ε-caprolactam

Endpoint summary

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

Reference

Reference 1

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.

Reference 2

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.

Reference 3

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. 

Reference 4

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.

Reference 5

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. 

Reference 6

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.

Reference 7

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. 

Reference 8

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

Reference

Reference 1

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 index^b
			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.

Reference 2

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)	Deletions^a		Chromosome rearrangement^a	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.

Reference 3

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.

Reference 4

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.

Reference 5

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)	% retention^a± SD	Conclusion^b
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)	NG^a± SD	% of cells with ≥ 5 NG	% of cells^bin 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.