Hexahydro-2H-azepin-2-one, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance (and the decomposition products) do not show any relevant mutagenic or cytotoxic effects.

Link to relevant study records

Reference

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data obtained by experimental results with sodium caprolactamate, as defined in section 1.2:

The test item didn't show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only).

Cytotoxity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased.

Therefore it can be stated, that under the test conditions, the test item is not mutagenic in the Bacteria Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102, and TA 1535.

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Data obtained by Read-Across from Caprolactam:

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).

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Data obtained by Read-Across from sodium hydroxide:

In vivo Studies

Valid in vivo genotoxicity studies are not available.

A mouse bone marrow micronucleus test using 15 mM NaOH at a dose of 10 mg/kg bw revealed no significant increase of nuclei (Aaron et al., 1989). The test compound was administered as a single i.p. dose to treatment groups (5 males and 5 females) at 30, 48 and 72h. Mouse oocytes of the Swiss strain were used to determine possible aneuploidy-inducing effects (Brook et al., 1985). Mice were injected intraperitoneally with 0.3-0.4 ml of 0.01 M NaOH and chromosome spreads were made 12 h after injection. NaOH was used as control substance. No evidence of non-disjunction was found in control groups up to the age of 40 weeks tested.

Both the in vitro and the in vivo genetic toxicity test indicated no evidence for a mutagenic activity. Furthermore NaOH is not expected to be systemically available in the body under normal handling and use conditions and for this reason additional testing is considered unnecessary (see section 3.1).

In vitro Studies

NaOH was assayed in the Ames reversion test with S. typhimurium strains TA1535, TA1537, TA1538, TA98, TA100 and in a DNA-repair test with E. coli strains WP2, WP67 and CM871 (De Flora et al., 1984). Based on the results of these tests NaOH was classified as non genotoxic.

The clastogenic activity of NaOH was studied in an in vitro chromosomal aberration test using Chinese hamster ovary (CHO) K1 cells (Morita et al., 1989). No clastogenic activity was found at NaOH concentrations of 0, 4, 8 and 16 mM NaOH, which corresponded with initial pH values of 7.4, 9.1, 9.7 and 10.6, respectively. Incubation of CHO-K1 cells with NaOH in the presence of rat liver S9 increased the clastogenic activity of S9, or induced new clastogens by breakdown of the S9. Therefore, testing at non-physiological pH might give false-positive responses, which means that the effect of sodium hydroxide is of a methodical kind and not valid to asses the genotoxicity under realistic conditions.

Justification for selection of genetic toxicity endpoint
Since sodium caprolactamate hydrolyses under physiological conditions with the formation of epsilon-Caprolactam and sodium hydroxide, cross-reading from toxicological studies of epsilon-Caprolactam is justified. For the substance’s endpoint assessment also the impact of sodium hydroxideis taken into consideration, however avaiable literature indicated that there is no contribution to be expected.

Justification for classification or non-classification

No indication for a genotoxic potential in vitro and in vivo was found. Therefore there is no indication for a genetic toxicity classification.