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Genetic toxicity in vitro

A number of studies are available where Caprolactam (CAP) 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.

CAP 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, CAP 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 according to current guidelines 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 CAP. 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 CAP 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 caprolactam is not genotoxic in vitro.

 

For a comprehensive overview, the large amount of additional genotoxicity studies was partly combined by assay read out (e.g. chromosomal aberration, gene mutation, SCE) in the IUCLID5 file. All of them supported the above mentioned results.

 

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).


Endpoint Conclusion:

Justification for classification or non-classification

No indication for a genotoxic potential was found in vitro and in vivo.