V of EEC Directive 79-831, Part B (1984).
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
mouse
Strain:
other: 1C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 25-28 g


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water for test substance, corn oil for positive control
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Caprolactam (CAP) was dissolved in distilled water and orally applied by stomach intubation in a volume of 0.1 ml/10 g body weight.

Duration of treatment / exposure:
single administration
Frequency of treatment:
1
Post exposure period:
24-48 h
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
benzo(a)pyrene
- Route of administration: gavage
- Doses / concentrations: 63 mg/kg bw
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
Bone marrow of CAP-treated animals was sampled after 24, 30 and 48 h.
The sampling time for benzo(a)pyrene (BP)-treated animals was 30 h after treatment.
Each experimental group consisted of 5 treated males and 5 treated females plus 2 animals of each sex given distilled water. BP was tested with 5 males only. A total of 100 cells at mitotic metaphase per animal were scored microscopically. Mitotic indices were determined by counting the number of mitoses among 500 cells in a given field on each slide, i.e., a total of 1000 cells per animal.


METHOD OF ANALYSIS: The aberrations scored were of the chromatid or isochromatid type, i.e., gaps, breaks and fragments.


Evaluation criteria:
not reported
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

 Tab. 1:

Treatment

Interval

(h)

Numbers of cells scored

Number of cells with

Aberrant cells

(%±SE)a

Mitotic indexb

Gaps

Breaks

Exchanges

Water

-

1000

16

5

0

0.5±0.2

3.1

 

CAP

24

1000

20

3

0

0.3±0.2

2.4

30

1000

4

0

0

0.0±0.0

2.1*

48

1000

4

3

0

0.3±0.2

2.1*

BP

30

500

20

10

4

2.4±0.9**

2.8

aExcluding cells with only gaps

bPercent cells at mitosis (based on 1000 cells counted per animal)

* p<0.05, ** p<0.01

Conclusions:
Based on the results, the test substance is negative for induction of chromosomal aberrations in bone marrow of mice.
Executive summary:

A study compareable to OECD testguideline 475 was performed. The test substance did not induce chromosomal aberrations in mouse bone marrow under the present experimental conditions. The frequencies of cells with breaks and gaps remained at the control level in all 3 sampling groups. The mitotic indices were reduced at the 30- and 48-h intervals indicating a cytotoxic effect. The positive control data with 63 mg/kg BP show a significant increase of chromosomal aberrations over the control. Based on the results, the test substance is negative for induction of chromosomal aberrations in bone marrow of mice.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable publication which meets basic scientific principles
Principles of method if other than guideline:
The test substance was tested in vivo for the ability to induce chromosome aberrations in mouse bone marrow cells. Single intraperitoneal injections were given to 8 mice each at dose rate of 175, 350 and 700 mg/kg bw.
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): Caprolactam
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12-14 weeks
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: phosphate-buffered saline
Details on exposure:
Chemical dilutions were prepared immediately before administration and adjusted so that 0.4 ml was administered intraperitoneally per animal in all cases.
Duration of treatment / exposure:
single
Frequency of treatment:
once
Post exposure period:
18 h
Dose / conc.:
700 mg/kg bw/day
Dose / conc.:
350 mg/kg bw/day
Dose / conc.:
175 mg/kg bw/day
No. of animals per sex per dose:
8 males
Control animals:
yes, concurrent vehicle
Positive control(s):
7,12-dimethylbenz[a]-anthracene (DMBA) dissolved in corn oil
- Route of administration: ip
- Doses / concentrations: 200 mg/kg bw
Tissues and cell types examined:
bone marrow cells
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
700 mg/kg bw
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
CAP was relatively toxic when injected into mice. 2 of the 3 animals given 800 mg/kg bw died within 24 h, although the average generation time among marrow cells of the survivor was not affected. Among the 8 mice given 700 mg/kg bw, 3 died within 24 h but the average generation time was not prolonged in the survivors.

Tab. 1:

Treatment (mg/kg bw)

Deletionsa

Chromosome rearrangementa

Aberrations/ cell

% Aberration cell

chromatid

chromosomal

175

4

2

0

0.015

1.3

350

14

2

0

0.040

3.5

700

12

1

0

0.033

3.3

DMBA

82

3

0

0.213

13.8

Corn oil

11

2

0

0.033

2.0

aTotal aberrations in 400 metaphases scored per treatment.

Conclusions:
Based on the results, the test substance is negative in chromosome aberration assay with mouse bone marrow cells.
Executive summary:

A study testing for the ability in vivo to induce chromosome aberrations in mouse bone marrow cells. Statistical analyses of the aberration data showed no significant difference among aberrations per cell or percent aberrant cells, regardless of whether gaps were included or excluded from the analyses. Similar analyses showed no significant difference among the average generation time values for animals in any of the treatment groups.Based on the results, the test substance is negative in chromosome aberration assay with mouse bone marrow cells.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
other: C57BL/6J
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-12 weeks
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
Details on exposure:
The test substance plus the positive and negative control (olive oil) were administered via the oral route, at a volume of 0.2 ml per 10 g bw (20 ml/kg bw).
Duration of treatment / exposure:
single dose
Frequency of treatment:
once
Post exposure period:
24-48 h
Dose / conc.:
700 mg/kg bw/day (actual dose received)
Dose / conc.:
435 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 46 mg/kg bw
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): The bone marrow smears were sampled at either 24, 36 or 48 h after dosing.


DETAILS OF SLIDE PREPARATION: The animals were killed by cervical dislocation and slides were prepared using the method described by Richardson et al. (Mutation Res. 124, 241-246, 1983).


METHOD OF ANALYSIS: 1000 polychromatic erythrocytes (PCEs) were observed per slide for presence of micronuclei. A ratio of PCEs to normocytes was also scored for each slide (200 cells read) as a measure of cytotoxicity. In the limited repeat study the slides were assessed in two ways, firstly as in the main study, and secondly where 5000 PCEs were observed for the presence of micronuclei, where slides were assessed starting at the beginning of the smear and proceeding to the leading edge, as recommended by Ashby and Mohammed (Mutation Res. 164, 217-235, 1986).

Evaluation criteria:
not reported
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Combining the results obtained (male and female) a statistically and biologically significant increase in polychromatic erythrocytes (PCEs) containing micronuclei was observed with cyclophosphamide at all the sampling times, thus verifying the sensitivity of the test system.

Tab. 1: Main study: Incidence of micronuclei/1000 PCEs

Dose

Sex

Mean incidence of micronuclei/1000 cells

24 h

36 h

48 h

Olive oil,

20 ml/kg bw

 

Male

4.4±1.1

4.8±3.56

4.8±3.89

Female

3.0±1.9

2.4±1.14

1.8±1.48

Combined

3.7±1.6

3.6±2.8

3.3±3.2

Cyclophosphamide,

46 mg/kg bw

Male

33.6±13.6**

49.0±9.74**

21.0±5.56**

Female

30.6±4.5**

36.0±6.63**

19.4±7.60**

Combined

32.3±9.7**

42.5±10.4**

20.2±6.3**

CAP,

435 mg/kg bw

Male

5.6±2.5

7.2±4.08

3.8±1.48

Female

3.8±0.44

4.0±2.12

3.4±1.67

Combined

4.7±1.9

5.6±3.5

3.6±1.5

CAP,

700 mg/kg bw

Male

6.3±1.52*

4.0±1.87

2.4±2.07

Female

5.6±1.67*

3.5±2.64

2.5±1.29

Combined

5.9±1.6**

3.8±2.1

2.4±1.7

* Statistically significantly different from the control group at the 5% level

** Statistically significantly different from the control group at the 1% level

Tab. 2: Repeat test: Incidence of micronuclei/1000 polychromatic erythrocytes at the 24-h sampling time following oral dosing

Dose (mg/kg bw)

Sex

Mean incidence of micronuclei/1000 cells (24 h)

Control, olive oil

(20 ml/kg bw)

Male

1.3 ± 0.57 (3)

Female

2.0 ± 1.58

Cyclophosphamide

(46 mg/kg bw)

Male

18.6 ± 6.34**

Female

5.8 ± 4.26*

Caprolactam

(700 mg/kg bw)

Male

3.4 ± 2.30**

Female

1.8 ± 1.09

* Statistically significantly different from the control group at the 5% level.

** Statistically significantly different from the control group at the 1% level.

All means based on 5 observations except where indicated in parentheses.

 

Tab. 3: Repeat test: Incidence of micronuclei/5000 polychromatic erythrocytes at the 24-h sampling time following oral dosing

Dose (mg/kg bw)

Sex

Mean incidence of micronuclei/1000 cells (24 h)

Control, olive oil

(20 ml/kg bw)

Male

0.7 ± 0.33

Female

0.6 ± 0.46

Caprolactam

(700 mg/kg bw)

Male

1.9 ± 1.0 (3)

Female

0.32 ± 0.30

Combined

0.9 ± 0.99

All means based on 5 observations except where indicated in parentheses.

Conclusions:
Based on the results, the test substance is not clastogenic in the mouse micronucleus test according to EU and GHS standards.
Executive summary:

A study compareable to OECD guideline 474 was performed. A statistically significant increase in PCEs containing micronuclei was only observed observed with the test substance at the 24-h sampling time and the 700 mg/kg bw dose-level. This effect was statisticaly significant at the 1% level when both sexes were combined, and at the 5% level when analysed separately.

In the repeat study where 1000 PCEs were assessed, no statistically significant increase was observed over control values when the sexes were combined, however when the sexes were analysed separately a small statistically significant increase over control values was observed in male mice.

In the repeat study where 5000 PCEs were assessed at dose level of 700 mg/kg bw, a small statistically significant effect was still observed only in male mice (control mean 0.7 versus CAP mean of 1.9), however these effects were within the range of historical controls.

Based on the results, the test substance is not clastogenic in the mouse micronucleus test according to EU and GHS standards.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication which meets basic scientific principles
Principles of method if other than guideline:
According to NTP standards protocols.
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
- Name of test material (as cited in study report): Caprolactam
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Toxicology Program production facility
- Age at study initiation: 9-14 weeks
- Weight at study initiation: 25-33 g
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Phosphate buffer saline
Details on exposure:
For the initial MN test, groups of 5-6 mice were injected IP on three consecutive days with the test chemical (at l x, 1/2 x, and 1/4 x, where x is the maximum dose determined in the dose determination experiments), a weakly active dose of the positive control chemical, or the appropriate solvent.
Duration of treatment / exposure:
once
Frequency of treatment:
3
Dose / conc.:
162.5 mg/kg bw/day
Remarks:
Initial test
Dose / conc.:
350 mg/kg bw/day
Remarks:
Initial test
Dose / conc.:
162.5 mg/kg bw/day
Remarks:
Repeat test
Dose / conc.:
350 mg/kg bw/day
Remarks:
Repeat test
Dose / conc.:
487.5 mg/kg bw/day
Remarks:
Repeat test
No. of animals per sex per dose:
5-6
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Route of administration: ip
- Doses / concentrations: 0.2 mg/kg bw
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
Mice were euthanized with CO2, 24 hr after the third treatment. Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol, and stained with acridine orange. For each animal, slides were evaluated at 1000x magnification for the number of MN-PCE among 2000 PCE and for the percentage of PCE among 200 erythrocytes.
Evaluation criteria:
Data are typically presented as the mean number of micronucleated cells per 1000 cells for each treatment group. A positive trend test is one in which the P value is equal to or less than 0.025. For the micronucleus frequency in any dose group to be considered significantly elevated over the control group, the P value must be equal to or less than 0.025 divided by the number of chemical-treatment groups.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

Tab. 1: Main test

 

Sample Collection Time

Sex

Cell

Dosing Regimen

Trend Test P-Value

24 Hours

Male

PCE

IP x 3, 72 Hours

0.112

 

Dose (mg/kg bw)

No. of Animals Scored

Mean MN-PCE/1000 PCE ± SEM

Pairwise P

Vehicle Control:

Phosphate Buffered Saline

 0         

5

2.10 ± 0.64

 

Test Chemical:

 

162.5       

5

2.00 ± 0.42

0.562

 

325         

3

3.17 ± 1.09

0.095

Positive Control:

Mitomycin-C

 0.2       

5

5.80 ± 0.78

< 0.0001

 

Tab. 2: Repeat test

 

Sample Collection Time

Sex

Cell

Dosing Regimen

Trend Test P-Value

24 Hours

Male

PCE

IP x 3, 72 Hours

0.330

 

Dose (mg/kg)

No. of Animals Scored

Mean MN-PCE/1000 PCE ± SEM

Pairwise P

Vehicle Control:

Phosphate Buffered Saline

 0         

5

2.60 ± 0.33

 

Test Chemical:

 

162.5       

5

3.80 ± 0.46

0.066

 

325         

5

2.80 ± 0.54

0.393

 

487.5       

5

3.30 ± 0.58

0.181

Positive Control:

Mitomycin-C

 0.2       

5

8.00 ± 0.95

< 0.0001

 

Conclusions:
Based on the test results, the test substance is negative in the present micronucleus test in mice.
Executive summary:

No significant increase was noted in the incidence of MN-PCE in the groups treated with 162.5, 350 or 487.5 mg/kg bw. Based on the test results, the test substance is negative in the present micronucleus test in mice.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication which meets basic scientific principles.
Principles of method if other than guideline:
The test substance was administered by gavage to Fischer 344 rats at a dose of 750 mg/kg bw and the hepatocytes were isolated 12, 24 or 48 h after treatment. The isolated hepatocytes were subsequently examined for the induction of DNA-strand breaks (SB) as described by Bermudez et al. (Environ. Mutagen. 4, 667-679, 1982), and unscheduled DNA synthesis (UDS) as described by Mirsalis et al. (Environ. Mutagen. 4, 553-562, 1982).
GLP compliance:
not specified
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Weight at study initiation: 200-280 g


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±1
- Humidity (%): 50±10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
Details on exposure:
The test substance was administered by gavage (1 ml/100 g body weight) using water as vehicle.
Control animals received only vehicle.
Duration of treatment / exposure:
single
Frequency of treatment:
once
Post exposure period:
48 h
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 males
Control animals:
yes, concurrent vehicle
Positive control(s):
mM dimethylnitrosamine (DMN)
- Route of administration: Control cells treated DMN in vitro for 18 h, in the presence of [3H]thymidine as the positive control for UDS
- Doses / concentrations: 1 mM
Tissues and cell types examined:
hepatocytes
Details of tissue and slide preparation:
As described by Bermudez et al. (Environ. Mutagen. 4, 667-679, 1982) and Mirsalis et al. (Environ. Mutagen. 4, 553-562, 1982). In case of UDS, control cells treated with 1 mM dimethylnitrosamine in-vitro for 18 h, in the presence of [3H]thymidine were used as the positive control.
Evaluation criteria:
In case of UDS, a positive response is assumed where the net grains/nucleus is equal to or greater than 5.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Tab. 1: Induction of strand breaks in hepatocytes of rats treated with CAP

Dose

(mg/kg bw)

Time

(h)

n

(animals)

% retentiona± SD

Conclusionb

750

12

3

81±6

NEG

750

24

3

71±4

NEG

750

48

3

76±3

NEG

a % retention = mean of eluted filters x 100/mean of reference filters

aNEG, where no significant (p<0.05) difference from controls was observed. The mean retention for all controls (n=4) was 78±4.

 

Tab. 2: Unscheduled DNA synthesis induction in hepatocytes of rats treated with CAP

Dose

(mg/kg bw)

Time

(h)

n

(animals)

NGa± SD

% of cells with ≥ 5 NG

% of cellsbin S-phase

control

 

4

-4±1

0

0.37

750

12

3

-3±1

0

0.03

750

24

3

-4±1

0

0.10

750

48

3

-4±1

0

0.10

DMN

18

3

46±28

98

-

aNG, net grains/nucleus . The number or grains over the nucleus is determined by subtracting the number of grains in an area of the cytoplasm, equal to that of the nucleus, from the nuclear counts. Each data point represents the mean of the net grains per nucleus of 75-150 cells. Net grains/nucleus of greater than 5 are considered a positive response .

b3000 cells were counted per animal

Conclusions:
Under the conditions of this test, no induction of SB or UDS was observed in hepatocytes following the administration to rats of the test substance.
Executive summary:

The test substance was administered by gavage to Fischer 344 rats at a dose of 750 mg/kg bw and the hepatocytes were isolated 12, 24 or 48 h after treatment. The isolated hepatocytes were subsequently examined for the induction of DNA-strand breaks (SB) and unscheduled DNA synthesis (UDS). Increases in SB were not detected 12, 24 or 48 h following the administration of the test substance. No significant (P < 0.05) difference from controls was observed. The mean retention for all controls (n = 4) was 78 ± 4, and in the test substance after 12, 24 and 48 h were 81±6, 71±4 and 76±3, respectively. UDS was not induced in hepatocytes from the livers of rats treated with the test substance when the cells were examined at 12, 24 or 48 h following treatment. The net grains/nucleus was not equal to or greater than 5. Dimethylnitrosamine treatment of hepatocytes in vitro resulted in the induction of UDS indicating that the system was operational.Under the conditions of this test, no induction of SB or UDS was observed in hepatocytes following the administration to rats of the test substance.

Additional information

Genetic toxicity in vitro

A number of studies are available where the test substance was tested for its mutagenic potential in-vitro.

 

Weight of evidence in vitro gene mutation in bacteria:

1) Allied Chemical Corp. (ACC), In Vitro Mutagenicity and Cell Transformation Screening of Caprolactam, MA-03-77-4, 1979.

2) Mueller W. et al. (1993), Environ. Health Persp. Suppl. 101, 33-36.

The test substance was not mutagenic in the Ames test with and without metabolic activation. 5 different Salmonella strains were tested negative but no test was performed with E.coli (ACC, 1979). In order to detect oxidizing mutagens or cross-linking agent S. typhimurium strain TA 102 was used in a separate assay (Mueller et al., 1993).

 

Weight of evidence in vitro cytogenicity in mammalian cells:

1) Chromosomal aberration in CHO-cells,

Gulati, D.K. et al. (1989), Environm. Molec. Mutag. 13, 133-193.

2) in vitro micronucleus assay in CHO-cells,

Douglas, G.R. et al. (1985), Prog. Mutat. Res. 5, 359-366.

3) Unscheduled DNA synthesis assay in rat hepatocytes,

Probst, G.S. & Hill, L.E. (1985), Prog. Mutat. Res. 5, 381-386.

4) Sister chromatid exchange assay in CHO-cells,

Gulati, D.K. et al. (1989), Environm. Molec. Mutag. 13, 133-193.

No signs of a genotoxic potential were identified in a chromosomal aberration test with CHO cells at doses of 16-5000 µg/ml (Gulati et al., 1989) and a UDS test with primary rat hepatocytes at doses of 0.056-1130 µg/ml (Probst et al., 1985). Additionally, the test substance was observed to be negative in the micronucleus assay with CHO cells at 566-11300 µg/ml (Douglas et al. 1985) and the SCE assay with CHO cells at doses of 16-5000 µg/ml (Gulati 1985). All assays were performed in the absence and presence of a metabolic activation system.

 

Weight of evidence in vitro gene mutation in mammalian cells

1) HGPRT assay in V79-cells,

Fox, M. & Delow, G.F. (1985), Prog. Mutat. Res. 5, 517-523.

2) In vitro gene mutation assay in mouse lymphoma L5178Y cells,

Myhr, B. et al. (1985), Prog. Mutat. Res. 5, 555-568.

No signs of a mutagenic activity were detected in the HPRT Test with V79 cells at doses of 300-4000 µg/ml (Fox et al., 1985) and the mouse lymphoma test at doses of 500-5000 µg/ml (Myhr et al., 1985). Both assays were performed with and without the presence of a metabolic activation system.

 

As part of an interlaboratory survey, many in-vitro tests were performed with the test substance. Most of these gave negative results. Although, few experiments with human lymphocytes yielded inconsistent results. At high cytotoxic concentrations (higher than the 10 mM recommended in the guideline) chromosomal aberrations were described (Norppa et al., 1989).

A very small but significant increase in the frequency of chromosomal aberrations was described at the highest tested test substance dose-levels (5.5mg/ml, Sheldon et al., 1989; 7.5mg/l, Kristiansen et al., 1989). A likewise small but dose dependent increase in chromosomal aberrations in human lymphocytes was described by Howard et al. (1985) at doses of 270-2750 µg/ml with and without auxiliary metabolic activation.

Summarizing the vast amount of negative in vitro assays and comparing them to the exclusive findings in human primary lymphocytes at high cytotoxic concentrations, by means of a weight of evidence it can be anticipated that the test substance is not genotoxic in vitro.

 

Genetic toxicity in vivo

Weight of evidence:

1) Chromosomal aberration assay in bone marrow cells of mice, gavage application.

Adler, I.D. & Ingwersen,(1989), Mutat. Res. 224, 343-345.

2) Chromosomal aberration assay in bone marrow cells of mice, intraperitoneal application.

McFee, A.F. & Lowe, K.W. (1989), Mutat. Res. 224, 347-350.

3) Mouse micronucleus assay in bone marrow cells, gavage application.

Sheldon, T. (1989), Mutat. Res. 224, 351-355.

4) Mouse micronucleus assay in bone marrow cells, 3 subsequent intraperitoneal applications.

Shelby et al. (1993), Environm. Molec. Mutagenesis 21, 160-179.

5) SCE-assay in bone marrow cells of mice, intraperitoneal application.

McFee, A.F. & Lowe, K.W. (1989), Mutat. Res. 224, 347-350.

6) Assay for unscheduled DNA synthesis (UDS) and DNA strand breaks (SB) in hepatocytes of rats, gavage application.

Bermudez, E. et al. (1989), Mutat. Res. 224, 361-364.

 

No chromosomal aberrations were induced in bone marrow cells of mice treated with caprolactam orally via gavage in dose levels up to 1000 mg/kg bw (Adler and Ingwersen, 1989) or intraperitoneally in dose levels up to 700 mg/kg bw (McFee and Lowe, 1989).

Simillarly, no micronuclei were induced in bone marrow cells of mice treated with caprolactam orally via gavage in dose levels up to 700 mg/kg bw (Sheldon, 1989) or 3 times intraperitoneally in dose levels up to ca. 500 mg/kg bw (Shelby et al., 1993).

Finally, no induction of DNA strand breaks or unscheduled DNA synthesis was observed in hepatocytes of rats gavaged with CAP (750 mg/kg bw, Bermudez et al., 1989) and no induction of SCE was observed in bone marrow cells of mice intraperitoneally dosed with CAP (up to 700 mg/kg bw, McFee and Lowe, 1989). 

 

The results of two mouse spot test performed in two independent laboratories with two different mouse strains were ambiguous. 

In one experiment treatment of heterozygous pigment precursor cells in embryos with up to 500 mg/kg bw resulted in a slight increase in the frequency of colored spots in adult animals (Fahrig, 1989). Statistical significance was only obtained in 1 of 3 experimental groups. Similar results were obtained in the second laboratory with maximal dose levels of 700 mg/kg bw (Neuhäuser-Klaus & Lehmacher, 1989). Again slight increase in the frequency of colored spots was observed only gaining statistical significance in 1 of 5 replicates and exhibiting no signs of dose dependency.

The nature of these spots in both experiments suggested that they may have been the result of the induction of mitotic recombination and not as a result of mutagenicity. Induction of mitotic recombination is presumably secondary to the high, dose dependent toxicity of caprolactam observed under the conditions of the assay (mortality, loss of litter). Therefore the relevance of this effect remains unclear.

 

Combining all results, CAP showed neither mutagenic nor clastogenic potential with respect to most of the different genetic endpoints tested. Few in-vitro and in-vivo tests show induction of mitotic recombination; however these effects remain unclear, especially taking into account the negative results in rats and mice carcinogenicity bioassays (NTP, 1982).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity was observed in valid in-vitro or in-vivo studies. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.